DNA AMPLIFICATION

Introduction

Advances in genetic studies was accelerated in 1970’s by discovery of a new technique called gene cloning (making
copies of the same gene). But this process was overtaken by a second revolutionary procedure of polymerase chain
reaction in 1980’s (making several short copies of a DNA molecule using DNA polymerase enzyme). This new
procedure opened new approaches in genetic research as well as the wider field of biology.

Amplification therefore deals with reproduction of multiple identical copies (replicates) of a DNA sequence (or a
gene). This can be carried out by various different methods, including cell cloning where host cells inserted into a
vector are replicated during the process of division of a vector. However, of great interest in DNA amplification and
very important in recombinant DNA technology is the process of PCR, which has a range of applications in medicine
and forensic science.

Polymerase chain reactions (PCR)

PCR, a procedure developed in 1987 by Kary Mullis et al., is capable of producing enormous amplification (i.e.
identical copies) of a selected 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 DNA molecule. The
amplification process is mediated by two short single stranded oligonucleotide primers (20-30 nucleotides), that
have complementary sequences to the flanking regions of the target DNA region. Primers attach to the flanking
regions by complementary-base pairing (G=C and A=T) using hydrogen bonding.

The amplified product called the amplicon is generally a small region of 100-1000 base pairs long. It is normally
difficult and complex to amplify any targets greater than 5,000 base pairs long.

For the process of PCR to be carried out the following materials are required:

 Thermal cycler/hot water bath

 PCR amplification mix typically containing:

 Sample of double stranded DNA with a target sequence

 Thermostable DNA polymerase (originally from Thermus aquaticus or currently from recombinant
technology)

 Two oligonucleotide primers which are complementary to the sequence flanking the target
sequence

 Deoxynucleotide triphosphates (dNTPs)

 Reaction buffer containing magnesium ions and other components

DNA amplification by PCR can be summarized into 3 main stages:

1. Heat denaturation (Double DNA strand breakage)

A double strand of a DNA molecule carrying a target sequence is broken down (denatured) into single strands by
heating at a temperature range of 90-95 oC. The two strands separate due to breakage of the hydrogen bonds
holding them together.

2. Primer annealing/hybridization

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 end of each target
strands. As the mixture cools at a lower temperature (50-65 oC), each strand of DNA molecule becomes attached
(annealed) to an oligonucleotide primer complementary to either end of the target sequence forming a hybrid.

3. Primer extension/synthesis

The enzyme (DNA polymerase) is then added and complementary strands are synthesized at a temperature of 60-
75oC. The polymerase causes synthesis of new DNA strand in the 5' to 3' direction away from each of the primers.
The enzyme (originally Taq polymerase) used is normally very stable at high temperatures hence is not inactivated by
the heat treatment like most enzymes.

The process/cycle of denaturation,- hybridization-synthesis is repeated several times (usually 30-35 cycles) with each
new strand acting as a template for the next cycle of synthesis. This result in exponential (logarithmic) increase in the
amount of DNA produced due to doubling in each cycle. 30-35 cycles of amplification can yield around 1μg DNA of
2000bp length from 10-6μg original template DNA ( a million-fold amplification).

Insert diagrams of amplification process/copies here

Initially the 3 different stages at 3 different temperatures were carried out in separate water baths but currently 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). For example, a typical thermal cycle for DNA
amplification, might be programmed 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 is approximately 4 minutes because of heating and cooling between each stage.

Analysis of PCR Product

PCR is just a beginning of a complex series of experiments in which the amplification product is studied in various
ways in order to learn more about the features of the molecule that acted originally as a template. There are several
ways of analyzing a PCR product including:

1. Agarose gel electrophoresis

2. Direct sequence analysis of the PCR product

3. Cloning of the PCR product.

Gel electrophoresis involves visualization of a band containing DNA fragments after Ethidium bromide staining, or if
the DNA yield is low, then Southern blotting could be used to detect the amplicon. Detection of a band of a particular
size indicates 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. Hence, PCR can be used in
combination with other techniques to amplify DNA as well as detect specific target sequences.

NB: PCR can be an extremely sensitive technique but is prone to contamination leading to false positive results.
Hence extra caution has to be practiced during PCR procedures from DNA extraction to amplification.

DNA Polymerases

Initially, DNA polymerase enzymes such as E.coli polymerase I, 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 there are 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 hence is 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 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 is produce by a bacterium called Thermus aquaticus which is adapted to hot springs and would not
survive in nature if it did not have special characteristics such as this temperature stable (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 since high temperaures allow only specific oligo-
binding to occur.
 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 leading to base mis-incorporation (error) rate that 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

Produced by recombinant technology and is better than the natural Taq enzyme due to greater batch conformity,
hence, higher reproducibility.

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.

c) Production of DNA for sequencing

 Target DNA in clone is amplified using appropriate primers and hybridized with radiolabelled 32 P primer and
directly sequenced. This avoids need for sub-cloning into sequencing vector.

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.

e) Diagnosis of monogenic diseases (single gene disorders)

PCR is applied in pre-natal diagnosis of single gene disorders and testing gene carriers. It has improved speed,
accuracy and technical flexibility over previous methods, e.g. pre-natal diagnosis of sickle-cell anaemia and beta-
thalassaemia. For example, diagnosis is now possible by PCR within 1 day vs. 2-4 weeks by Southern blotting, e.g.
cystic fibrosis mutation.

f) Detection of microorganisms

Generally, PCR is often faster, more specific and more sensitive than other conventional methods. It is used to
detect:

 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.

For example, PCR can detect DNA target sequences diagnostic of Mycobacterium tuberculosis in a matter of hours
hence patients get early treatment.

h) 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 is also possible on extensively degraded DNA. Examples include: DNA from single dried blood spot, saliva,
semen, tissue from under fingernails, hair root, etc.

The main problem with PCR is that identification is made from copied DNA rather than original material. This may
lead to serious problems in identity of the origin of the sample hence victimizing the wrong person. For example due
to cross-contamination between samples the laboratory worker may '"prove" himself, or herself, to be the criminal!

i) Amplification of RNA

By use of reverse transcriptase, RNA molecules could be converted into single stranded cDNA which can be amplified
using the normal PCR process. The useful application of RT-PCR is in measuring the relative amount of the mRNA in
different tissues or in same tissues at different times .The amount of RNA in a cell is a reflection of the activity of a
parent gene hence its quantification enables monitoring of changes in gene expression.

j) Comparison of genomes

Use of short primers is important in understanding the evolutionary origin of various animal and plant species. The
basis of this distinction is the characteristic banding pattern of the DNA template used which represents the
organization of the cells genome. Two closely related organisms would be expected to have similar banding patterns
than two distantly related organisms. This technique of using random short sequence is called random amplified
polymorphic DNA (RAPD) analysis.