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Contents and supplementary information for: Principles of
Gene Manipulation
Chapter 1 Gene manipulation: an all-embracing technique
Chapter 2
Basic techniques - (POGC02.pdf, 1,560KB)
Chapter 3 Cutting and joining DNA molecules
Chapter 4 Basic biology of plasmid and phage vectors
Chapter 5 Cosmids, phasmids and other advanced vectors
Chapter 6 Cloning strategies
Additional updated information on Cloning strategies
Chapter 7 Sequencing and mutagenesis
Chapter 8 Cloning in bacteria other than E. coli
Chapter 9 Cloning in Saccharomyces cerevisiae and other fungi
Chapter 10 Gene transfer to animal cells
Additional updated information on Gene transfer to animal
cells
Chapter 11 Genetic manipulation of animals
Additional updated information on Genetic manipulation of
animals
Chapter 12 Gene transfer to plants
Additional updated information on Gene transfer to plants
Chapter 13 Advances in transgenic technology
Additional updated information on Advances in transgenic
technology or (POGC13.pdf - size: 353KB)

Chapter 14 Applications of recombinant DNA tecnology

Overview / Supplememtary Material / Related Titles / Related Websites / Ordering Information / Examination Copies / MCQ's /
New Edition
POGC01 9/11/2001 11:02 AM Page 1

CH A P T E R 1
Gene manipulation:
an all-embracing technique

Introduction Sequence analysis

Occasionally technical developments in science Cloning permits the isolation of discrete pieces of a
occur that enable leaps forward in our knowledge genome and their amplification. This in turn enables
and increase the potential for innovation. Molecular the DNA to be sequenced. Analysis of the sequences
biology and biomedical research experienced such of some genetically well-characterized genes led to
a revolutionary change in the mid-70s with the the identification of the sequences and structures
development of gene manipulation. Although the which characterize the principal control elements
initial experiments generated much excitement, it is of gene expression, e.g. promoters, ribosome bind-
unlikely that any of the early workers in the field ing sites, etc. As this information built up it became
could have predicted the breadth of applications to possible to scan new DNA sequences and identify
which the technique has been put. Nor could they potential new genes, or open reading frames, because
have envisaged that the methods they developed they were bounded by characteristic motifs. Initially
would spawn an entire industry comprising several this sequence analysis was done manually but to
hundred companies, of varying sizes, in the USA the eye long runs of nucleotides have little meaning
alone. and patterns evade recognition. Fortunately such
The term gene manipulation can be applied to analyses have been facilitated by rapid increases
a variety of sophisticated in vivo genetics as well as in the power of computers and improvements in soft-
to in vitro techniques. In fact, in most Western ware which have taken place contemporaneously
countries there is a precise legal definition of gene with advances in gene cloning. Now sequences can
manipulation as a result of government legisla- be scanned quickly for a whole series of structural
tion to control it. In the UK, gene manipulation is features, e.g. restriction enzyme recognition sites,
defined as start and stop signals for transcription, inverted
the formation of new combinations of heritable palindromes, sequence repeats, Z-DNA, etc., using
material by the insertion of nucleic acid molecules, programs available on the Internet.
produced by whatever means outside the cell, From the nucleotide sequence of a gene it is easy
into any virus, bacterial plasmid or other vector to deduce the protein sequence which it encodes.
system so as to allow their incorporation into a Unfortunately, we are unable to formulate a set of
host organism in which they do not naturally general rules that allows us to predict a protein’s
occur but in which they are capable of continued three-dimensional structure from the amino acid
propagation. sequence of its polypeptide chain. However, based
The definitions adopted by other countries are sim- on crystallographic data from over 300 proteins,
ilar and all adequately describe the subject-matter certain structural motifs can be predicted. Nor does
of this book. Simply put, gene manipulation per- an amino acid sequence on its own give any clue to
mits stretches of DNA to be isolated from their host function. The solution is to compare the amino acid
organism and propagated in the same or a different sequence with that of other better-characterized pro-
host, a technique known as cloning. The ability to teins: a high degree of homology suggests similarity
clone DNA has far-reaching consequences, as will in function. Again, computers are of great value
be shown below. since algorithms exist for comparing two sequences
or for comparing one sequence with a group of other
POGC01 9/11/2001 11:02 AM Page 2

2 CHAPTER 1

sequences simultaneously. The Internet has made Gene cloning provides elegant solutions to the above
such comparisons easy because researchers can problems. Once isolated, entire genes or groups of
access all the protein sequence data that are stored genes can be introduced back into the cell type whence
in central databases, which are updated daily. they came or into different cell types or completely new
organisms, e.g. bacterial genes in plants or animals.
The levels of gene expression can be measured directly
In vivo biochemistry
or through the use of reporter molecules and can
Any living cell, regardless of its origin, carries out a be modulated up or down at the whim of the experi-
plethora of biochemical reactions. To analyse these menter. Also, specific mutations, ranging from a
different reactions, biochemists break open cells, single base-pair to large deletions or additions, can
isolate the key components of interest and measure be built into the gene at any position to permit all
their levels. They purify these components and try kinds of structural and functional analyses. Function
to determine their performance characteristics. For in different cell types can also be analysed, e.g. do
example, in the case of an enzyme, they might deter- those structural features of a protein which result in
mine its substrate specificity and kinetic parameters, its secretion from a yeast cell enable it to be exported
such as Km and Vmax, and identify inhibitors and their from bacteria or higher eukaryotes? Experiments like
mode of action. From these data they try to build up these permit comparative studies of macromolecu-
a picture of what happens inside the cell. However, lar processes and, in some cases, gene cloning and
the properties of a purified enzyme in a test-tube sequencing provides the only way to begin to under-
may bear little resemblance to its behaviour when stand such events as mitosis, cell division, telomere
it shares the cell cytoplasm or a cell compartment structure, intron splicing, etc. Again, the Internet has
with thousands of other enzymes and chemical com- made such comparisons easy because researchers can
pounds. Understanding what happens inside cells access all the protein sequence data that are stored
has been facilitated by the use of mutants. These in central databases, which are updated daily.
permit the determination of the consequences of The original goal of sequencing was to determine
altered regulation or loss of a particular compon- the precise order of nucleotides in a gene. Then the
ent or activity. Mutants have also been useful in goal became the sequence of a small genome. First it
elucidating macromolecule structure and function. was that of a small virus (φX174, 5386 nucleotides).
However, the use of mutants is limited by the fact Then the goal was larger plasmid and viral genomes,
that with classical technologies one usually has then chromosomes and microbial genomes until
little control over the type of mutant isolated and/or ultimately the complete genomes of higher eukaryotes
location of the mutation. (humans, Arabidopsis) were sequenced (Table 1.1).

Table 1.1 Increases in sizes of genomes sequenced.

Genome sequenced Year Genome size Comment

Bacteriophage fX174 1977 5.38 kb First genome sequenced

Plasmid pBR322 1979 4.3 kb First plasmid sequenced
Bacteriophage l 1982 48.5 kb
Epstein–Barr virus 1984 172 kb
Yeast chromosome III 1992 315 kb First chromosome sequenced
Haemophilus influenzae 1995 1.8 Mb First genome of cellular organism to be sequenced
Saccharomyces cerevisiae 1996 12 Mb First eukaryotic genome to be sequenced
Ceanorhabditis elegans 1998 97 Mb First genome of multicellular organism to be sequenced
Drosophila melanogaster 2000 165 Mb
Homo sapiens 2000 3000 Mb First mammalian genome to be sequenced
Arabidopsis thaliana 2000 125 Mb First plant genome to be sequenced
POGC01 9/11/2001 11:02 AM Page 3

Gene manipulation 3

Now the sequencing of large genomes has become recombinant DNA technology been greater than on
routine, albeit in specialist laboratories. Having the the practice of medicine.
complete genome sequence of an organism provides The first medical benefit to arise from recombinant
us with fascinating insights into certain aspects DNA technology was the availability of significant
of its biology. For example, we can determine the quantities of therapeutic proteins, such as human
metabolic capabilities of a new microbe without growth hormone (HGH). This protein is used to treat
knowing anything about its physiology. However, adolescents suffering from pituitary dwarfism to enable
there are many aspects of cellular biology that can- them to achieve a normal height. Originally HGH was
not be ascertained from sequence data alone. For purified from pituitary glands removed from cadavers.
example, what RNA species are made when in the However, a very large number of pituitary glands
cell or organism life cycle and how fast do they are required to produce sufficient HGH to treat just
turn over? What proteins are made when and how one child. Furthermore, some children treated with
do the different proteins in a cell interact? How does pituitary-derived HGH have developed Creutzfeld–
environment affect gene expression? The answers to Jakob syndrome. Following the cloning and expres-
these questions are being provided by the new dis- sion of the HGH gene in Escherichia coli, it is possible
ciplines of genomics, proteomics and environomics to produce enough HGH in a 10 litre fermenter to
which rely heavily on the techniques of gene mani- treat hundreds of children. Since then, many differ-
pulation, which are discussed in later chapters. A ent therapeutic proteins have become available for
detailed presentation of whole-genome sequencing, the first time. Many of these proteins are also manu-
genomics and proteomics can be found in Primrose factured in E. coli but others are made in yeast or
and Twyman (2002). animal cells and some in plants or the milk of animals.
The only common factor is that the relevant gene
has been cloned and overexpressed using the tech-
The new medicine
niques of gene manipulation.
The developments in gene manipulation that have Medicine has benefited from recombinant DNA
taken place in the last 25 years have revolutionized technology in other ways (Fig. 1.1). New routes to
the study of biology. There is no subject area within vaccines have been developed. The current hepatitis
biology where recombinant DNA is not being used B vaccine is based on the expression of a viral anti-
and as a result the old divisions between subject areas gen on the surface of yeast cells and a recombinant
such as botany, genetics, zoology, biochemistry, etc. vaccine has been used to eliminate rabies from foxes
are fast breaking down. Nowhere has the impact of in a large part of Europe. Gene manipulation can

Genetic
Profiling Cloned P450s
disease
Infectious
disease
Animal models Diagnostic
or human disease Pharamacogenomics nucleic
acids
Plants Gene therapy

Gene repair
Therapeutic Therapeutic
small molecules MEDICINE nucleic
acids Anti-sense drugs
Microbes
DNA
Vaccines
Diagnostic Therapeutic Vaccines
proteins proteins
Microbes
Plants
Microbes Animals

Fig. 1.1 The impact of gene manipulation on the practice of medicine.

POGC01 9/11/2001 11:02 AM Page 4

4 CHAPTER 1

also be used to increase the levels of small molecules exploit the potential of recombinant DNA technology.
within microbial cells. This can be done by cloning Thus was Genentech born (Box 1.1). Since then
all the genes for a particular biosynthetic pathway thousands of biotechnology companies have been
and overexpressing them. Alternatively, it is pos- formed worldwide. As soon as major new develop-
sible to shut down particular metabolic pathways ments in the science of gene manipulation are
and thus redirect particular intermediates towards reported, a rash of new companies are formed to
the desired end-product. This approach has been commercialize the new technology. For example,
used to facilitate production of chiral intermediates many recently formed companies are hoping the
and antibiotics. Novel antibiotics can also be created data from the Human Genome Sequencing Project
by mixing and matching genes from organisms pro- will result in the identification of a large number
ducing different but related molecules in a technique of new proteins with potential for human therapy.
known as combinatorial biosynthesis. Others are using gene manipulation to understand
Gene cloning enables nucleic acid probes to be the regulation of transcription of particular genes,
produced readily and such probes have many uses arguing that it would make better therapeutic sense
in medicine. For example, they can be used to to modulate the process with low-molecular-weight,
determine or confirm the identity of a microbial orally active drugs.
pathogen or to diagnose pre- or perinatally an Although there are thousands of biotechno-
inherited genetic disease. Increasingly, probes are logy companies, fewer than 100 have sales of their
being used to determine the likelihood of adverse products and even fewer are profitable. Already
reactions to drugs or to select the best class of drug many biotechnology companies have failed, but
to treat a particular illness (pharmacogenomics). A the technology advances at such a rate that there
variant of this technique is to use cloned cytochrome is no shortage of new company start-ups to take
P450s to determine how a new drug will be meta- their place. One group of biotechnology companies
bolized and if any potentially toxic by-products will that has prospered is those supplying specialist
result. reagents to laboratory workers engaged in gene
Nucleic acids are also being used as therapeutic manipulation. In the very beginning, researchers
entities in their own right. For example, antisense had to make their own restriction enzymes and
nucleic acids are being used to down-regulate gene this restricted the technology to those with pro-
expression in certain diseases. In other cases, nucleic tein chemistry skills. Soon a number of com-
acids are being administered to correct or repair panies were formed which catered to the needs of
inherited gene defects (gene therapy/gene repair) researchers by supplying high-quality enzymes for
or as vaccines. In the reverse of gene repair, animals DNA manipulation. Despite the availability of these
are being generated that have mutations iden- enzymes, many people had great difficulty in clon-
tical to those found in human disease. Note that ing DNA. The reason for this was the need for care-
the use of antisense nucleic acids and gene therapy/ ful quality control of all the components used in
repair depends on the availability of information the preparation of reagents, something researchers
on the exact cause of a disease. For most medical are not good at! The supply companies responded
conditions such information is lacking and cur- by making easy-to-use cloning kits in addition to
rently available drugs are used to treat symptoms. enzymes. Today, these supply companies can pro-
This situation will change significantly in the next vide almost everything that is needed to clone,
decade. express and analyse DNA and have thereby accel-
erated the use of recombinant DNA technology
in all biological disciplines. In the early days of
Biotechnology: the new industry
recombinant DNA technology, the development of
The early successes in overproducing mammalian methodology was an end in itself for many academic
proteins in E. coli suggested to a few entrepreneurial researchers. This is no longer true. The researchers
individuals that a new company should be formed to have gone back to using the tools to further our
POGC01 9/11/2001 11:02 AM Page 5

Gene manipulation 5

Box 1.1 The birth of an industry

Biotechnology is not new. Cheese, bread and yoghurt of gene manipulation. Swanson’s enthusiasm for the
are products of biotechnology and have been known technology and his faith in it was contagious. By
for centuries. However, the stock-market excitement the close of the meeting the decision was taken to
about biotechnology stems from the potential of found Genentech (Genetic Engineering Technology).
gene manipulation, which is the subject of this book. Though Swanson and Boyer faced scepticism from
The birth of this modern version of biotechnology both the academic and business communities they
can be traced to the founding of the company forged ahead with their idea. Successes came thick
Genentech. and fast (see Table B1.1) and within a few years
In 1976, a 27-year-old venture capitalist called they had proved their detractors wrong. Over
Robert Swanson had a discussion over a few beers 1000 biotechnology companies have been set up in
with a University of California professor, Herb Boyer. the USA alone since the founding of Genentech
The discussion centred on the commercial potential but very, very few have been as successful.

