MOLECULAR

DIAGNOSTICS
MODULE
Molecular Techniques
Unit 1. Amplification Techniques: Polymerase
Chain Reaction
Introduction

Early analyses of nucleic acids were limited by the

availability of material to be analysed. Generating enough copies of a
single gene sequence required propagation of millions of cells in culture or
isolation of large amounts of genomic DNA. If a gene had been cloned,
many copies could be generated on bacterial plasmids, but this preparation
was laborious, and some sequences were resistant to propagation in this
manner.

The advent of the ability to amplify a specific DNA sequence

opened the possibility to analyse at the nucleotide level virtually any piece
of DNA in nature. The first specific amplification method of any type was
the polymerase chain reaction (PCR). Other amplification methods have
been developed based on making modifications of PCR. The methods that
have been developed to amplify nucleic acids can be divided into three
groups, based on whether the target nucleic acid itself, a probe specific for
the target sequence, or the signal used to detect the target nucleic acid is
amplified. These methods are discussed in this Unit.

Learning Objectives
1. Know the brief history of PCR
2. Describe the principles of PCR
3. Determine the basic requirements and Instrumentation of PCR
4. Differentiate the different types of PCR
5. Enumerate the applications of PCR
 Presentation of Contents

POLYMERASE CHAIN REACTION

History
Kary Banks Mullis developed PCR in 1985 and was awarded nobel prize
in 1993. The first successful amplification was a short fragment of the
Escherichia coli plasmid, pBR322. The first paper describing a practical
application, the amplification of beta-globin and analysis for diagnosis of patients
with sickle cell anemia, was published 2 years later. He called the method a
“polymerase-catalyzed chain reaction” because DNA polymerase was the enzyme
he used to drive the replication of DNA, and once it started the replication
continued in a chain reaction. The name was quickly shortened to PCR. Since
PCR was conceived and first performed, it has become increasingly user-friendly,
more automated, and more amenable to use in a clinical laboratory, with infinite
applications possible.

Principles
PCR is an in vitro technique that takes specific sequence of DNA of small
amount (eg. drops of blood, semen strains, single hair, vaginal swabs etc.) and
amplifies it to be used for further testing. Two methods currently exist for
amplifying the DNA or making copies: Cloning and PCR. However, cloning takes
a long time for enough clones to reach maturity while PCR works on even a
single molecule quickly.

Basic Requirements and Instrumentation

The basic requirements for PCR reaction (table 1) comprises of 1) DNA

sequence of target region must be known, 2) Primers which is typically 20-30
bases in size, 3) DNA polymerase (eg. Taq) which is thermo-stable or not
inactivated by heat at 95C, 4) Deoxyribonucleoside triphosphates (dNTPs), 5)
Buffer solution and 6) Divalent cations (eg Mg2+).

Table 1. Components of a Typical PCR Reaction (taken from page 124 of

___)
Component Purpose
0.25 mM each primer Directs DNA synthesis to the desired region
(oligodeoxynucleotides)
0.2 mM each dATP, dCTP, Building blocks that extend the primers
dGTP, dTTP
50 mM KCl Monovalent cation (salt), for optimal

10 mM Tris, pH 8.4
1.5 mM MgCl2
2.5 units polymerase
102–105 copies of template
hybridization of primers to template
Buffer to maintain optimal pH for the
enzyme reaction
Divalent cation, required by the enzyme
The polymerase enzyme that extends the
primers (adds dNTPs)
Sample DNA that is being tested.

PCR primers is a strand of nucleic acid that serves as a starting point for
DNA replication. A primer for each target sequence on the end of your DNA is
needed. This allows both strands to be copied simultaneously in both forward and
reverse directions. However, there are few primer problems;
1. Primer should flank the sequence of interest
2. Primers that match multiple sequences will give multiple products
3. Repeated sequences can be amplified – but only if unique flanking
regions can be found where primers can bind
4. A primer may form a dimer with itself or with the other primer
5. Primers can have self-annealing regions within each primer (ie hairpin
and foldbackloops)

PCR Taq DNA polymerase stands for Thermus aquaticus, a

microorganism found in 176F hot springs in Yellow stone National Forest. It is
stable in high temperatures and acts in the presence of Magesium. The optimum
temperature of Taq polymerase is 72C. The disdvantages of Taq polymerase is
that it lacks 3’ to 5’ exonuclease proof reading activity, commonly present in
other polymerases, It mis-incorporates 1 base in 10 4., a 400 base pair target will
contain an error in 33% of molecules after 20 cycles and error distribution will be
random.

