8.

DNA AMPLIFICATION
1) Introduction

Amplification means making multiple identical copies (replicates) of a

DNA sequence. This can be carried out by various different methods,
including cell cloning where host cells (manipulated using a vector to
contain a DNA insert of interest) are allowed to divide and, as they do
so, the insert is replicated also. However, one particular method of DNA
amplification has proved very important in recombinant DNA technology
and is used in a range of applications in medicine and forensic science.
That method is PCR.

a) What is PCR?

PCR stands for the Polymerase Chain Reaction and was developed in
1987 by Kary Mullis and associates.

PCR is capable of producing enormous amplification (i.e. identical

copies) of a short DNA sequence from a single molecule of starter DNA.

It is used to amplify a specific DNA (target) sequence lying between

known positions (flanks) on a double-stranded (ds) DNA molecule.

The amplification process is mediated by oligonucleotide primers that,

typically, are 20-30 nucleotides long. The primers are single-stranded
(ss) DNA that have sequences complementary to the flanking regions of
the target sequence. Primers anneal to the flanking regions by
complementary-base pairing (G=C and A=T) using hydrogen bonding.

The amplified product is known as an amplicon.

Generally, PCR amplifies smallish DNA targets 100-1000 base pairs

(bp) long.
(It is technically difficult to amplify targets >5000 bp long.)

PCR has many applications in research, medicine and forensic science.

b) How does it work?

Requirements:
 • thermal cycler
• PCR amplification mix typically containing:

• sample dsDNA with a target sequence

• thermostable DNA polymerase
• two oligonucleotide primers which are complementary to the
sequence flanking the target sequence
• deoxynucleotide triphosphates (dNTPs)
• reaction buffer containing magnesium ions and other components

3 stages:

1. Heat denaturation

A DNA molecule carrying a target sequence is denatured by heat at 90-

95oC. The two strands separate due to breakage of the hydrogen bonds
holding them together.

2. Primer annealing

In the presence of an excess of dNTPs (the 'building blocks' of new

DNA material), oligonucleotide primers are added. The primers are
complementary to either end of the target sequence but lie on opposite
strands. As the mixture cools at a lower temperature (50-65oC), each
strand of DNA molecule becomes annealed with an oligonucleotide
primer complementary to either end of the target sequence.

3. Primer extension

DNA polymerase is then added and complementary strands are

synthesized at a temperature of 60-75oC. The polymerase causes
synthesis of new material in the 5' to 3' direction away from each of the
primers.

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Following primer extension, the mixture is heated (again at 90-95oC) to

denature the molecules and separate the strands and the cycle
repeated.
Each new strand then acts as a template for the next cycle of synthesis.
Thus amplification proceeds at an exponential (logarithmic) rate, i.e.
amount of DNA produced doubles at each cycle.

30-35 cycles of amplification can yield around 1μg DNA of 2000bp

length from 10-6μg original template DNA. This is a million-fold
amplification!

PCR DIAGRAMS
(click for larger image)

Initially the 3 different stages at 3 different temperatures were carried

out in separate water baths but nowadays a thermal cycler is used (a
machine that automatically changes the temperature at the correct time
for each of the stages and can be programmed to carry out a set
number of cycles).

A typical thermal cycle might be as follows:

• Heat denaturation at 94oC for 20 seconds

• Primer annealing at 55oC for 20 seconds
• Primer extension at 72oC for 30 seconds

Total time for one cycle = approx. 4 minutes (You can't simply add up
the different times for the stages above because heating and cooling
between each stage also have to be considered!!!)

Following PCR, the amplification product can be detected using gel

electrophoresis where visualization of a band containing DNA fragments
of a particular size can be indicate the presence of the target sequence
in the original starter DNA sample. Similarly, absence of a band may
indicate that the target sequence was not present in the original starter
DNA sample. In this way, PCR can be used in combination with other
techniques to not just simply amplify DNA (which, in essence, is all it
does!) but also to detect specific target sequences.

PCR can be an extremely sensitive technique but is prone to

contamination (unless scrupulous precautions are taken) leading to
false positive results.

c) Fidelity and Taq DNA polymerase

Initially, DNA polymerase enzymes such as E.coli polymerase I

(including Klenow fragment), and T4 polymerase were used for primer
extension.

These DNA polymerases possess very high fidelity (accuracy of

copying) due to proof-reading exonuclease activity (in 3'--->5' direction).

But two main drawbacks with these types of polymerase:

• Optimum working temperature of 37oC produces some

oligonucleotide mis-priming due to non-specific hybridization of
primers (i.e. primers anneal to wrong sequence of DNA).
• High temperature needed for DNA dissociation between cycles of
amplification causes inactivation of the enzyme, so fresh enzyme
needs to be added every cycle - very inconvenient and time
consuming.