Table B1.1 Key events at Genentech.

1976 Genentech founded

1977 Genentech produced first human protein (somatostatin) in a microorganism
1978 Human insulin cloned by Genentech scientists
1979 Human growth hormone cloned by Genentech scientists
1980 Genentech went public, raising $35 million
1982 First recombinant DNA drug (human insulin) marketed (Genentech product licensed to Eli Lilly & Co.)
1984 First laboratory production of factor VIII for therapy of haemophilia. Licence granted to Cutter Biological
1985 Genentech launched its first product, Protropin (human growth hormone), for growth hormone deficiency in children
1987 Genentech launched Activase (tissue plasminogen activator) for dissolving blood clots in heart-attack patients
1990 Genentech launched Actimmune (interferon-g1b ) for treatment of chronic granulomatous disease
1990 Genentech and the Swiss pharmaceutical company Roche complete a $2.1 billion merger

knowledge of biology, and the development of cloning experiments were undertaken in E. coli.
new methodologies has largely fallen to the supply Subsequently, cloning techniques were extended to
companies. a range of other microorganisms, such as Bacillus
subtilis, Pseudomonas sp., yeasts and filamentous
fungi, and then to higher eukaryotes. Curiously,
The central role of E. coli
cloning in E. coli is technically easier than in any
E. coli has always been a popular model system other organism. As a result, it is rare for researchers
for molecular geneticists. Prior to the development to clone DNA directly in other organisms. Rather,
of recombinant DNA technology, there existed a DNA from the organism of choice is first mani-
large number of well-characterized mutants, gene pulated in E. coli and subsequently transferred back
regulation was understood and there was a ready to the original host. Without the ability to clone
availability of a wide selection of plasmids. Com- and manipulate DNA in E. coli, the application of
pared with other microbial systems it was match- recombinant DNA technology to other organisms
less. It is not surprising, therefore, that the first would be greatly hindered.
POGC01 9/11/2001 11:02 AM Page 6

6 CHAPTER 1

The role of vectors Chapter 2

Basic Agarose gel electrophoresis
Techniques Blotting (DNA, RNA, protein)
Nucleic acid hybridization
DNA transformation & electroporation
Polymerase chain reaction (PCR)

Cutting & Restriction enzymes Chapter 3

Joining DNA Methods of joining DNA

Basic properties of plasmids Chapters 4 & 5

Desirable properties of vectors
Plasmids as vectors
Vectors Bacteriophage λ vectors
Single-stranded DNA vectors
Vectors for cloning large DNA molecules
Specialist vectors
Over-producing proteins

Cloning strategies Chapter 6

Putting it Cloning genomic DNA
all together: cDNA cloning
Cloning in Screening strategies
Practice Expression cloning
Difference cloning

Analysing & Basic DNA sequencing Chapter 7

Changing Analysing sequence data Fig. 1.2 ‘Roadmap’ outlining the basic
Cloned Site-directed mutagenesis
Genes Phage display techniques in gene manipulation and
their relationships.

vectors was the propagation of cloned DNA but

Outline of the rest of the book
today vectors fulfil many other roles, such as facil-
As noted above, E. coli has an essential role in recom- itating DNA sequencing, promoting expression of
binant DNA technology. Therefore, the first half of cloned genes, facilitating purification of cloned gene
the book is devoted to the methodology for manipu- products, etc. The specialist vectors for these tasks
lating genes in this organism (Fig. 1.2). Chapter 2 are described in Chapter 5. With this background in
covers many of the techniques that are common place it is possible to describe in detail how to clone
to all cloning experiments and are fundamental to the particular DNA sequences that one wants. There
the success of the technology. Chapter 3 is devoted are two basic strategies. Either one clones all the
to methods for selectively cutting DNA molecules DNA from an organism and then selects the very
into fragments that can be readily joined together small number of clones of interest or one amplifies
again. Without the ability to do this, there would the DNA sequences of interest and then clones these.
be no recombinant DNA technology. If fragments Both these strategies are described in Chapter 6.
of DNA are inserted into cells, they fail to replicate Once the DNA of interest has been cloned, it can
except in those rare cases where they integrate into be sequenced and this will yield information on
the chromosome. To enable such fragments to be the proteins that are encoded and any regulatory
propagated, they are inserted into DNA molecules signals that are present. There might also be a wish
(vectors) that are capable of extrachromosomal re- to modify the DNA and/or protein sequence and
plication. These vectors are derived from plasmids determine the biological effects of such changes.
and bacteriophages and their basic properties are The techniques for sequencing and changing cloned
described in Chapter 4. Originally, the purpose of genes are described in Chapter 7.
POGC01 9/11/2001 11:02 AM Page 7

Gene manipulation 7

Cloning in
Getting DNA into bacteria Chapter 8
Bacteria
Cloning in Gram-negative bacteria Other Than
Cloning in Gram-positive bacteria E.coli

Why clone in fungi Chapter 9 Cloning in

Vectors for use in fungi Yeast &
Expression of cloned DNA Other
Two hybrid system Fungi
Analysis of the whole genome

Transformation of animal cells Chapter 10 Gene

Use of non-replicating DNA Transfer
Replication vectors To Animal
Viral transduction Cells

Transgenic mice Chapter 11 Genetic

Other transgenic mammals
Manipulation
Transgenic birds, fish, Xenopus of Animals
Transgenic invertebrates

Handling plant cells Chapter 12 Genetic

Agrobacterium-mediated transformation
Manipulation
Direct DNA transfer
of Plants
Plant viruses as vectors

Inducible expression systems Chapter 13 Advanced

Site-specific recombination Techniques
Fig. 1.3 ‘Roadmap’ of the advanced Gene inhibition for Gene
techniques in gene manipulation and Insertional mutagenesis Manipulation
Gene tagging in Plant and
their application to organisms other Entrapment constructs Animals
than E. coli.

In the second half of the book the specialist tech- and plant representatives of higher eukaryotes and
niques for cloning in organisms other than E. coli Chapter 13 covers some cutting-edge techniques for
are described (Fig. 1.3). Each of these chapters can these same systems.
be read in isolation from the other chapters in this The concluding chapter is a survey of the dif-
section, provided that there is a thorough under- ferent applications of recombinant DNA techno-
standing of the material from the first half of the logy that are being exploited by the biotechnology
book. Chapter 8 details the methods for cloning in industry. Rather than going through application
other bacteria. Originally it was thought that some after application, we have opted to show the inter-
of these bacteria, e.g. B. subtilis, would usurp the play of different technologies by focusing on six
position of E. coli. This has not happened and gene themes:
manipulation techniques are used simply to better • Nucleic acid sequences as diagnostic tools.
understand the biology of these bacteria. Chapter 9 • New drugs and new therapies for genetic diseases.
focuses on cloning in fungi, although the emphasis • Combating infectious disease.
is on the yeast Saccharomyces cerevisiae. Fungi are • Protein engineering.
eukaryotes and are useful model systems for invest- • Metabolic engineering.
igating topics such as meiosis and mitosis, control of • Plant breeding in the twenty-first century.
cell division, etc. Animal cells can be cultured like By treating the topic in this way we have been able to
microorganisms and the techniques for cloning in show the interplay between some of the basic tech-
them are described in Chapter 10. Chapters 11 and niques and the sophisticated analysis now possible
12 are devoted to the intricacies of cloning in animal with genome sequence information.
POGC02 9/11/2001 11:01 AM Page 8

CHAPTER 2
Basic techniques

and in the absence of replication will be diluted out of

Introduction
their host cells. It should be noted that, even if a DNA
The initial impetus for gene manipulation in vitro molecule contains an origin of replication, this may
came about in the early 1970s with the simultan- not function in a foreign host cell.
eous development of techniques for: There is an additional, subsequent problem. If the
• genetic transformation of Escherichia coli; early experiments were to proceed, a method was
• cutting and joining DNA molecules; required for assessing the fate of the donor DNA. In
• monitoring the cutting and joining reactions. particular, in circumstances where the foreign DNA
In order to explain the significance of these devel- was maintained because it had become integrated in
opments we must first consider the essential require- the host DNA, a method was required for mapping the
ments of a successful gene-manipulation procedure. foreign DNA and the surrounding host sequences.

The basic problems The solutions: basic techniques

Before the advent of modern gene-manipulation If fragments of DNA are not replicated, the obvious
methods there had been many early attempts at solution is to attach them to a suitable replicon.
transforming pro- and eukaryotic cells with foreign Such replicons are known as vectors or cloning
DNA. But, in general, little progress could be made. vehicles. Small plasmids and bacteriophages are the
The reasons for this are as follows. Let us assume most suitable vectors for they are replicons in their
that the exogenous DNA is taken up by the recipient own right, their maintenance does not necessarily
cells. There are then two basic difficulties. First, require integration into the host genome and their
where detection of uptake is dependent on gene DNA can be readily isolated in an intact form. The
expression, failure could be due to lack of accurate different plasmids and phages which are used as
transcription or translation. Secondly, and more vectors are described in detail in Chapters 4 and 5.
importantly, the exogenous DNA may not be main- Suffice it to say at this point that initially plasmids
tained in the transformed cells. If the exogenous and phages suitable as vectors were only found in E.
DNA is integrated into the host genome, there is no coli. An important consequence follows from the use
problem. The exact mechanism whereby this integ- of a vector to carry the foreign DNA: simple methods
ration occurs is not clear and it is usually a rare become available for purifying the vector molecule,
event. However this occurs, the result is that the complete with its foreign DNA insert, from trans-
foreign DNA sequence becomes incorporated into formed host cells. Thus not only does the vector
the host cell’s genetic material and will subsequently provide the replicon function, but it also permits the
be propagated as part of that genome. If, however, easy bulk preparation of the foreign DNA sequence,
the exogenous DNA fails to be integrated, it will free from host-cell DNA.
probably be lost during subsequent multiplication of Composite molecules in which foreign DNA has
the host cells. The reason for this is simple. In order been inserted into a vector molecule are sometimes
to be replicated, DNA molecules must contain an called DNA chimeras because of their analogy with
origin of replication, and in bacteria and viruses there the Chimaera of mythology – a creature with the
is usually only one per genome. Such molecules are head of a lion, body of a goat and tail of a serpent.
called replicons. Fragments of DNA are not replicons The construction of such composite or artificial
POGC02 9/11/2001 11:01 AM Page 9

Basic techniques 9

recombinant molecules has also been termed genetic

engineering or gene manipulation because of the po-
kb pairs
tential for creating novel genetic combinations by
21.226
biochemical means. The process has also been termed
molecular cloning or gene cloning because a line of
genetically identical organisms, all of which contain 7.421
the composite molecule, can be propagated and grown
5.804
in bulk, hence amplifying the composite molecule
5.643
and any gene product whose synthesis it directs. 4.878
Although conceptually very simple, cloning of
a fragment of foreign, or passenger, or target DNA
in a vector demands that the following can be 3.530
accomplished.
• The vector DNA must be purified and cut open.
• The passenger DNA must be inserted into the
–
vector molecule to create the artificial recombinant.
DNA joining reactions must therefore be performed.
Methods for cutting and joining DNA molecules are
now so sophisticated that they warrant a chapter of
their own (Chapter 3).
• The cutting and joining reactions must be read-
ily monitored. This is achieved by the use of gel +
electrophoresis.
Fig. 2.1 Electrophoresis of DNA in agarose gels. The direction
• Finally, the artificial recombinant must be trans-
of migration is indicated by the arrow. DNA bands have been
formed into E. coli or another host cell. Further details visualized by soaking the gel in a solution of ethidium bromide
on the use of gel electrophoresis and transformation (see Fig. 2.3), which complexes with DNA by intercalating
of E. coli are given in the next section. As we have between stacked base-pairs, and photographing the orange
noted, the necessary techniques became available at fluorescence which results upon ultraviolet irradiation.
about the same time and quickly led to many cloning
experiments, the first of which were reported in
1972 ( Jackson et al. 1972, Lobban & Kaiser 1973). electrophoresis is not well understood (Holmes
& Stellwagen 1990). The migration of the DNA
molecules through the pores of the matrix must play
Agarose gel electrophoresis
an important role in molecular-weight separations
The progress of the first experiments on cutting and since the electrophoretic mobility of DNA in free
joining of DNA molecules was monitored by velocity solution is independent of molecular weight. An
sedimentation in sucrose gradients. However, this agarose gel is a complex network of polymeric
has been entirely superseded by gel electrophoresis. molecules whose average pore size depends on the
Gel electrophoresis is not only used as an analytical buffer composition and the type and concentration
method, it is routinely used preparatively for the of agarose used. DNA movement through the gel
purification of specific DNA fragments. The gel is was originally thought to resemble the motion of a
composed of polyacrylamide or agarose. Agarose is snake (reptation). However, real-time fluorescence
convenient for separating DNA fragments ranging microscopy of stained molecules undergoing elec-
in size from a few hundred base pairs to about 20 kb trophoresis has revealed more subtle dynamics
(Fig. 2.1). Polyacrylamide is preferred for smaller (Schwartz & Koval 1989, Smith et al. 1989). DNA
DNA fragments. molecules display elastic behaviour by stretching in
The mechanism responsible for the separation the direction of the applied field and then contract-
of DNA molecules by molecular weight during gel ing into dense balls. The larger the pore size of the
POGC02 9/11/2001 11:01 AM Page 10