Self-Assessment Activity

Draw a sample of exponential amplification of DNA in PCR

 PCR involves 3 process: Denaturation, Annealing and Elongation.
Denaturation is the first step in PCR, in which the DNA strands are separated by
heating to 95C. The hydrogen bonds between the two strands breaks down and the
two strands separates. Annealing is the process of allowing two sequences of
DNA to form hydrogen bonds. The annealing of the target sequences and primers
is done by cooling the DNA to 55C. Elongation is now shifted to 72C which is
ideal for polymerase. Primers are extended by joining the bases complementary to
DNA strands. Taq polymerase binds to the template DNA and starts adding
nucleotides that are complementary to the first strand. Now the first cycle is over
and next cycle is continued.

Self-Assessment Activity

Summarize the steps in PCR

Process Temperature Time

Denaturation
Annealing
Elongation

Types

PCR is highly versatile technique and has been modified in variety of way to suit
specific applications

Inverse PCR
In this method, amplification of DNA of unknown sequence is carried out
from known sequence. This is especially useful in identifying flanking sequences
of various genomic inserts.
 Figure 1. Inverse PCR (taken from
https://upload.wikimedia.org/wikipedia/en/thumb/d/da/Inverse_PCR_2.png/250px
-Inverse_PCR_2.png)

Reverse Transcription PCR (RT-PCR)

RT-PCR is employed for amplification of RNA molecules. It is widely
used in expression profiling, to determine the expression of a gene or to identify
the sequence of an RNA transcript.
 Figure 2. Reverse transcription PCR (taken from
https://upload.wikimedia.org/wikipedia/commons/e/e7/RT_PCR_Model.jpg)

Quantitative real time PCR (Q-RT PCR)

It is used to amplify and also for quantification and detection of DNA,
copies of DNA or RNA. It is commonly used to determine whether a DNA
sequence is present in a sample and the number of its copies in the sample. It uses
fluorescent dyes to measure the amount of amplified product in real time.
Fluorescence detected is directly proportional to the fluorophore released and the
amount of DNA template present in the PCR.

Figure 3. Quantitative real-time PCR (taken from

https://d3i71xaburhd42.cloudfront.net/2703a4f3f178bf4c76a6b44364cd815a9965
6d71/3-Figure1-1.png)

Self-Assessment Activity

Differentiate other types of PCR

Type Detects Unique Feature

Applications

PCR is not only vital in the clinical laboratory by amplifying small

amounts of DNA for STD detection, but it is also important for genetic
predisposing for defects such as Factor V leiden. The PCR technology can be
employed in law enforcement, genetic testing of animal stocks and vegetable
hybrids, and drug screening along with many more areas. Below are as follows:

Molecular Identification
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
DNA Fingerprinting
Classification of organisms
Genotyping
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogen
Sequencing
Bioinformatics
Genomic cloning
Human genome project
Genetic Engineering
Site-directed mutagenesis
Gene expression studies
 Application
Instructions: Discuss how COVID-19 is tested using PCR. Start with
specimen collection to releasing of results.
NOTE: BE CREATIVE!

References:

Buckingham, Lela & Flaws, Maribeth L., (2007) Molecular Diagnostics

Fundamentals, Methods and Clinical Applications, FA Davis: Philadelphia
Kalsoom, Aisha (2012). Polymerase Chain Reaction taken from
https://www.slideshare.net/AYSHA007/copy-of-pcr
Bansal, Anshika (2016) PCR-Slidshare taken from
https://www.slideshare.net/AnshikaBansal4/polymerase-chain-reaction-
62980249
Sujathar23 (2012) https://www.slideshare.net/sujathar23/pcr-and-types
Shanthilal j (2008) https://www.slideshare.net/jshanthilal/pcr-presentation-657596