An important breakthrough was the use of thermostable DNA

polymerases. These do not denature at the temperatures used to cause
denaturation of the DNA and, therefore, fresh aliquots of enzyme do not
have to be added after each cycle. This also meant that the entire
process could more easily be automated.

The most well-known of these thermostable DNA polymerases is Taq.

This enzyme has a molecular size of 94kD and an optimum reaction
temperature of 75-80oC. But it is also stable at the higher temperature
used for heat denaturation of the sample DNA (i.e. 90-95oC).

Taq polymerase comes from the bacterium Thermus aquaticus which

lives in hot springs and would not survive in nature if it did not have
special adaptations such as this thermostable DNA polymerase.
Taq allows oligonucleotide annealing and primer extension to occur at
high temperatures without itself being denatured.

Advantages of Taq:

• Great reduction in mis-priming by oligonucleotide primers. Why?

(If you don't understand the reason, see Stringency in Section 6).
• Enzyme survives high temperature so no fresh aliquots are
required. This saves time and allows easier automation of the
process.

Disadvantage of Taq:

• Lack of proof-reading activity means base mis-incorporation

(error) rate is 2-4 times higher than that of 'conventional'
polymerase enzymes.

Other thermostable DNA polymerases:

Stoffel fragment
61kD fragment of Taq polymerase but approximately two-times more
thermostable and with optimal activity over a wider range of magnesium
concentration.

Recombinant Taq polymerase

This has the advantage over 'natural' Taq enzyme of greater batch
conformity and, hence, higher reproducibility.

2) Technical applications of PCR

a) Generation of probes

Cloned DNA can be amplified using primers complementary to known

vector sequences flanking an insert

• Amplified fragment used directly for probing or sequencing.

• Fragment length controlled by use of primers complementary to
internal sequences.
• Can, therefore, produce a range of deletion mutants for sub-
cloning and analysis.
Also, uncloned genes can be amplified from 1st strand cDNA if portion
of amino acid sequence at either end of protein product is already
known.

• Requires degenerate pool of oligonucleotide primers of all

possible sequences.
• PCR at 37oC results in primer mis-matches.
• Produces amplified target DNA with variable termini.
• Use of Taq polymerase and higher annealing temperature only
produces fragments with correct terminal sequences.

b) Generation of cDNA libraries

Eukaryote mRNA has poly A tail at 3' terminus.

• Small amount of cDNA can be made by reverse transcription from

only 1 or 2 mammalian cells by priming mRNA with oligo-dT.
• 1st strand cDNA then undergoes 3' homopolymer tailing with G
residues.
• So molecules have polyT and polyG at either end.
• PCR proceeds using oligo-dT and oligo-dC primers.

However, error rate of 0.25% means the accumulation of a significant

number of errors.

Therefore, need to sequence several independently-isolated clones of a

gene of interest to confirm correct sequence.

c) Production of DNA for sequencing

• Target DNA in clone is amplified using appropriate primers.

• Amplified product then annealed with 32P labelled primer and
directly sequenced.
• Avoids need for sub-cloning into sequencing vector.
• problem arises when same primer used for PCR and sequencing
reactions because primers left over from PCR may compete with
labelled sequencing primers.

d) Analysis of mutations

Deletions and insertions in a gene can be detected by differences in

size of amplified product.
 • Location of mutation determined by selective use of primers for
different regions of target DNA.
• Or by failure to amplify i.e. when mutation lies within region
complementary to one primer.

Point mutations can be detected by using competitive nucleotide

priming:

• 2 or more labelled primers with single base changes used in

separate reactions.
• Only perfectly matched primers yield product with high specific
activity.

3) PCR in medicine and forensic science

a) Diagnosis of monogenic diseases (single gene disorders)

Since1987, PCR has had a major impact on pre-natal diagnosis of

single gene disorders. PCR has also proved very important in carrier
testing.

• Improved speed, accuracy and technical flexibility over previous

methods, e.g. pre-natal diagnosis of sickle-cell anaemia and beta-
thalassaemia.
• Diagnosis is now possible by PCR in 1-7 days vs. 2-4 weeks by
Southern blotting, e.g. cystic fibrosis mutation can be detected
within one day using PCR.

For pre-natal diagnosis, PCR is used to amplify DNA from foetal cells
obtained from amniotic fluid.

Single base changes then detected by one or more of following:

• Dot blot (spot hybridization) with oligonucleotides specific for

known mutation.
• Restriction enzyme analysis (RFLP - restriction fragment length
polymorphism).
• Direct sequencing.