10 CHAPTER 2

gel, the greater the ball of DNA which can pass

through and hence the larger the molecules which A– B–
can be separated. Once the globular volume of the
DNA molecule exceeds the pore size, the DNA

Migration of DNA
molecule can only pass through by reptation. This
120°
occurs with molecules about 20 kb in size and it is
difficult to separate molecules larger than this with-
out recourse to pulsed electrical fields.
In pulsed-field gel electrophoresis (PFGE) (Schwartz
B+ A+
& Cantor 1984) molecules as large as 10 Mb can be
separated in agarose gels. This is achieved by caus-
ing the DNA to periodically alter its direction of
migration by regular changes in the orientation of Fig. 2.2 Schematic representation of CHEF (contour-clamped
the electric field with respect to the gel. With each homogeneous electrical field) pulsed-field gel electrophoresis.
change in the electric-field orientation, the DNA
must realign its axis prior to migrating in the new
NH2
direction. Electric-field parameters, such as the
direction, intensity and duration of the electric field,
are set independently for each of the different fields
Br –
and are chosen so that the net migration of the DNA N⊕
is down the gel. The difference between the direction H2 N C2H5

of migration induced by each of the electric fields is

the reorientation angle and corresponds to the angle
that the DNA must turn as it changes its direction of
migration each time the fields are switched. Fig. 2.3 Ethidium bromide.
A major disadvantage of PFGE, as originally
described, is that the samples do not run in straight
lines. This makes subsequent analysis difficult. This line over a wider range than the semilogarithmic
problem has been overcome by the development of plot. In any event, gel electrophoresis is frequently
improved methods for alternating the electrical field. performed with marker DNA fragments of known
The most popular of these is contour-clamped homo- size, which allow accurate size determination of an
geneous electrical-field electrophoresis (CHEF) (Chu unknown DNA molecule by interpolation. A par-
et al. 1986). In early CHEF-type systems (Fig. 2.2) ticular advantage of gel electrophoresis is that the
the reorientation angle was fixed at 120°. However, DNA bands can be readily detected at high sensitiv-
in newer systems, the reorientation angle can be ity. The bands of DNA in the gel are stained with
varied and it has been found that for whole-yeast the intercalating dye ethidium bromide (Fig. 2.3),
chromosomes the migration rate is much faster with and as little as 0.05 µg of DNA in one band can be
an angle of 106° (Birren et al. 1988). Fragments of detected as visible fluorescence when the gel is
DNA as large as 200–300 kb are routinely handled illuminated with ultraviolet light.
in genomics work and these can be separated in a In addition to resolving DNA fragments of differ-
matter of hours using CHEF systems with a reori- ent lengths, gel electrophoresis can be used to separ-
entation angle of 90° or less (Birren & Lai 1994). ate different molecular configurations of a DNA
Aaij and Borst (1972) showed that the migration molecule. Examples of this are given in Chapter 4
rates of the DNA molecules were inversely propor- (see p. 44). Gel electrophoresis can also be used for
tional to the logarithms of the molecular weights. investigating protein–nucleic acid interactions in
Subsequently, Southern (1979a,b) showed that the so-called gel retardation or band shift assay. It is
plotting fragment length or molecular weight based on the observation that binding of a protein
against the reciprocal of mobility gives a straight to DNA fragments usually leads to a reduction in
POGC02 9/11/2001 11:01 AM Page 11

Basic techniques 11

electrophoretic mobility. The assay typically involves • blotting of nucleic acids from gels;
the addition of protein to linear double-stranded DNA • dot and slot blotting;
fragments, separation of complex and naked DNA • colony and plaque blotting.
by gel electrophoresis and visualization. A review of Colony and plaque blotting are described in detail on
the physical basis of electrophoretic mobility shifts and pp. 104–105 and dot and slot blotting in Chapter 14.
their application is provided by Lane et al. (1992).
Southern blotting
Nucleic acid blotting
The original method of blotting was developed by
Nucleic acid labelling and hybridization on mem- Southern (1975, 1979b) for detecting fragments in
branes have formed the basis for a range of experi- an agarose gel that are complementary to a given
mental techniques central to recent advances in our RNA or DNA sequence. In this procedure, referred to
understanding of the organization and expression as Southern blotting, the agarose gel is mounted on
of the genetic material. These techniques may be a filter-paper wick which dips into a reservoir con-
applied in the isolation and quantification of specific taining transfer buffer (Fig. 2.5). The hybridization
nucleic acid sequences and in the study of their membrane is sandwiched between the gel and a
organization, intracellular localization, expression stack of paper towels (or other absorbent material),
and regulation. A variety of specific applications which serves to draw the transfer buffer through the
includes the diagnosis of infectious and inherited gel by capillary action. The DNA molecules are car-
disease. Each of these topics is covered in depth in ried out of the gel by the buffer flow and immobilized
subsequent chapters. on the membrane. Initially, the membrane material
An overview of the steps involved in nucleic acid used was nitrocellulose. The main drawback with
blotting and membrane hybridization procedures is this membrane is its fragile nature. Supported nylon
shown in Fig. 2.4. Blotting describes the immobiliza- membranes have since been developed which have
tion of sample nucleic acids on to a solid support, greater binding capacity for nucleic acids in addition
generally nylon or nitrocellulose membranes. The to high tensile strength.
blotted nucleic acids are then used as ‘targets’ in For efficient Southern blotting, gel pretreatment is
subsequent hybridization experiments. The main important. Large DNA fragments (> 10 kb) require a
blotting procedures are: longer transfer time than short fragments. To allow

Immobilization of nucleic acids

• Southern blot
• Northern blot
• Dot blot
• Colony/plaque lift

Pre-hybridization

Labelled DNA
or RNA probe

Hybridization Removal of probe

prior to reprobing

Stringency washes
Fig. 2.4 Overview of nucleic acid
blotting and hybridization (reproduced
courtesy of Amersham Pharmacia
Detection
Biotech).
POGC02 9/11/2001 11:01 AM Page 12

12 CHAPTER 2

Weight < 0.75 kg After transfer, the nucleic acid needs to be fixed to
the membrane and a number of methods are avail-
Glass plate
able. Oven baking at 80°C is the recommended
Paper tissues method for nitrocellulose membranes and this can
also be used with nylon membranes. Due to the
3 sheets filter paper flammable nature of nitrocellulose, it is important
Membrane that it is baked in a vacuum oven. An alternative
Gel fixation method utilizes ultraviolet cross-linking. It
is based on the formation of cross-links between a
small fraction of the thymine residues in the DNA
Plastic tray
and positively charged amino groups on the surface
of nylon membranes. A calibration experiment must
Fig. 2.5 A typical capillary blotting apparatus. be performed to determine the optimal fixation period.
Following the fixation step, the membrane is placed
in a solution of labelled (radioactive or non-radioactive)
uniform transfer of a wide range of DNA fragment RNA, single-stranded DNA or oligodeoxynucleotide
sizes, the electrophoresed DNA is exposed to a short which is complementary in sequence to the blot-
depurination treatment (0.25 mol/l HCl) followed by transferred DNA band or bands to be detected.
alkali. This shortens the DNA fragments by alkaline Conditions are chosen so that the labelled nucleic
hydrolysis at depurinated sites. It also denatures the acid hybridizes with the DNA on the membrane.
fragments prior to transfer, ensuring that they are in Since this labelled nucleic acid is used to detect and
the single-stranded state and accessible for probing. locate the complementary sequence, it is called the
Finally, the gel is equilibrated in neutralizing solution probe. Conditions are chosen which maximize the
prior to blotting. An alternative method uses posit- rate of hybridization, compatible with a low back-
ively charged nylon membranes, which remove the ground of non-specific binding on the membrane
need for extended gel pretreatment. With them the (see Box 2.1). After the hybridization reaction has
DNA is transferred in native (non-denatured) form been carried out, the membrane is washed to remove
and then alkali-denatured in situ on the membrane. unbound radioactivity and regions of hybridization

Box 2.1 Hybridization of nucleic acids on membranes

The hybridization of nucleic acids on membranes is a to the probe. Prolonged incubations may not generate
widely used technique in gene manipulation and any significant increase in detection sensitivity.
analysis. Unlike solution hybridizations, membrane The composition of the hybridization buffer can
hybridizations tend not to proceed to completion. greatly affect the speed of the reaction and the
One reason for this is that some of the bound nucleic sensitivity of detection. The key components of these
acid is embedded in the membrane and is inaccessible buffers are shown below:

Rate enhancers Dextran sulphate and other polymers act as volume excluders to increase both the rate and the
extent of hybridization
Detergents and blocking agents Dried milk, heparin and detergents such as sodium dodecyl sulphate (SDS) have been used
to depress non-specific binding of the probe to the membrane. Denhardt’s solution
(Denhardt 1966) uses Ficoll, polyvinylpyrrolidone and bovine serum albumin
Denaturants Urea or formamide can be used to depress the melting temperature of the hybrid so that reduced
temperatures of hybridization can be used
Heterologous DNA This can reduce non-specific binding of probes to non-homologous DNA on the blot

continued
POGC02 9/11/2001 11:01 AM Page 13

Basic techniques 13

Box 2.1 continued

oligonucleotides. It is standard practice to use

Stringency control
oligonucleotides to analyse putative mutants
Stringency can be regarded as the specificity with following a site-directed mutagenesis experiment
which a particular target sequence is detected by where the difference between parental and mutant
hybridization to a probe. Thus, at high stringency, progeny is often only a single base-pair change
only completely complementary sequences will be (see p. 132 et seq.).
bound, whereas low-stringency conditions will allow The availability of the exact sequence of
hybridization to partially matched sequences. oligonucleotides allows conditions for hybridization
Stringency is most commonly controlled by the and stringency washing to be tightly controlled so
temperature and salt concentration in the post- that the probe will only remain hybridized when
hybridization washes, although these parameters it is 100% homologous to the target. Stringency is
can also be utilized in the hybridization step. commonly controlled by adjusting the temperature
In practice, the stringency washes are performed of the wash buffer. The ‘Wallace rule’ (Lay Thein &
under successively more stringent conditions Wallace 1986) is used to determine the appropriate
(lower salt or higher temperature) until the desired stringency wash temperature:
result is obtained.
Tm = 4 × (number of GC base pairs) + 2 × (number
The melting temperature (Tm) of a probe–target
of AT base pairs)
hybrid can be calculated to provide a starting-point
for the determination of correct stringency. The In filter hybridizations with oligonucleotide probes,
Tm is the temperature at which the probe and the hybridization step is usually performed at 5°C
target are 50% dissociated. For probes longer than below Tm for perfectly matched sequences. For every
100 base pairs: mismatched base pair, a further 5°C reduction is
necessary to maintain hybrid stability.
Tm = 81.5°C + 16.6 log M + 0.41 (% G + C)
The design of oligonucleotides for hybridization
where M = ionic strength of buffer in moles/litre. experiments is critical to maximize hybridization
With long probes, the hybridization is usually carried specificity. Consideration should be given to:
out at Tm − 25°C. When the probe is used to • probe length – the longer the oligonucleotide, the
detect partially matched sequences, the less chance there is of it binding to sequences other
hybridization temperature is reduced by 1°C than the desired target sequence under conditions
for every 1% sequence divergence between of high stringency;
probe and target. • oligonucleotide composition – the GC content
Oligonucleotides can give a more rapid will influence the stability of the resultant hybrid
hybridization rate than long probes as they can and hence the determination of the appropriate
be used at a higher molarity. Also, in situations stringency washing conditions. Also the presence
where target is in excess to the probe, for example of any non-complementary bases will have an effect
dot blots, the hybridization rate is diffusion-limited on the hybridization conditions.
and longer probes diffuse more slowly than

are detected autoradiographically by placing the stringency (i.e. higher temperature or, more com-
membrane in contact with X-ray film (see Box 2.2). monly, lower ionic strength). Autoradiography
A common approach is to carry out the hybridiza- following each washing stage will reveal any DNA
tion under conditions of relatively low stringency bands that are related to, but not perfectly comple-
which permit a high rate of hybridization, followed mentary with, the probe and will also permit an
by a series of post-hybridization washes of increasing estimate of the degree of mismatching to be made.
POGC02 9/11/2001 11:01 AM Page 14

