PCR: An Overview

By
Dr. Nassra Dabour

Contents
History of PCR
Polymerase Chain Reaction
PCR Reaction Components
Standard PCR Reaction
Avoiding Contamination
Thermal Cycling Profile for Standard
PCR
Gel Electrophoresis
Variants of PCR (multiplex, Tm PCR)
Primers design (practical part)

History OF PCR

As is the photo copier a basic

requirement in an office, so is the
PCR machine in a molecular
biology Laboratory !!!!!!!!!
PCR is DNA replication in a test
tube..

 Great mind behind this PCR :Kary Banks Mullis

Developed PCR in 1985 and was awarded nobel
prize in 1993.
PCR machine otherwise called Thermocycler.

DNA ?

DNA is a nucleic acid that is composed

of two complementary nucleotide
building block chains.
The nucleotides are made up of
a phosphate group,
a five carbon sugar,
and a nitrogen base.

 DNA Sugar
Deoxyribonucleic acid
DNA has four nitrogen bases.
Two are purines , Adenine ( A ), Guanine ( G )
Two are pyrimidines , Cytosine ( C ), Thymine ( T )

AATGGGATGC

Primary

Polymerase Chain Reaction

PCR targets and amplifies a specific region of a
DNA strand.
It is an in vitro technique to generate large
quantities of a

specified DNA.

Often, only a small amount of DNA is available

(EX: A drop of blood, Semen strains, Single hair,
vaginal swabs etc).
Two methods currently exist for amplifying the
DNA or making copies
Cloningtakes a long time for enough clones

What does PCR

Template (the DNA you are exploring)
need?
Sequence-specific primers, Forward & Reverse.
Polymerases
Nucleotides (dATP, dCTP, dGTP, dTTP) (dNTP)
Magnesium chloride (enzyme cofactor)
Buffer
Water, mineral oil
PCR machine

DNA Template
Integrity & Purity
High molecular weight
Pure
Amount
Human genomic DNA should be up to
500ng
Bacterial DNA 1-10ng
Plasmid DNA 0.1-1ng

Primers
Primer length17-30 bases in length (ideal 16-20).
Balance G/C and A/T-rich domains.20-70% G+C,
- Ideally a primer should have a near
random mix of nucleotides, a 50% GC content
Make 3 end of primers a G, C, GC, or CG. Or make GC
content equal in both primers

 Melting temperature (Tm) at which 2 strands

of the duplex dissociate.
- It can be determined experimentally or
calculated from formula
Tm = (4(G+C)) +
(2(A+T))
- Set Tm between 55-80C (Ideal 50-60)
Avoid creating complimentary 3-end base pairs

How Does
Primer-Dimer
Form?

Gel Electrophoresis
Primer-dimer range can vary from 50 bp up to 150 bps

Gel Electrophoresis

rimer-Dimer Effect
Reduced amplification efficiency
Inaccurate quantification of PCR products and potentially
misleading expression levels
Failure of amplification is dependent on downstream
application sensitivity of primer dimer (Sequencing, and
genotyping)
Inefficient use of amplification reagents, enzymes, labor,
and time

Solution for primers

dimer reaction

Gel-based Purification

xcise band from Gel with a razor

or
Filtration Columnbased Clean-up

 Avoid runs of three or more Cs or Gs at the

3-end of primers.
- ideally a primer should have a near random
mix of nucleotides, a 50% GC content
the PolyG or PolyC stretches that can
promote non-specific annealing
Avoid primer self-complementary

Four Normal Deoxynucleosides

Triphosphate
Final concentration of dNTPs should be 50-500
M (each dNTP). Usually included at conc. of
200 M for each nucleotide.
Always use balanced solution of all four dNTPs
to minimize polymerase error rate.

DNA Polymerase
The most widely characterized polymerase is
that
from
Thermus
aquaticus
(Taq),
Thermophilic bacterium lives in hot springs and
capable of growing at 70 -75 C ,
Consist of a single polypeptide chain has a
molecular weight of 95 Kd, and has an optimum
polymerization temperature of 70 80 C (72
C).
0.5 2 units to 50 l reaction. Too little will
limit the amount of products, while

Disadvantages of Taq
Polymerase
Taq Pol lacks
3 to 5 exonuclease proof
reading activity, commonly present in
other polymerases.
Taq mis-incorporates 1 base in 104.
A 400 bp target will contain an error in
33% of molecules after 20 cycles.
Error distribution will be random.