Important to be certain of result so combination of two methods provides

confirmation.
Other conditions which can be detected with the same approach
include:

• Tay-Sachs disease
• phenylketonurea
• cystic fibrosis (CF)
• haemophilia
• Huntingdon's disease
• Duchenne muscular dystrophy (DMD)

In DMD, affected gene is very large - 2Mb; codes for a cytoskeletal

protein, dystrophin.

• Gene composed of coding sequences (exons) interspersed with

non-coding introns of up to 35kb.
• In 60% of cases, DMD arises from deletions in any of 9 specific
exons.
• So multiplex (simultaneous) amplification of all 9 exons needed to
detect change.
• 40% of cases involve sequence polymorphisms e.g. point
mutation.
• Detected by RFLP analysis following PCR.

b) Detection of microorganisms

Generally, PCR and other nucleic acid-based methods such as probes

are often faster, more specific and more sensitive than conventional
methods. However, because nucleic acid-based methods are not
available for all microorganisms, not appropriate for some, and too
expensive for others, they tend to be used for:

• fastidious microorganisms (difficult, or impossible, to grow on

artificial growth media, e.g. Chlamydia species, Rickettsia
species, Trypanosoma species, Treponema pallidum,
Pneumocystis carinii, all viruses).
• slow growing microorganisms, e.g. Mycobacterium species.
• microorganisms present in small numbers in some specimens or
patients and/or at certain stages in the disease, e.g.
Mycobacterium tuberculosis, HIV.
• detection of microbial genes responsible for some aspect of
pathogenesis, e.g. toxin production, antibiotic resistance, pili
formation, capsule production.
 • extremely hazardous microorganisms (where culture is especially
risky), e.g. Category 4 pathogens such as Ebola virus.

Note that to use nucleic-acid-based methods to detect characterize and

identify microorganisms, a DNA or RNA target sequence unique (or
certainly very rare in other microorganisms) must be known in order to
produce primers or probes complementary to it. Also, this sequence
must be highly conserved, i.e. present in all/most strains and variants
of the particular species. The 16S gene (which codes for one of the
ribosomal sub-units) is often used as a target since the DNA sequence
is generally unique to a particular species and it is highly conserved.
Alternatively, the gene coding for an unusual phenotypic character (e.g.
a biochemical reaction) can be targeted. For instance, the gene product
(say an enzyme) could be amino-acid sequenced and then the DNA
sequence coding for the product deduced by "Reverse Genetics" (i.e.
working backwards from the protein product to the DNA sequence using
our knowledge of the genetic code, codon usage, etc.). The process is
called "Reverse Genetics" because it is the reverse of what happens in
nature where the starting point is the DNA sequence and the product
(after transcription and translation) is an amino acid sequence and,
finally, a protein.

It should also be noted that most nucleic-acid-based methods cannot

distinguish live (viable) cells from dead (non-viable) cells. This may not
matter with some infectious diseases where the mere presence of the
pathogen in a specimen indicates disease, e.g. syphilis (Treponema
pallidum), but is not so useful with specimens where only the presence
of viable (live) cells may be considered significant, e.g. Salmonella food
poisoning species in food. However, in some cases live microbial cells
can be detected by NA-based methods, e.g. by targeting microbial
mRNA in a specimen. Since mRNA has a short half life compared to
DNA, its presence in a specimen may indicate viable cells as its source.

Similarly, if NA-based methods such as PCR are used to detect a gene

responsible for the pathogenicity of a microorganism (e.g. toxin
production or antibiotic resistance) the mere presence of the gene is not
necessarily indicative of the presence of the gene product since gene
expression may not be occurring at the time. Again, though one could
target the mRNA which is, at least, indicative of the initial stage of gene
expression, i.e. transcription.

e.g. Retroviruses
Rapid diagnosis may pre-date appearance of antibodies in blood of
patient.

• However, great sensitivity required due to low numbers of viral

copies present in cells.
• Viral DNA/RNA only represents a minute proportion of total cell
DNA.
• Also, may only be one infected cell per 10,000 and, therefore too
little viral DNA for Southern blotting.
• May need to discriminate one specific member from large family
of related viruses. So also require high degree of specificity while
also targeting conserved regions of DNA to guard against high
level of genetic variability characteristic of retroviruses.
• Appropriate selection of primers can distinguish HIV1 from HIV2.
• High risk of cross-contaminating sample with small amounts of
amplified DNA from previous sample requires extra precautions to
prevent false-positives.
• PCR can detect 10-20 copies of viral DNA from 150,000 human
cells.
• Sensitivity for HIV1 and HTLV I of 80% and 100% respectively.