14 CHAPTER 2

Box 2.2 The principles of autoradiography

The localization and recording of a radiolabel within autoradiograph. It is best suited to detection of
a solid specimen is known as autoradiography weak- to medium-strength b-emitting radionuclides
and involves the production of an image in a (3H, 14C, 35S). Direct autoradiography is not suited
photographic emulsion. Such emulsions consist of to the detection of highly energetic b-particles,
silver halide crystals suspended in a clear phase such as those from 32P, or for g-rays emitted from
composed mainly of gelatin. When a b-particle or isotopes like 125I. These emissions pass through and
g-ray from a radionuclide passes through the beyond the film, with the majority of the energy
emulsion, the silver ions are converted to silver atoms. being wasted. Both 32P and 125I are best detected
This results in a latent image being produced, which by indirect autoradiography.
is converted to a visible image when the image is Indirect autoradiography describes the technique
developed. Development is a system of amplification by which emitted energy is converted to light by
in which the silver atoms cause the entire silver halide means of a scintillator, using fluorography or
crystal to be reduced to metallic silver. Unexposed intensifying screens. In fluorography the sample
crystals are removed by dissolution in fixer, giving is impregnated with a liquid scintillator. The
an autoradiographic image which represents the radioactive emissions transfer their energy to the
distribution of radiolabel in the original sample. scintillator molecules, which then emit photons which
In direct autoradiography, the sample is placed expose the photographic emulsion. Fluorography
in intimate contact with the film and the radioactive is mostly used to improve the detection of weak
emissions produce black areas on the developed b-emitters (Fig. B2.1). Intensifying screens are

35S 3H

+ − + −

Fig. B2.1 Autoradiographs showing the detection of 35S- and 3H-labelled proteins in acrylamide gels with (+) and without
(−) fluorography. (Photo courtesy of Amersham Pharmacia Biotech.)
continued
POGC02 9/11/2001 11:01 AM Page 15

Basic techniques 15

Box 2.2 continued

sheets of a solid inorganic scintillator which are This means that the probability of a second photon
placed behind the film. Any emissions passing being captured before the first silver atom has
through the photographic emulsion are absorbed reverted is greater for large amounts of radioactivity
by the screen and converted to light, effectively than for small amounts. Hence small amounts of
superimposing a photographic image upon the radioactivity are under-represented with the use of
direct autoradiographic image. fluorography and intensifying screens. This problem
The gain in sensitivity which is achieved by use of can be overcome by a combination of pre-exposing a
indirect autoradiography is offset by non-linearity film to an instantaneous flash of light (pre-flashing)
of film response. A single hit by a b-particle or g-ray and exposing the autoradiograph at −70°C.
can produce hundreds of silver atoms, but a single Pre-flashing provides many of the silver halide
hit by a photon of light produces only a single silver crystals of the film with a stable pair of silver atoms.
atom. Although two or more silver atoms in a silver Lowering the temperature to −70°C increases the
halide crystal are stable, a single silver atom is stability of a single silver atom, increasing the time
unstable and reverts to a silver ion very rapidly. available to capture a second photon (Fig. B2.2).

A B C

Fig. B2.2 The improvement in sensitivity of detection of 125I-labelled IgG by autoradiography obtained by using an
intensifying screen and pre-flashed film. A, no screen and no pre-flashing; B, screen present but film not pre-flashed;
C, use of screen and pre-flashed film. (Photo courtesy of Amersham Pharmacia Biotech.)
POGC02 9/11/2001 11:01 AM Page 16

16 CHAPTER 2

Long DNA –
fragments
Gene X Restriction Gel Fig. 2.6 Mapping restriction sites
endo- electro- around a hypothetical gene sequence
nuclease phoresis DNA
fragments in total genomic DNA by the Southern
blot method.
Short DNA
Genomic DNA is cleaved with a
fragments restriction endonuclease into hundreds
+
of thousands of fragments of various
Genomic DNA Genomic DNA Agarose gel sizes. The fragments are separated
according to size by gel electrophoresis
and blot-transferred on to nitrocellulose
(1) Denature in alkali
(2) Blot-transfer, bake paper. Highly radioactive RNA or
denatured DNA complementary in
sequence to gene X is applied to the
(1) Hybridize nitrocellulose Nitrocellulose nitrocellulose paper bearing the blotted
Autoradio- with radioactive probe DNA. The radiolabelled RNA or DNA
graphy
will hybridize with gene X sequences
(2) Wash
and can be detected subsequently by
Photographic autoradiography, so enabling the sizes
film of restriction fragments containing
gene X sequences to be estimated from
their electrophoretic mobility. By
Images correspond only to Radioactive RNA or using several restriction endonucleases
fragments containing gene X denatured DNA containing singly and in combination, a map of
sequences – estimate sequences complementary Single stranded
fragment sizes from mobility to gene X (radioactive probe) DNA fragments restriction sites in and around gene
X can be built up.

The Southern blotting methodology can be extre- ization with radiolabelled DNA probes. As before,
mely sensitive. It can be applied to mapping restric- hybridizing bands are located by autoradiography.
tion sites around a single-copy gene sequence in a Alwine et al.’s method thus extends that of Southern
complex genome such as that of humans (Fig. 2.6), and for this reason it has acquired the jargon term
and when a ‘mini-satellite’ probe is used it can be northern blotting.
applied forensically to minute amounts of DNA (see Subsequently it was found that RNA bands can
Chapter 14). indeed be blotted on to nitrocellulose membranes
under appropriate conditions (Thomas 1980) and
suitable nylon membranes have been developed.
Northern blotting
Because of the convenience of these more recent
Southern’s technique has been of enormous value, methods, which do not require freshly activated paper,
but it was thought that it could not be applied the use of DBM paper has been superseded.
directly to the blot-transfer of RNAs separated by gel
electrophoresis, since RNA was found not to bind to
Western blotting
nitrocellulose. Alwine et al. (1979) therefore devised
a procedure in which RNA bands are blot-transferred The term ‘western’ blotting (Burnette 1981) refers
from the gel on to chemically reactive paper, where to a procedure which does not directly involve nucleic
they are bound covalently. The reactive paper is acids, but which is of importance in gene manipula-
prepared by diazotization of aminobenzyloxymethyl tion. It involves the transfer of electrophoresed
paper (creating diazobenzyloxymethyl (DBM) paper), protein bands from a polyacrylamide gel on to a
which itself can be prepared from Whatman 540 membrane of nitrocellulose or nylon, to which they
paper by a series of uncomplicated reactions. Once bind strongly (Gershoni & Palade 1982, Renart &
covalently bound, the RNA is available for hybrid- Sandoval 1984). The bound proteins are then avail-
POGC02 9/11/2001 11:01 AM Page 17

Basic techniques 17

able for analysis by a variety of specific protein–ligand electrophoretic transfer methods: transfer is very
interactions. Most commonly, antibodies are used to rapid and gel treatment can be performed in situ on
detect specific antigens. Lectins have been used to the vacuum apparatus. This ensures minimal gel
identify glycoproteins. In these cases the probe may handling and, together with the rapid transfer, pre-
itself be labelled with radioactivity, or some other vents significant DNA diffusion.
‘tag’ may be employed. Often, however, the probe
is unlabelled and is itself detected in a ‘sandwich’
Transformation of E. coli
reaction, using a second molecule which is labelled,
for instance a species-specific second antibody, or Early attempts to achieve transformation of E. coli
protein A of Staphylococcus aureus (which binds were unsuccessful and it was generally believed that
to certain subclasses of IgG antibodies), or strept- E. coli was refractory to transformation. However,
avidin (which binds to antibody probes that have Mandel and Higa (1970) found that treatment with
been biotinylated). These second molecules may CaC12 allowed E. coli cells to take up DNA from bac-
be labelled in a variety of ways with radioactive, teriophage λ. A few years later Cohen et al. (1972)
enzyme or fluorescent tags. An advantage of the showed that CaC12-treated E. coli cells are also effect-
sandwich approach is that a single preparation of ive recipients for plasmid DNA. Almost any strain of
labelled second molecule can be employed as a E. coli can be transformed with plasmid DNA, albeit
general detector for different probes. For example, with varying efficiency, whereas it was thought that
an antiserum may be raised in rabbits which reacts only recBC− mutants could be transformed with lin-
with a range of mouse immunoglobins. Such a ear bacterial DNA (Cosloy & Oishi 1973). Later,
rabbit anti-mouse (RAM) antiserum may be radio- Hoekstra et al. (1980) showed that recBC+ cells can
labelled and used in a number of different applica- be transformed with linear DNA, but the efficiency is
tions to identify polypeptide bands probed with only 10% of that in otherwise isogenic recBC− cells.
different, specific, monoclonal antibodies, each mono- Transformation of recBC− cells with linear DNA is
clonal antibody being of mouse origin. The sand- only possible if the cells are rendered recombination-
wich method may also give a substantial increase proficient by the addition of a sbcA or sbcB muta-
in sensitivity, owing to the multivalent binding of tion. The fact that the recBC gene product is an
antibody molecules. exonuclease explains the difference in transforma-
tion efficiency of circular and linear DNA in recBC+
cells.
Alternative blotting techniques
As will be seen from the next chapter, many bac-
The original blotting technique employed capillary teria contain restriction systems which can influence
blotting but nowadays the blotting is usually accom- the efficiency of transformation. Although the com-
plished by electrophoretic transfer of polypeptides plete function of these restriction systems is not yet
from an SDS-polyacrylamide gel on to the membrane known, one role they do play is the recognition and
(Towbin et al. 1979). Electrophoretic transfer is also degradation of foreign DNA. For this reason it is
the method of choice for transferring DNA or RNA usual to use a restriction-deficient strain of E. coli as
from low-pore-size polyacrylamide gels. It can also a transformable host.
be used with agarose gels. However, in this case, Since transformation of E. coli is an essential step
the rapid electrophoretic transfer process requires in many cloning experiments, it is desirable that it be
high currents, which can lead to extensive heating as efficient as possible. Several groups of workers
effects, resulting in distortion of agarose gels. The have examined the factors affecting the efficiency of
use of an external cooling system is necessary to transformation. It has been found that E. coli cells
prevent this. and plasmid DNA interact productively in an en-
Another alternative to capillary blotting is vacuum- vironment of calcium ions and low temperature
driven blotting (Olszewska & Jones 1988), for which (0–5°C), and that a subsequent heat shock (37–45°C)
several devices are commercially available. Vacuum is important, but not strictly required. Several other
blotting has several advantages over capillary or factors, especially the inclusion of metal ions in
POGC02 9/11/2001 11:01 AM Page 18

18 CHAPTER 2

addition to calcium, have been shown to stimulate sources (see Chapter 3) and reduce the transforma-
the process. tion efficiency. Large DNAs transform less effici-
A very simple, moderately efficient transforma- ently, on a molar basis, than small DNAs. Even with
tion procedure for use with E. coli involves resus- such improved transformation procedures, certain
pending log-phase cells in ice-cold 50 mmol/l potential gene-cloning experiments requiring large
calcium chloride at about 1010 cells/ml and keeping numbers of clones are not reliable. One approach
them on ice for about 30 min. Plasmid DNA (0. 1 µg) which can be used to circumvent the problem of low
is then added to a small aliquot (0.2 ml) of these now transformation efficiencies is to package recombin-
competent (i.e. competent for transformation) cells, ant DNA into virus particles in vitro. A particular
and the incubation on ice continued for a further form of this approach, the use of cosmids, is described
30 min, followed by a heat shock of 2 min at 42°C. in detail in Chapter 5. Another approach is electro-
The cells are then usually transferred to nutrient poration, which is described below.
medium and incubated for some time (30 min to
1 h) to allow phenotypic properties conferred by the
Electroporation
plasmid to be expressed, e.g. antibiotic resistance
commonly used as a selectable marker for plasmid- A rapid and simple technique for introducing cloned
containing cells. (This so-called phenotypic lag genes into a wide variety of microbial, plant and ani-
may not need to be taken into consideration with mal cells, including E. coli, is electroporation. This
high-level ampicillin resistance. With this marker, technique depends on the original observation by
significant resistance builds up very rapidly, and Zimmerman & Vienken (1983) that high-voltage
ampicillin exerts its effect on cell-wall biosynthesis electric pulses can induce cell plasma membranes to
only in cells which have progressed into active fuse. Subsequently it was found that, when sub-
growth.) Finally the cells are plated out on selective jected to electric shock, the cells take up exogenous
medium. Just why such a transformation procedure DNA from the suspending solution. A proportion of
is effective is not fully understood (Huang & Reusch these cells become stably transformed and can be
1995). The calcium chloride affects the cell wall and selected if a suitable marker gene is carried on the
may also be responsible for binding DNA to the cell transforming DNA. Many different factors affect the
surface. The actual uptake of DNA is stimulated by efficiency of electroporation, including temperature,
the brief heat shock. various electric-field parameters (voltage, resistance
Hanahan (1983) has re-examined factors that and capacitance), topological form of the DNA,
affect the efficiency of transformation, and has devised and various host-cell factors (genetic background,
a set of conditions for optimal efficiency (expressed growth conditions and post-pulse treatment). Some
as transformants per µg plasmid DNA) applicable to of these factors have been reviewed by Hanahan
most E. coli K12 strains. Typically, efficiencies of 107 et al. (1991).
to 109 transformants/µg can be achieved depending With E. coli, electroporation has been found to give
on the strain of E. coli and the method used (Liu & plasmid transformation efficiencies (109 cfu/µg DNA)
Rashidbaigi 1990). Ideally, one wishes to make a comparable with the best CaC12 methods (Dower et al.
large batch of competent cells and store them frozen 1988). More recently, Zhu and Dean (1999) have
for future use. Unfortunately, competent cells made reported 10-fold higher transformation efficiencies
by the Hanahan procedure rapidly lose their com- with plasmids (9 × 109 transformants/µg) by co-
petence on storage. Inoue et al. (1990) have optimized precipitating the DNA with transfer RNA (tRNA)
the conditions for the preparation of competent cells. prior to electroporation. With conventional CaCl2-
Not only could they store cells for up to 40 days at mediated transformation, the efficiency falls off
−70°C while retaining efficiencies of 1–5 × 109 cfu/µg, rapidly as the size of the DNA molecule increases
but competence was affected only minimally by salts and is almost negligible when the size exceeds 50 kb.
in the DNA preparation. While size also affects the efficiency of electroporation
There are many enzymic activities in E. coli which (Sheng et al. 1995), it is possible to get transforma-
can destroy incoming DNA from non-homologous tion efficiencies of 106 cfu/µg DNA with molecules
POGC02 9/11/2001 11:01 AM Page 19