DNA polymerases adds nucleotides to

the 5' end of a strand of DNA. If a
mismatch is accidentally incorporated,
the polymerase is inhibited from
further
extension.
Proofreading
removes the mismatched nucleotide
and extension continues

STEPS INVOLVED for PCR

DENATURATION:

The reaction mixture is heated to a

temperature between 90-98C so that the ds
DNA is denatured into single strands by
disrupting the hydrogen bonds between
complementray bases.
Duration of this step is 1-2 min

ANNEALING:

Temperature of reaction mixture is

cooled to 45-60 C
Primers base pair with the
complementary sequence in the
DNA.
Hydrogen bonds reform.

EXTENSION:

The temperature is now shifted to 72 C

which is ideal for polymerase.
Primers are extended by joining the bases
complementary to DNA strands.

 Elongation step continues where the

polymerase adds dNTP's from 5' to 3', reading
the template from 3' to 5' side, bases are
added complementary to the template.
Now first cycle is over and next cycle is
continued ,as PCR machine is automated
thermo-cycler the same cycle is repeated up to
30-40 times.

PCR cycle

Exponential
Amplification

As amplification proceeds, the DNA sequence between primers

doubles after each cycle. (The amplification of the target sequence
proceeding in an exponential fashion ( 1 2 4 8 16.) up to
million of times the starting amount until enough is present to be

Agarose Gel Electrophoresis

It is a method used in biochemistry and
molecular biology to separate DNA, or RNA
molecules based upon charge, size and shape.
Agarose is a polysaccharide derivative of
agar.

 Amplified fragments can be visualized easily

following staining with a chemical stain such as
ethidium bromide.
The DNA fragments are separated by charge
and the relative sizes of fragments are

Factors for Optimal PCR:

PCR Primers
- correctly designed pair of primers is
required
- primer dimer,hairpin formation should be
prevented
- length of primer
DNA Polymerase
- It lacks proof reading exonuclease activity
- Other polymerases can be used .eg: hot
starter polymerase, high fidelity

 Annealing Temperature
- Very important since the success and
specificity of PCR depend on it
because DNA-DNA hybridization is a
temperature dependent process.
- Ideal Annealing temperature must be
low enough to enable hybridization
between primer and template but high
enough to prevent amplification of
non target sites.
- Should be usually 1-2 C or 5 C
lower than melting temperature (Tm)
of the template-primer duplex

Limitations of PCR
Need for target DNA sequence information
Primer Designing for unexplored ones.
Boundary regions of DNA to be amplified
must be known.
Infidelity of DNA replication.
Taq Pol no Proof reading mech Error 40%
after 20 cycles
Short size and limiting amounts of PCR product
Up to 5kb can be easily amplified .
Up to 40kb can be amplified with some
modifications.
Cannot amplify gene >100kb

Applications of PCR
Screening human DNA samples for mutations
associated with genetic diseases.
Can obtain sequences from hair, blood stain,
bones, other forensic specimens, other
remains preserved at archaeological sites.
Sequencing
Cloning and consequence OGM

Variations of PCR:

Other types of PCR

Overlap extension PCR
Assemble PCR
Helicase dependent amplication
Intersequence-specific PCR(ISSR)
Ligation-mediated PCR
Methylation specifin PCR
Miniprimer PCR
Multiplex PCR
Nested PCR
Temperature gradient PCR
Solid phase PCR
Touch down PCR and so on..

Gradient PCR
Definition: Gradient PCR is a technique that allows the
empirical determination of an optimal annealing
temperature using the least number of steps. This
optimization can often be achieved in one experiment.
The selection of the annealing temperature is possibly the
most critical component for optimizing the specificity of a
PCR reaction.
In most cases, this temperature must be empirically tested.
The PCR is normally started at 5C below the calculated
temperature of the primer melting point (Tm).

Multiplex PCR
Definition
Multiplex PCR is defined as the simultaneous amplification
of two or more DNA targets in a single reaction vessel

 Multiplex-PCR consists of multiple primer sets within a

single PCR mixture to produce amplicons of varying
sizes that are specific to different DNA sequences.
By targeting multiple genes at once, additional
information may be gained from a single test run that
otherwise would require several times the reagents and
more time to perform.
Annealing temperatures for each of the primer sets
must be optimized to work correctly within a single
reaction, and amplicon sizes.

Application of multiplex PCR

Selected applications
Mutation analysis
Gene deletion analysis
Pathogen detection
GMO
STR analysis
Quantitative analysis
Gene expression analysis
Multiplex pre- amplification
Software
PrimerPlex - Software for Multiplex PCR Primer design
MPprimer

MULTIPLEX ADVANTAGES
1) Increases the number of genes that can be analyzed
2) Reduced sample requirements
3) Saves time, effort and potentially money
4) The same reaction conditions for several targets
5) Eliminate pipetting differences between targets
6) Internal control within the same well
7) Convenient for screening