e.g. Mycobacterium tuberculosis

The bacterium that causes tuberculosis (TB) is conventionally identified

by:

• Microscopy (e.g. acid-fast, auromine O, or fluorescent antibody

staining of the bacterial cells in the patient's sputum followed by
visualization and detection using microscopy. However, the
number of cells in the specimen is often very low (in some
patients effectively zero) meaning that false negatives are
common.
• Culture on solid, artificial growth media, e.g. Lowenstein-Jensen
(LJ) or Dorset's Egg. However, it can take up to 6 weeks for
colonies of Mycobacterium tuberculosis to become visible.
[Some other Mycobacterium species are even slower growers,
e.g. Mycobacterium ulcerans can take 6 months!!!]

PCR can detect DNA target sequences diagnostic of Mycobacterium

tuberculosis in a matter of hours. The saving in time over conventional
methods means that patient treatment and tracing of contacts can begin
much sooner.
c) PCR in forensic science

Crucial forensic evidence may often be present in very small quantities,

e.g. one human hair, body fluid stain (blood, saliva, semen). Often there
is too little material for direct DNA typing and other analyses. But PCR
can generate sufficient DNA from a single cell!

PCR also possible on extensively degraded DNA. Examples include:

DNA from single dried blood spot, saliva, semen, tissue from under
fingernails, hair root, etc.

Other advantages of PCR in forensic science are:

• Relatively simple to perform and therefore to standardize.

• Fast- results obtainable within 24 hours.

Main legal problem with PCR is that identification is made from copied
DNA rather than original material.

• Therefore must demonstrate that errors due to mis-incorporation

are below significant level. PCR can only show the probability of a
DNA sample matching a suspect. Particularly in the past, defence
lawyers took advantage of (some?) jurors lack of knowledge of
statistics (think how many people buy National Lottery tickets and
are sure they are going to win!) to persuade them to acquit a
suspect because the DNA evidence wasn't 100% conclusive.
Nowadays, most juries will convict if the probability of the DNA
specimen not originating from the accused can be proved to be
millions to one.
• Another potential problem is due to cross-contamination between
samples. Unless great care is taken, the laboratory worker may
'"prove" himself, or herself, to be the murderer or rapist!
• A one-way line of flow from sample preparation to PCR to DNA
typing is essential.

There are not only many variants of PCR, but also alternative
amplification techniques in use. For instance, amplification can be
carried out by the use of a host-vector system and cell cloning. Before
the development of PCR, this was the main method used but it is not as
powerful nor as convenient for most purposes as PCR.
Another example of an alternative amplification technique is the Ligase
Chain Reaction (LCR).

This is a DNA amplification technique which can be used to detect trace

levels of known nucleic acid sequences. LCR involves a cyclic two-step
reaction:

1. A high-temperature melting step in which double-stranded target

DNA unwinds to become single-stranded.
2. A cooling step in which two sets of adjacent, complementary
oligonucleotides anneal to the single-stranded target molecules
and ligate together.

The products of the ligation from one cycle serve as templates for the
next cycle’s ligation reaction. LCR results in the exponential
amplification of the ligation products in a manner analogous to the
exponential amplification of template in the PCR reaction. An example
of an application of LCR is the detection of DNA sequences specific to
particular microorganisms (e.g. Chlamydia trachomatis) to aid
identification and diagnosis of the disease caused.

An example of a variant of PCR used to detect mutations is the

Amplification Refractory Mutation System (ARMS).

Also known as: Allele Specific PCR (ASPCR); PCR Amplification of

Specific Alleles (PASA).

This is an amplification technique used for the detection of known

single-base substitutions or microdeletions/insertions. Two
complementary reactions are used. One contains a primer specific for
the normal allele and the other reaction contains a primer for the mutant
allele (both have a common 2nd primer). One PCR primer perfectly
matches one allelic variant of the target but is mismatched to the other.
The mismatch is located at/near the 3' end of the primer leading to
preferential amplification of the perfectly matched allele. Genotyping is
based on whether there is amplification in one or in both reactions. A
band in the normal reaction only indicates a normal allele. A band in the
mutant reaction only indicates a mutant allele. Bands in both reactions
indicate a heterozygote. ARMS can detect a mutant allele in the
presence of 40 copies of the normal allele. ARMS is claimed to be: rapid
(1 working day), reproducible, inexpensive, automatable.
ARMS can be used to screen for homozygous and heterozygous
(carrier) states for: cystic fibrosis, alpha-1-antitrypsin deficiency, sickle-
cell anaemia, phenylketonuria, apolipoprotein E, B-thalassaemia, etc.