Basic techniques 19

as big as 240 kb. Molecules three to four times this et al. 1987). Small unilamellar (single-bilayer) ves-
size also can be electroporated successfully. This is icles are produced. DNA in solution spontaneously
important because much of the work on mapping and efficiently complexes with these liposomes (in
and sequencing of genomes demands the ability contrast to previously employed liposome encapsida-
to handle large fragments of DNA (see p. 64 and tion procedures involving non-ionic lipids). The
p. 126). positively charged liposomes not only complex with
DNA, but also bind to cultured animal cells and are
efficient in transforming them, probably by fusion
Transformation of other organisms
with the plasma membrane. The use of liposomes as
Although E. coli often remains the host organism of a transformation or transfection system is called
choice for cloning experiments, many other hosts lipofection.
are now used, and with them transformation may
still be a critical step. In the case of Gram-positive
The polymerase chain reaction (PCR)
bacteria, the two most important groups of organ-
isms are Bacillus spp. and actinomycetes. That B. The impact of the PCR upon molecular biology has
subtilis is naturally competent for transformation been profound. The reaction is easily performed, and
has been known for a long time and hence the gen- leads to the amplification of specific DNA sequences
etics of this organism are fairly advanced. For this by an enormous factor. From a simple basic prin-
reason B. subtilis is a particularly attractive alternat- ciple, many variations have been developed with
ive prokaryotic cloning host. The significant features applications throughout gene technology (Erlich
of transformation with this organism are detailed 1989, Innis et al. 1990). Very importantly, the PCR
in Chapter 8. Of particular relevance here is that has revolutionized prenatal diagnosis by allowing
it is possible to transform protoplasts of B. subtilis, a tests to be performed using small samples of fetal tis-
technique which leads to improved transformation sue. In forensic science, the enormous sensitivity of
frequencies. A similar technique is used to transform PCR-based procedures is exploited in DNA profiling;
actinomycetes, and recently it has been shown that following the publicity surrounding Jurassic Park,
the frequency can be increased considerably by first virtually everyone is aware of potential applica-
entrapping the DNA in liposomes, which then fuse tions in palaeontology and archaeology. Many other
with the host-cell membrane. processes have been described which should pro-
In later chapters we discuss ways, including elec- duce equivalent results to a PCR (for review, see
troporation, in which cloned DNA can be introduced Landegran 1996) but as yet none has found wide-
into eukaryotic cells. With animal cells there is no spread use.
great problem as only the membrane has to be In many applications of the PCR to gene mani-
crossed. In the case of yeast, protoplasts are required pulation, the enormous amplification is secondary
(Hinnen et al. 1978). With higher plants one strat- to the aim of altering the amplified sequence. This
egy that has been adopted is either to package the often involves incorporating extra sequences at the
DNA in a plant virus or to use a bacterial plant ends of the amplified DNA. In this section we shall
pathogen as the donor. It has also been shown that consider only the amplification process. The applica-
protoplasts prepared from plant cells are competent tions of the PCR will be described in appropriate places.
for transformation. A further remarkable approach
that has been demonstrated with plants and animals
Basic reaction
(Klein & Fitzpatrick-McElligott 1993) is the use of
microprojectiles shot from a gun (p. 238). First we need to consider the basic PCR. The
Animal cells, and protoplasts of yeast, plant and principle is illustrated in Fig. 2.7. The PCR involves
bacterial cells are susceptible to transformation by two oligonucleotide primers, 17–30 nucleotides in
liposomes (Deshayes et al. 1985). A simple transforma- length, which flank the DNA sequence that is to be
tion system has been developed which makes use of amplified. The primers hybridize to opposite strands
liposomes prepared from a cationic lipid (Felgner of the DNA after it has been denatured, and are
POGC02 9/11/2001 11:01 AM Page 20

20 CHAPTER 2

Cycle 1 orientated so that DNA synthesis by the polymerase

5’+ 3’ Double stranded proceeds through the region between the two
3’–
Denaturation by
5’ DNA target primers. The extension reactions create two double-
heat followed by stranded target regions, each of which can again be
primer annealing
5’+ 3’
denatured ready for a second cycle of hybridization
and
3’ 5’ and extension. The third cycle produces two double-
5’ 3’ stranded molecules that comprise precisely the
3’– 5’
DNA synthesis target region in double-stranded form. By repeated
(primer extension) cycles of heat denaturation, primer hybridization
5’ 3’
3’ 5’
and extension, there follows a rapid exponential
and accumulation of the specific target fragment of
5’ 3’
3’ 5’ DNA. After 22 cycles, an amplification of about 106-
fold is expected (Fig. 2.8), and amplifications of this
Denaturation by heat followed by primer
annealing and DNA synthesis order are actually attained in practice.
Cycle 2
In the original description of the PCR method
(Mullis & Faloona 1987, Saiki et al. 1988, Mullis
5’ 3’
3’ 5’
1990), Klenow DNA polymerase was used and,
+
5’ 3’
because of the heat-denaturation step, fresh enzyme
3’ 5’ had to be added during each cycle. A breakthrough
+
5’ 3’ came with the introduction of Taq DNA polymerase
3’ 5’
+ (Lawyer et al. 1989) from the thermophilic bacterium
5’ 3’ Thermus aquaticus. The Taq DNA polymerase is
3’ 5’
resistant to high temperatures and so does not need
Denaturation by heat followed by primer
annealing and DNA synthesis to be replenished during the PCR (Erlich et al. 1988,
Cycle 3
Sakai et al. 1988). Furthermore, by enabling the
extension reaction to be performed at higher tem-
5’ 3’ peratures, the specificity of the primer annealing is
3’ 5’
not compromised. As a consequence of employing
5’ 3’
3’ 5’ the heat-resistant enzyme, the PCR could be auto-
mated very simply by placing the assembled reaction
5’ 3’
3’ 5’ in a heating block with a suitable thermal cycling
5’ 3’ programme (see Box 2.3).
3’ 5’

5’ 3’
3’ 5’
5’ 3’
3’ 5’ Fig. 2.7 (left) The polymerase chain reaction. In cycle 1 two
primers anneal to denatured DNA at opposite sides of the
5’ 3’ target region, and are extended by DNA polymerase to give
3’ 5’
new strands of variable length. In cycle 2, the original strands
5’ 3’ and the new strands from cycle 1 are separated, yielding a
3’ 5’
total of four primer sites with which primers anneal. The
primers that are hybridized to the new strands from cycle 1
are extended by polymerase as far as the end of the template,
Repeated cycles lead to exponential leading to a precise copy of the target region. In cycle 3,
doubling of the target sequence double-stranded DNA molecules are produced (highlighted in
colour) that are precisely identical to the target region.
Further cycles lead to exponential doubling of the target
region. The original DNA strands and the variably extended
strands become negligible after the exponential increase of
target fragments.
POGC02 9/11/2001 11:01 AM Page 21

Basic techniques 21

Cycle number Number of double-stranded fere with the PCR, and ways of eliminating them
target molecules
have been reviewed by Bickley and Hopkins (1999).
1 0
2 0
3 2 RT-PCR
4 4
5 8
The thermostable polymerase used in the basic PCR
6 16
7 32 requires a DNA template and hence is limited to the
8 64 amplification of DNA samples. There are numerous
9 128
10 256
instances in which the amplification of RNA would
11 512 be preferred. For example, in analyses involving the
12 1024 diffierential expression of genes in tissues during
13 2048
14 4096 development or the cloning of DNA derived from an
15 8192 mRNA (complementary DNA or cDNA), particularly
16 16,384 a rare mRNA. In order to apply PCR methodology
17 32,768
18 65,536 to the study of RNA, the RNA sample must first be
19 131,072 reverse-transcribed to cDNA to provide the necessary
20 262,144
21 524,288
DNA template for the thermostable polymerase. This
22 1,048,576 process is called reverse transcription (RT), hence
23 2,097,152 the name RT-PCR.
24 4,194,304
25 8,388,608 Avian myeloblastosis virus (AMV) or Moloney
26 16,777,216 murine leukaemia virus (MuLV) reverse transcrip-
27 33,554,432 tases are generally used to produce a DNA copy of
28 67,108,864
29 134,217,728 the RNA template. Various strategies can be adopted
30 268,435,456 for first-strand cDNA synthesis (Fig. 2.9).

Fig. 2.8 Theoretical PCR amplification of a target fragment

with increasing number of cycles. Long accurate PCR (LA-PCR)
Amplification of long DNA fragments is desirable for
Recent developments have sought to minimize numerous applications of gene manipulation. The
amplification times. Such systems have used small basic PCR works well when small fragments are
reaction volumes in glass capillaries to give large amplified. The efficiency of amplification and there-
surface area-to-volume ratios. This results in almost fore the yield of amplified fragments decrease signi-
instantaneous temperature equilibration and minimal ficantly as the size of the amplicon increases over 5 kb.
annealing and denaturation times. This, accompan- This decrease in yield of longer amplified fragments
ied by temperature ramp rates of 10–20°C/s, made is attributable to partial synthesis across the desired
possible by the use of turbulent forced hot-air sys- sequence, which is not a suitable substrate for the
tems to heat the sample, results in an amplification subsequent cycles. This is demonstrated by the pres-
reaction completed in tens of minutes. ence of smeared, as opposed to discrete, bands on a gel.
While the PCR is simple in concept, practically Barnes (1994) and Cheng et al. (1994) examined
there are a large number of variables which can the factors affecting the thermostable polymeriza-
influence the outcome of the reaction. This is espe- tion across larger regions of DNA and identified key
cially important when the method is being used with variables affecting the yield of longer PCR frag-
rare samples of starting material or if the end result ments. Most significant of these was the absence of
has diagnostic or forensic implications. For a detailed a 3′–5′ exonuclease (proofreading) activity in Taq
analysis of the factors affecting the PCR, the reader polymerase. Presumably, when the Taq polymerase
should consult McDowell (1999). There are many misincorporates a dNTP, subsequent extension of
substances present in natural samples (e.g. blood, the strand either proceeds very slowly or stops
faeces, environmental materials) which can inter- completely. To overcome this problem, a second
POGC02 9/11/2001 11:01 AM Page 22

22 CHAPTER 2

Box 2.3 The polymerase chain reaction achieves enormous amplifications,

of specific target sequence, very simply

The reaction is assembled in a single tube, and then The reaction is cycled 25–35 times, with the
placed in a thermal cycler (a programmable following temperature programme:
heating/cooling block), as described below. Denaturation 94°C, 0.5 min
A typical PCR for amplifying a human genomic Primer annealing 55°C,1.5 min
DNA sequence has the following composition. The Extension 72°C, 1 min
reaction volume is 100 ml. Typically, the reaction takes some 2–3 h overall.
Input genomic DNA, 0.1–1 mg Notes:
Primer 1, 20 pmol • The optimal temperature for the annealing step
Primer 2, 20 pmol will depend upon the primers used.
20 mmol/l Tris-HCl, pH 8.3 (at 20°C) • The pH of the Tris-HCl buffer decreases markedly
1.5 mmol/l magnesium chloride with increasing temperature. The actual pH varies
25 mmol/l potassium chloride between about 6.8 and 7.8 during the thermal cycle.
50 mmol/l each deoxynucleoside triphosphate • The time taken for each cycle is considerably
(dATP, dCTP, dGTP, dTTP) longer than 3 min (0.5 + 1.5 + 1 min), depending
2 units Taq DNA polymerase upon the rates of heating and cooling between steps,
A layer of mineral oil is placed over the reaction but can be reduced considerably by using turbo
mix to prevent evaporation. systems (p. 21).
• The standard PCR does not efficiently amplify
sequences much longer than about 3 kb.

Random primer

5’ A A A A A A A 3’ mRNA
3’ 1st strand cDNA

random primer

Oligo (dT) primer

5’ A A A A A A A 3’ mRNA
3’ T T T T T T T 5’ 1st strand cDNA

Sequence-specific primer
Fig. 2.9 Three strategies for synthesis of
5’ A A A A A A A 3’ mRNA first-strand cDNA. (a) Random primer;
3’ 1st strand cDNA
(b) oligo (dT) primer; (c) sequence-specific
primer primer.

thermostable polymerase with proofreading capab- • Primers should be 17 to 30 nucleotides in length.

ility is added. Thermostable DNA polymerases with • A GC content of about 50% is ideal. For pri-
proofreading capabilities are listed in Table 2.1. mers with a low GC content, it is desirable to
choose a long primer so as to avoid a low melting
temperature.
Key factors affecting the PCR
• Sequences with long runs (i.e. more than three or
The specificity of the PCR depends crucially upon the four) of a single nucleotide should be avoided.
primers. The following factors are important in • Primers with significant secondary structure are
choosing effective primers. undesirable.
POGC02 9/11/2001 11:01 AM Page 23

Basic techniques 23

merase substrate and should not interfere with the

Table 2.1 Sources of thermostable DNA polymerases amplification primers (Dang & Jayasena 1996).
with proofreading (3′–5′ exonuclease) activity. In order to minimize further the amplification of
spurious products, the strategy of nested primers may
DNA polymerase Source
be deployed. Here the products of an initial PCR
Tma Thermotoga maritima amplification are used to seed a second PCR ampli-
Deep VentTM Pyrococcus sp. fication, in which one or both primers are located
Tli Thermococcus litoralis internally with respect to the primers of the first
Pfu Pyrococcus furiosus
PCR. Since there is little chance of the spurious prod-
Pwo Pyrococcus woesi
ucts containing sequences capable of hybridizing
with the second primer set, the PCR with these
nested primers selectively amplifies the sought-after
DNA.
• There should be no complementarity between the As noted above, the Taq DNA polymerase lacks a
two primers. The great majority of primers which 3′–5′ proofreading exonuclease. This lack appears
conform with these guidelines can be made to work, to contribute to errors during PCR amplification due
although not all comparable primer sets are equally to misincorporation of nucleotides (Eckert & Kunkel
effective even under optimized conditions. 1990). Partly to overcome this problem, other
In carrying out a PCR it is usual to employ a thermostable DNA polymerases with improved
hot-start protocol. This entails adding the DNA fidelity have been sought, although the Taq DNA
polymerase after the heat-denaturation step of the polymerase remains the most widely used enzyme
first cycle, the addition taking place at a temperature for PCR. In certain applications, especially where
at or above the annealing temperature and just prior amplified DNA is cloned, it is important to check the
to the annealing step of the first cycle. The hot start nucleotide sequence of the cloned product to reveal
overcomes the problem that would arise if the DNA any mutations that may have occurred during the
polymerase were added to complete the assembly PCR. The fidelity of the amplification reaction can be
of the PCR reaction mixture at a relatively low assessed by cloning, sequencing and comparing
temperature. At low temperature, below the desired several independently amplified molecules.
hybridization temperature for the primer (typically
in the region 45–60°C), mismatched primers will
Real-time quantitative PCR
form and may be extended somewhat by the poly-
merase. Once extended, the mismatched primer is There are many applications of the PCR where it
stabilized at the unintended position. Having been would be advantageous to be able to quantify the
incorporated into the extended DNA during the amount of starting material. Theoretically, there is
first cycle, the primer will hybridize efficiently in a quantitative relationship between the amount of
subsequent cycles and hence may cause the ampli- starting material (target sequence) and the amount
fication of a spurious product. of PCR product at any given cycle. In practice,
Alternatives to the hot-start protocol include the replicate reactions yield different amounts of prod-
use of Taq polymerase antibodies, which are inactiv- uct, making quantitation unreliable. Higuchi et al.
ated as the temperature rises (Taylor & Logan 1995), (1992, 1993) pioneered the use of ethidium bromide
and AmpliTaq GoldTM, a modified Taq polymerase to quantify PCR products as they accumulate. Ampli-
that is inactive until heated to 95°C (Birch 1996). fication produces increasing amounts of double-
Yet another means of inactivating Taq DNA stranded DNA, which binds ethidium bromide,
polymerase at ambient temperatures is the SELEX resulting in an increase in fluorescence. By plotting
method (systematic evolution of ligands by expo- the increase in fluorescence versus cycle number it is
nential enrichment). Here the polymerase is possible to analyse the PCR kinetics in real time. This
reversibly inactivated by the binding of nanomolar is much more satisfactory than analysing product
amounts of a 70-mer, which is itself a poor poly- accumulation after a fixed number of cycles.
POGC02 9/11/2001 11:01 AM Page 24

24 CHAPTER 2

Reporter Quencher
Forward R Q
primer Probe
5’
3’ 5’ Binding of
primers and probe
5’ 3’
5’
Reverse
primer

R Q
5’
3’ 5’ Polymerization
5’ 3’
5’

R Q
5’
3’ 5’ Strand
5’ 3’ displacement
5’

R
Q
5’
3’ 5’ Release of
5’ 3’ reporter
5’

R Q

5’
3’ 5’ Polymerization
5’ 3’ complete Fig. 2.10 Real-time quantitative PCR. See
5’ text for details.

The principal drawback to the use of ethidium (Fig. 2.10). This cleavage of the probe separates
bromide is that both specific and non-specific prod- the reporter dye from the quencher dye, thereby
ucts generate a signal. This can be overcome by the increasing the reporter-dye signal. Cleavage removes
use of probe-based methods for assaying product the probe from the target strand, allowing primer
accumulation (Livak et al. 1995). The probes used extension to continue to the end of the template
are oligonucleotides with a reporter fluorescent dye strand. Additional reporter-dye molecules are cleaved
attached at the 5′ end and a quencher dye at the 3′ from their respective probes with each cycle, effect-
end. While the probe is intact, the proximity of ing an increase in fluorescence intensity proportional
the quencher reduces the fluorescence emitted by to the amount of amplicon produced.
the reporter dye. If the target sequence is present, the Instrumentation has been developed which
probe anneals downstream from one of the primer combines thermal cycling with measurement of
sites. As the primer is extended, the probe is cleaved fluorescence, thereby enabling the progress of the
by the 5′ nuclease activity of the Taq polymerase PCR to be monitored in real time. This revolutionizes
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prevail; and although the powers of the earth rose up against them,
and used all their power and policy, both priests and people, to
overthrow them, yet they were never permitted to prevail; for the
Lord Jehovah, on whose almighty arm they had placed their entire
trust and confidence for support and defence, delivered them from
all their tribulations, and set them above their persecutors, and
caused them to rejoice on the banks of deliverance. And he is the
same God of power that he ever was, and a present helper in every
needful time; and although many in the present day, who have left
their first love, are rising up, and charging their fellow-professors
with holding unsound doctrines, and are endeavouring, by
unfounded and reproachful epithets, to destroy and undermine their
religious and moral character among men, and have separated from
their brethren, and set up separate meetings, giving them the
names of the meetings of Friends; and in their usurped authority
undertake to disown their fellow-members, who could not submit to
their usurpation: yet all their formal disownments being altogether
out of the order of the gospel, our meetings consider them of no
effect.
From Galen we proceeded to South Farmington, and attended a
meeting there on seventh day, at the third hour in the afternoon;
and the next day being first day, we attended North Farmington
meeting. The three following days we had meetings at Macedon,
Palmyra, and Williamson. These were likewise large favoured
meetings, in which truth was exalted over all, and we parted with
them in true peace of mind, and proceeded on our journey to
Rochester, and had a meeting on sixth day, by appointment: we also
staid and attended their meeting on first day.
After this we proceeded to Wheatland, and had an appointed
meeting there on second day, the 14th of 9th month; on third day
we were at Henrietta, and on fourth day at Mendon. These were all
favoured opportunities; the people’s minds seemed to be open to
receive us and our testimony with gladness. From this place we
turned back through Farmington and Scipio, to Skeneateles, and
attended a meeting by our appointment at a village about five miles
from the village of Skeneateles, on the east side of the lake. On first
day we attended Friends’ meeting at Skeneateles; on second day we
proceeded to Verona; and the next day had a meeting there, held in
a meeting-house occupied by the Baptists, Friends’ meeting-house
being too small to contain the people who assembled.
The next day being fourth day, we proceeded to Utica, and had an
appointed meeting in the evening. Here we remained over the next
day, and attended Friends’ meeting as it came in course. From this
place we proceeded to Charleston, and attended their meeting on
first day. These meetings, in like manner, were all solemn seasons;
and, I trust, profitable and comfortable to many; and I left them
with peace of mind. After the last meeting, we rode about thirteen
miles, and lodged with our kind friend Zacheus Mead. The following
day we proceeded on our journey to Newtown; and the next day
attended Friends’ meeting there. After this we proceeded to
Saratoga, and attended Friends’ preparative meeting at that place;
and not feeling clear to leave it, we had an appointed meeting there
the day after, of which public notice was given. It was very large;
and it proved a highly favoured season; the Lord’s presence was
manifested for our help, and truth was raised into dominion, and ran
like oil over the assembly. Many hearts were broken and contrited,
and the upright in heart were made to rejoice for the unmerited
favour.
The following day we had an appointed meeting at Milton. After this
we proceeded to Galway, and lodged with our ancient friend Philip
Macomber, who was in the ninety-first year of his age. Here we had
a meeting on seventh day. The next day, the 4th of 10th month, we
attended Providence meeting, which was very large. On second day
we had an appointed meeting at Mayfield. These were all seasons of
favour, particularly that at Providence, in which truth was exalted
over all opposition, and many hearts were contrited; from a sense of
which, our minds were bowed in reverence and humiliation before
Him, who is the author of all our sure mercies.
We proceeded from Mayfield to Greenfield, and on fourth day
attended Friends’ monthly meeting at that place, which was
composed of that and Milton preparative meetings. We had good
satisfaction in sitting with our Friends, and in observing their
commendable order, and the harmony and condescension manifest
in conducting the affairs of the Church.
From thence we proceeded to Easton, and had an appointed
meeting there on sixth day. On seventh day we were at Cambridge,
on first day at White Creek, and on second day we rode to Danby,
and the next day had a meeting there. These were all large and very
solemn seasons, in which the great Head of the Church manifested
his gracious presence, convicting and contriting many minds, and
the upright in heart were edified. From Danby we proceeded to
Granville, and had an appointed meeting there the next day, which
was a large solemn opportunity. The day after we proceeded on our
journey to Shoram, a town on the eastern shore of Lake Champlain.
Here we had a meeting the next day with the few Friends of that
place, and some of the neighbouring inhabitants. It was a
comfortable opportunity, and we left them with peace of mind. We
then rode to Ferrisburgh, and on first day, the 18th of 10th month,
had a very large favoured meeting at that place. On second day we
had an appointed meeting at Monkton. This was likewise a large
favoured opportunity, in which truth reigned over all opposition, to
the praise of his own excellent name, who is over all, God blessed
for ever.
As I was somewhat unwell, we rested on third day with our kind
friends Thomas and Rowland T. Robinson; and feeling my mind now
clear from any further service in these parts, on fourth day we
turned our faces homewards, and proceeded back to Shoram. On
fifth day we had an appointed meeting in that village, principally for
those not members of our society. Although the people came
together in a negligent manner as respected the appointed time, yet
they generally behaved orderly, and appeared to give good attention
to what they heard; and my mouth was opened by Him who opens
and none can shut, in a large effective testimony to the truths of the
gospel; which brought a precious solemnity over the assembly, and
they appeared to go away satisfied, and we left them with the
answer of peace in our own minds.
The next day we proceeded on our journey to Granville, and from
thence the following day to Queensbury. On first day, the 25th of
10th month, we attended Friends’ meeting there; and notice being
given to the neighbouring inhabitants of our attendance, they came
in until the house was filled, and a number had to stand without for
want of room; and a blessed meeting we had, in which the power of
truth ran as oil over the assembly, tendering and contriting many
minds, and to the comfort and rejoicing of the upright in heart.
We had an appointed meeting on the following day at Moreau, which
was a large favoured meeting. From thence we proceeded to
Saratoga, and lodged with our kind friend Thomas Wilbur, and the
next day Thomas accompanied us to Pittstown, where we had an
appointed meeting on fourth day. This was truly an humbling
season, in which truth was exalted over all; great brokenness and
contrition of spirit were manifested among the people, and we were
edified together in love, which inspired our minds with thanksgiving
and gratitude for the unmerited favour.
From this place we proceeded to Troy, and as I was somewhat
unwell, we rested the following day with our kind friend Isaac
Merritt. On sixth day we proceeded on our journey to the
neighbourhood of Hudson, and put up with our kind friend Thomas
Wright; and seventh day being very rainy and inclement we
continued here, and attended Hudson meeting on first day, which
was a large satisfactory meeting.
On second day we proceeded on our journey to Stanford, and
lodged with our kind friend John Hull. The two following days we
attended the quarterly meeting at Nine Partners. At this place there
is a very large body of Friends united together in gospel fellowship,
and they were favoured to conduct the business of the quarterly
meeting in harmony and condescension. The public meeting was
very large. It was attended by a great number who were not in
membership with us, and who behaved orderly, and it was indeed a
very solemn edifying season.
After the close of this meeting, we returned that evening to
Stanford, in order to attend the quarterly meeting at that place,
which opened the next day with a meeting of Ministers and Elders. I
attended this, and the following day, the meeting for discipline. A
large number of the neighbouring inhabitants attended this meeting,
and sat with Friends, until the partition between the men and
women was closed. They behaved very orderly, and a precious
solemnity spread over the assembly, and many essential doctrines of
the gospel were opened to the people in the demonstration of the
spirit, truth was raised into victory over all, and the upright in heart
were edified and comforted. The meeting of Ministers and Elders
was likewise a precious opportunity, in which comfort and
encouragement were freely administered to them.
From Stanford we proceeded on our journey to Cornwall, crossing
the Hudson river on our way. We arrived here on seventh day
evening, and attended their meeting on first day, the 8th of 11th
month. This meeting was large, and a truly baptizing season, in
which many hearts were humbled and contrited, and truth reigned
over all; thanks be to God, who giveth us the victory, nothing due to
man.
The two following days we had meetings appointed at the Lower
and Upper Clove. These were well attended, and, I trust, profitable
edifying seasons to many present. They were composed of people of
various professions, conditions, and states; yet all appeared to be
brought down and subjected by the solemnizing influence and power
of truth, that reigned victoriously over all. Surely it was the Lord’s
doing, and it was marvellous in our eyes. These meetings closed my
labour and exercise in the gospel, to Friends and others in the yearly
meetings of Philadelphia, Baltimore, Ohio, Indiana, and New-York, as
expressed in a certificate of unity and concurrence, given me by the
monthly meeting of Jericho, and quarterly meeting of Westbury.
From the latter meeting we proceeded the next day directly to New-
York, where we arrived on fourth day evening. The day after we
attended Friends’ meeting at Hester-street, it being their usual
meeting day; and a marriage being accomplished at the close of it, it
was larger than usual, as many of the neighbouring inhabitants
attended. Way opened for me to declare the truth among them, to
the peace of my own mind, and to the mutual comfort and
encouragement of the upright in heart. I rested here until first day,
and attended Friends’ meeting at Rose-street in the morning, and
that held at Hester-street in the afternoon. They were both very
large solemn meetings. On second day evening I had an appointed
meeting at Brooklyn, likewise a large and very favoured season. In
all of these meetings the word preached had free course, and I had,
in the openings of truth, to declare to these large mixed
assemblages many things concerning the kingdom of God; and the
only sure way by which an admittance into his kingdom of peace and
joy may be obtained by the children of men.
The foregoing meetings were times of favour, and as a seal from the
hand of our gracious and never-failing Helper, to the labour and
travail which he has led me into, and enabled me to perform, for the
promotion of his great and noble cause of truth and righteousness in
the earth, as set forth in the foregoing account, and not suffering
any weapon formed against me to prosper. “This is the heritage of
the servants of the Lord, and their righteousness is of me, saith the
Lord.” For all these unmerited favours and mercies, in deep
humiliation my soul doth magnify the Lord, and return thanksgiving
and glory to his great and excellent name; for his mercy endureth
for ever.
On third day we proceeded homeward, and attended Westbury
monthly meeting on fourth day, on our way. After this I rode home,
and found my family well, to our mutual rejoicing; and we greeted
each other with thankful hearts for the unmerited favour.
We travelled in this journey nearly fifteen hundred miles.
ELIAS HICKS.
E. H. here closed his Journal, and signed his name; after which he
lived a little more than two months.
 APPENDIX.
LETTER TO HUGH JUDGE, OF OHIO.
Jericho, 2d mo. 14th, 1830.
Dear Hugh,
Thy very acceptable letter of the 21st ultimo, was duly received, and
read with interest, tending to excite renewed sympathetic, and
mutual fellow-feeling; and brought to my remembrance the cheering
salutation of the blessed Jesus, our holy and perfect pattern and
example, to his disciples, viz: “Be of good cheer, I have overcome
the world.” By which he assured his disciples that, by walking in the
same pathway of self-denial and the cross which he trod to
blessedness, they might also overcome the world; as nothing has
ever enabled any rational being, in any age of the world, to
overcome the spirit of the world, which lieth in wickedness, but the
cross of Christ.
Some may query, what is the cross of Christ? To these I answer, it is
the perfect law of God written on the tablet of the heart, and in the
heart of every rational creature, in such indelible characters that all
the power of mortals cannot erase nor obliterate. Neither is there
any power or means given or dispensed to the children of men, but
this inward law and light by which the true and saving knowledge of
God can be obtained. And by this inward law and light, all will be
either justified or condemned, and all be made to know God for
themselves, and be left without excuse, agreeably to the prophecy
of Jeremiah, and the corroborating testimony of Jesus in his last
counsel and command to his disciples, not to depart from Jerusalem
until they should receive power from on high; assuring them that
they should receive power, when they had received the pouring forth
of the spirit upon them, which would qualify them to bear witness of
him in Judea, Jerusalem, Samaria, and to the uttermost parts of the
earth; which was verified in a marvellous manner on the day of
Pentecost, when thousands were converted to the Christian faith in
one day. By which it is evident, that nothing but this inward light and
law, as it is heeded and obeyed, ever did, or ever can make a true
and real Christian and child of God. And until the professors of
Christianity agree to lay aside all their non-essentials in religion, and
rally to this unchangeable foundation and standard of truth, wars
and fightings, confusion and error will prevail, and the angelic song
cannot be heard in our land, that of “glory to God in the highest,
and on earth peace and good will to men.” But when all nations are
made willing to make this inward law and light, the rule and
standard of all their faith and works, then we shall be brought to
know and believe alike, that there is but one Lord, one faith, and but
one baptism; one God and Father, that is above all, through all, and
in all; and then will all those glorious and consoling prophecies,
recorded in the scriptures of truth be fulfilled. Isaiah ii. 4, “He,” the
Lord, “shall judge among the nations, and rebuke many people: and
they shall beat their swords into ploughshares and their spears into
pruning hooks: nation shall not lift up sword against nation; neither
shall they learn war any more.” Isaiah xi. “The wolf also shall dwell
with the lamb, and the leopard shall lie down with the kid; and the
calf, and the young lion, and the fatling together; and a little child
shall lead them. And the cow and the bear shall feed; their young
ones shall lie down together; and the lion shall eat straw like the ox.
And the sucking child shall play on the hole of the asp, and the
weaned child put his hand on the cockatrice’s den. They shall not
hurt nor destroy in all my holy mountain: for the earth,” that is our
earthly tabernacles, “shall be full of the knowledge of the Lord, as
the waters cover the sea.”
These scripture testimonies give a true and correct description of the
gospel state, and no rational being can be a real Christian and true
disciple of Christ, until he comes to know all these things verified in
his own experience, as every man and woman has more or less of all
those different animal propensities and passions in their nature; and
they predominate and bear rule, and are the source and fountain
from whence all wars, and every evil work proceed, and will continue
as long as man remains in his first nature, and is governed by his
animal spirit and propensities, which constitute the natural man,
which Paul tells us “receiveth not the things of the spirit of God, for
they are foolishness unto him, neither can he know them, because
they are spiritually discerned.” This corroborates the declaration of
Jesus to Nicodemus, “that, except a man be born again, he cannot
see the kingdom of God;” for “that which is born of the flesh is flesh,
and that which is born of the spirit is spirit.” Here Jesus assures us,
beyond all doubt, that nothing but spirit can either see or enter into
the kingdom of God; and this confirms Paul’s doctrine, that “as many
as are led by the spirit of God are the sons of God,” and “joint heirs
with Christ.” And Jesus assures us, by his declaration to his disciples,
John xiv. 16, 17, “If ye love me, keep my commandments; and I will
pray the Father, and he shall give you another comforter, that he
may abide with you for ever, even the spirit of truth, whom the
world cannot receive;” that is, men and women in their natural
state, who have not given up to be led by this spirit of truth, that
leads and guides into all truth; “because they see him not, neither
do they know him, but ye know him, for he dwelleth with you, and
shall be in you.” And as these give up to be wholly led and guided by
him, the new birth is brought forth in them, and they witness the
truth of another testimony of Paul’s, even that of being created
anew in Christ Jesus unto good works, which God had foreordained
that all his new-born children should walk in them, and thereby
show forth by their fruits and good works, that they were truly the
children of God, born of his spirit, and taught of him; agreeably to
the testimony of the prophet, that “the children of the Lord are all
taught of the Lord, and in righteousness they are established, and
great is the peace of his children.” And nothing can make them
afraid that man can do unto them; as saith the prophet in his appeal
to Jehovah, “Thou wilt keep him in perfect peace, whose mind is
staid on thee, because he trusteth in thee.” Therefore, let every one
that loves the truth, for God is truth, “trust in the Lord for ever, for in
the Lord Jehovah there is everlasting strength.”
I write these things to thee, not as though thou didst not know
them, but as a witness to thy experience, as “two are better than
one, and a threefold cord is not quickly broken.”
I will now draw to a close, with just adding, for thy encouragement,
be of good cheer, for no new thing has happened to us; for it has
ever been the lot of the righteous to pass through many trials and
tribulations, in their passage to that glorious, everlasting, peaceful,
and happy abode, where all sorrow and sighing come to an end—the
value of which is above all price; for when we have given all that we
have and can give, and suffered all that we can suffer, it is still
infinitely below its real value. And if we are favoured to gain an
inheritance in that blissful and peaceful abode, “where the wicked
cease from troubling, and the weary are at rest,” we must ascribe it
all to the unmerited mercy and loving-kindness of our heavenly
Father, who remains to be God over all, blessed for ever.
I will now conclude; and in the fulness of brotherly love to thee and
thine, in which my family unite, subscribe thy affectionate friend,
ELIAS HICKS.
To Hugh Judge.
Please present my love to all my friends, as way opens.

The writing of the preceding letter was the last act in the life of this
eminent individual, and the attentive reader will not fail to regard it
as an act of peculiar interest. It was as a seal to the labours of a
long life, and evinced the abiding and lively efficacy of that internal
principle which he had uniformly sought as his director and
preserver. But the work of this faithful servant was now
accomplished; “the silver cord was loosed,” and that spirit which had
been so diligently active in the service of its Divine Master, was now
to rest from its labours, and to reap its reward. Just when he had
finished the letter alluded to, he was attacked with a paralytic
affection, under the effects of which he became gradually weaker;
but his mind remained established in great peace and serenity, and
on the 27th of 2d month, 1830, he calmly expired, aged nearly
eighty-two years.
Of the character of this extraordinary man, it is not necessary now to
speak. The preceding pages describe the nature of his
engagements; and an estimate may thence be formed, of the
fervency of his spirit, and the brightness of his example. In his
general deportment, and in the expression of his countenance, there
was a remarkable union of gentleness and dignity, indicating the
habitual benevolence and solemnity of his feelings; and his public
communications were accompanied with a power and an authority
which demonstrated the purity of the source from whence they were
derived. The promotion of spiritual holiness and practical
righteousness in the earth, were the objects of his constant
solicitude; and he endeavoured, through divine assistance, to
exemplify in his own daily experience, the comprehensive command
of the prophet, “To do justly, to love mercy, and to walk humbly with
thy God.”
 THE MEMORIAL OF JERICHO MONTHLY MEETING OF
FRIENDS CONCERNING OUR ANCIENT FRIEND ELIAS
HICKS.
We believe the example exhibited in the life and religious exercise of
this our beloved Friend, is eminently calculated to set forth the
efficacy and sufficiency of that divine grace, which, when believed in
and obeyed, bringeth salvation.
He was born in the town of Hempstead, Queens county, Long Island,
state of New-York, the 19th day of the 3d month, 1748. His parents’
names were John and Martha Hicks. At the age of seventeen he was
placed as an apprentice to learn the trade of a carpenter; on the
expiration of his term, he returned to his father, with whom he lived
until the time of his marriage, which took place about the twenty-
third year of his age, to Jemima, daughter of Jonathan and Elizabeth
Seaman, of Jericho, in said county, where he resided the remainder
of his life.
From his own account we learn, that when very young, he was
favoured with clear and powerful impressions of divine grace
operating on his mind as a reprover for evil, which not duly
regarding, and being naturally of a lively and active disposition, he
associated with those who indulged in the vanities and amusements
too common in the world, though mostly in things deemed innocent
by the generality of mankind. But the gift of divine grace, which was
so early manifested, did not forsake him, though he often strove to
stifle its convictions, but followed him in judgment and in mercy,
until a willingness was wrought in him to give up all to follow Christ,
in the regeneration. On one occasion, when preparing to join in the
dance, and surrounded by his jovial companions, the pure witness
rose so powerfully in his mind, and so clearly set before him the evil
tendency of the course he was pursuing, that he reasoned not with
flesh and blood, but gave up to the heavenly vision, and in deep
contrition and prostration of soul, entered into covenant with the
God of his life, that if he would be pleased to furnish him with
strength, he would endeavour not to be again found in the like
disobedience; which covenant, through mercy, he was favoured to
keep inviolate. Thus, submitting to the purifying operation of the
Holy Ghost and fire, he was, in due time, qualified and called to
declare to others what God had done for his soul; under the divine
anointing, he was enabled to unfold the truths of the gospel, in the
demonstration of the spirit and with power. And, through a faithful
obedience to that which had begun the good work in him, he
became an eminent instrument in the Lord’s hand, for the promotion
of truth and righteousness in the earth.
He first appeared in the ministry, about the twenty-seventh year of
his age, and from this period, his time and talents were devoted to
the cause of his Divine Master, labouring diligently for its
advancement, not only at home, and in his own neighbourhood, but
in most parts of this continent where there are settlements of
Friends, and also, in many places amongst those not of our society.
In declaring what he believed to be the counsel of God, he was bold
and fearless, and his ministry, though unadorned with the
embellishments of human learning, was clear and powerful. In
argument he was strong and convincing, and his appeals to the
experience and convictions of his hearers, were striking and
appropriate. He saw, and deeply lamented the great departure of
many in the society of Friends, from that plainness and simplicity,
and that godly sincerity, which characterized it in the beginning.
Hence he felt himself called upon, under the influence of the love of
the gospel, to admonish his brethren in religious profession, to rally
to the ancient standard, the light of truth manifested in the heart,
and to follow no man any further, than he should be found a follower
of Christ. He assailed the strong holds of superstition and bigotry
with great boldness, which sometimes alarmed the timid, and roused
the prejudices of others. Yet to the candid inquirer and sincere
seeker after truth, he breathed the language of encouragement, of
consolation and of comfort. His great and primary concern was to
draw the minds of the people to practical righteousness—from all
outward dependance to the sure foundation, the rock of ages, the
spirit of truth, the comforter, “Christ within, the hope of glory.” He
generally corroborated the doctrines which he preached, by
appropriate references to the testimonies and experience of those
who have gone before us, as recorded in the scriptures of truth.
Through the efficacy of that power which enabled him to say, “By
the grace of God, I am what I am,” many were convinced of the
truth, through his ministry.
So full and pointed was his testimony against a hireling ministry,
which he held to be, not only in direct violation of the great gospel
precept “Freely ye have received, freely give,” but fraught with
incalculable injury to the best interests of mankind, that he
sometimes gave offence to those, whose minds were strongly biased
in its favour. Yet such was the general kindness and benevolence of
his character, that he did not willingly give offence to any. While he
condemned the practice, he was kind and charitable to those, who,
through the influence of education and early prejudice, differed from
him on this subject. Such was his concern that his examples should
comport with his testimony, that he was scrupulously careful to
defray his own expenses when travelling as a minister.
When his meetings were attended by a large concourse of persons
of various denominations, the solemnity and stillness that prevailed,
were often very remarkable, reminding us of the testimony of
primitive Friends, that the power accompanying their gospel labours
so overshadowed the assemblies, that truth reigned over all. Being
deeply sensible of his own inability to promote the cause of truth
and righteousness, without divine aid, he was engaged to dwell near
the fountain of light and life, and to minister as this opened and
gave ability. He was indeed an example of Christian humility, and
eminently preserved from being elated by the applause of men, or
depressed by their censure. Many were the exercises which he felt
on account of the evils which abound in the world, and the
oppressed condition of the African race excited his tenderest
sympathy. Their cause engaged his earnest solicitude for the greater
part of his life, and he was often led feelingly and powerfully to
advocate it. We believe that many were convinced, through his
labours, of the cruelty and injustice of holding them in bondage. He
bore for many years a faithful testimony against slavery, by carefully
abstaining from the use of articles which he believed to be produced
by the labour of slaves. When at home, and not engaged in services
more strictly of a religious character, he laboured diligently with his
own hands, believing it the duty of all to be usefully employed in
obtaining the necessaries of life; and when acquired, he acted as a
steward under the direction of the bountiful Giver, being restrained
from using them for selfish gratification. In the various relations of
life he was a bright example, worthy of imitation: he was an
affectionate husband; and as a father and guardian, his concern for
the religious and moral education of his children, and those placed
under his care, was very great, that they might be brought up in the
fear and admonition of the Lord. For these ends he exercised the
authority of a parent with firmness, but in much tenderness and
love. His tender sympathy was excited for the poor, to whom he was
a kind and liberal friend, often supplying their necessities. It may be
truly said of him, that he was a man fearing God and hating
covetousness. He was a peace-maker, endeavouring, both by
precept and example, to promote harmony in his neighbourhood;
and in this respect he was very useful, his Christian deportment
having gained the confidence and affection of his neighbours. He
was very diligent in the attendance of religious meetings, and often
led to encourage others, assuring them, from his own experience,
that none could expect to increase in the divine life, until they
considered that important duty paramount to temporal concerns: he
was also conspicuously useful in supporting the discipline of the
society.
At a very advanced age he continued to labour in the Lord’s
vineyard, with the same fervent zeal, the same dedication of heart,
for which he had been so eminently distinguished in the earlier
stages of his life; and in the exercise of his gift in the ministry, he
was as lively, clear, and cogent, as at any former period. Having
been long taught in the school of Christ, and being deeply
experienced in the things which concern our eternal well-being, he
was well qualified to administer counsel and encouragement to
others; and was frequently led, feelingly and forcibly, to impress
upon the minds of the rising generation, the importance and
necessity of early attention to the inward discoveries of divine light;
cautioning them not to rest in the tradition of their fathers, but to
walk by the same rule, and to mind the same thing, which has led
the righteous in all ages safely through time; nor to depend upon
the teachings of men, for that knowledge which brings life and
immortality to light in the soul; declaring that faithfulness and
obedience to the influence of divine grace in their own hearts, could
only qualify them to advance the standard of truth and
righteousness in the earth. His dedication to the law of the spirit of
life in Christ Jesus, his firmness in the support of those testimonies
which he felt himself called upon to maintain, and his plainness in
reproving unfaithfulness in others, and bearing testimony against
every appearance of evil, gave offence to some; yet none of these
things moved him, neither counted he his life dear to himself, so that
he might finish his course with joy, and the ministry he had received,
to testify the gospel of the grace of God; and we are persuaded that
his feet were established upon that rock, against which the powers
of darkness shall never be able to prevail. He was favoured, in times
of the greatest trial, to experience the truth of the prophetic
declaration, “Thou wilt keep him in perfect peace, whose mind is
stayed on thee, because he trusteth in thee.”
He was favoured with a good constitution, and in the decline of life,
was still actively engaged in the concerns of society, and
industriously employed in his temporal avocations.
His mental powers continued strong and vigorous to the end of his
labours. His comprehensive and energetic mind was apparently but
very little impaired by the revolution of more than fourscore years.
Within the last two years of his life, he travelled extensively in the
work of the ministry.
When he was eighty years of age, he opened in this monthly
meeting a concern to pay a religious visit to Friends and others in
some parts of the yearly meetings of New-York, Philadelphia,
Baltimore, Virginia, Ohio, and Indiana. He obtained a certificate of
unity and concurrence from this monthly meeting, endorsed by
Westbury quarterly meeting. In this visit he experienced many deep
probations on account of the unsettled state of society. “For the
divisions of Reuben, there were great searchings of heart.” Yet he
was enabled to accomplish his visit to the southern and western
yearly meetings, agreeably to his prospect. Shortly after his return
from this journey, he met with a severe affliction in the loss of his
beloved companion, with whom he had lived in near union and
affection for fifty-eight years.
In the summer of 1829, in pursuance of his prospect as before
mentioned, he visited most of the meetings of Friends in the
northern and western parts of our yearly meeting. His gospel
labours, during these arduous and extensive visits, were productive
of satisfaction and peace to his own mind, and were peculiarly
seasonable and acceptable to his friends, as appears by numerous
certificates of near unity, which he produced to this meeting on his
return home; after which, he attended all the meetings of Friends in
the city of New-York, and on this island, very much to their
satisfaction. In these last visits, as heretofore, his gospel labours
were remarkably clear and powerful, and we trust are profitably
remembered by many. He seemed renewedly concerned on account
of the deviations from that plainness and simplicity into which the
truth would lead; and expressed the comfort it would be to him to
see a reformation in these respects.
On first day morning, the 14th of 2d month last, he was engaged in
his room, writing to a friend, until a little after ten o’clock, when he
returned to that occupied by the family, apparently just attacked by
a paralytic affection, which nearly deprived him of the use of his
right side, and of the power of speech. Being assisted to a chair near
the fire, he manifested by signs, that the letter which he had just
finished, and which had been dropped by the way, should be taken
care of; and on its being brought to him, appeared satisfied, and
manifested a desire that all should sit down and be still, seemingly
sensible that his labours were brought to a close, and only desirous
of quietly waiting the final change. The solemn composure at this
time manifest in his countenance, was very impressive, indicating
that he was sensible the time of his departure was at hand, and that
the prospect of death brought no terrors with it. During his last
illness, his mental faculties were occasionally obscured, yet he was
at times enabled to give satisfactory evidence to those around him,
that all was well, and that he felt nothing in his way.
His dependance had long been upon that arm of power alone, which
supported him under every probation, and near the conclusion of the
letter above alluded to, he thus expressed himself: “And if we are
favoured to gain an inheritance in that blissful and peaceful abode,
where the wicked cease from troubling, and the weary are at rest,
we must ascribe it all to the unmerited mercy and loving kindness of
our heavenly Father, who remains to be God over all, blessed for
ever.” He continued gradually to decline until the evening of the
27th, when he quietly passed from the trials of time, we doubt not,
to receive the reward of the righteous.
His funeral took place on fourth day, the 3d of 3d month. It was
attended by a large concourse of Friends and others, and a solid
meeting was held on the occasion; after which, his remains were
interred in Friends’ burial ground at this place.
Signed by direction and on behalf of Jericho Monthly Meeting, held
4th month, 15th, 1830.
WILLET ROBBINS, }
Clerks.
ABIGAIL HICKS, }
At Westbury Quarterly Meeting held at Westbury, the 22d of 4th
month, 1830.
A memorial of Jericho Monthly Meeting, concerning our late beloved
friend Elias Hicks, was produced and read, and being satisfactory to
the meeting, was approved, directed to be endorsed, and forwarded
to the Meeting for Sufferings.
Signed on behalf of the meeting by
STEPHEN UNDERHILL, }
Clerks.
SARAH COCK, }

At a Meeting for Sufferings held in New-York, 5th month, 26th, 1830,

The memorial from Jericho Monthly Meeting, endorsed by the
Quarterly Meeting of Westbury, concerning our beloved friend Elias
Hicks, deceased, being deliberately attended to, was approved and
directed to the Yearly Meeting.
Extract from minutes of said meeting.
JOHN BARROW, Clerk.
At the Yearly Meeting of New-York, held by adjournments, from the
24th of the 5th month, to the 28th of the same inclusive, 1830,
A testimony of Jericho Monthly Meeting, endorsed by Westbury
Quarterly Meeting, and approved by the Meeting for Sufferings,
concerning our ancient beloved friend Elias Hicks, was read and
approved. Much solicitude was felt and expressed that it may, with
the remembrance of his exemplary life, encourage us to walk by the
same rule, and to mind the same thing, which enabled him to
become so eminently useful in his day and generation.
SAMUEL MOTT, }
Clerks.
ANN M. COMSTOCK, }
 TRANSCRIBER’S NOTE.
Archaic and obsolete spellings and usage were left as originally printed, however obvious
typos were fixed. Place names are often misspelled by current standards, but are easily
recognizable.
Details of the changes are below, the correction being inside square braces. In the text,
changes are indicated like this.
Page 19 proceeded to Oswego and Appoquague[Apoquague], and then to
Page 37 at Little Esopus, Marlborough, and Newburg[Newburgh] Valley;
Page 38 near Salem, Purchase, Apoquage[Apoquague], Mamaroneck, and
Page 51 Champlain, and got to Ferrisburg[Ferrisburgh] just in time
Page 54 occasioned by the many obvious deficiences[deficiencies] and
Page 61 travail among them, being baptised[baptized] into their low
Page 72 following days we attended meetings at Monallen[Menallen]
Page 79 15th we were at Moore’s Town and Rancocus[Rancocas]; in the
Page 114 at Chesnut Ridge, and Poquague[Poughquague], we proceeded
Page 210 manner, the way and means of man’s salvavation[salvation];
Page 217 own experience, those works of righeousness[righteousness]
Page 220 it made his heart glad, and he took Micha’s[Micah’s] ephod,
Page 223 attend Friend’s[Friends’] meetings: and towards his close,
Page 232 business, not feeeling[feeling] any particular religious
Page 275 the excellency of the gospel dispenpensation[dispensation],
Page 290 covering over the meeeting[meeting].
Page 301 to-day, to morrow[to-morrow] is dead. This subject very
Page 301 funeral, and led to an awkening[awakening] communication,
Page 301 our aproaching[approaching] yearly meeting. Left home early
Page 306 more faithfulnes[faithfulness] and attention to the inward
Page 311 attended Friend’s[Friends’] meeting at Plainfield. Notice
Page 315 Huntington and Monallin[Menallen]. In these opportunities my
Page 315 From Monallin[Menallen] we rode to Baltimore, in order to
Page 320 seven following days we attended meetings at Mulica[Mullica]
Page 352 the Saw Pitts[Pits]. Here we had a meeting the next day at
Page 359 Creek, and attended Friend’s[Friends’] meeting as it came in
Page 360 day we proceeded towards Ferrisburg[Ferrisburgh], where we
Page 369 return from the service he he[duplicate] had sent them out
Page 373 that my proceedure[procedure] was under right direction; for
Page 376 Concord, St. Clairville[Clairsville], Plainfield, Flushing,
Page 402 Huntington and Monallin[Menallen]. These were all favoured
Page 424 11th, we attended Friends’ meeting at Rancocus[Rancocas].
Page 429 in this Friends’[Friend’s] house. On fourth day we attended
*** END OF THE PROJECT GUTENBERG EBOOK JOURNAL OF THE
LIFE AND RELIGIOUS LABOURS OF ELIAS HICKS ***

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