Polymerase chain reaction

HISTORY

Kary Banks Mullis, Ph.D.

(born December 28, 1944) is an American

biochemist and was awarded the Nobel Prize in
Chemistry in 1993 for his development of the
Polymerase Chain Reaction (PCR).
•Spiegelman and Edward Hall discovered the hydrogen bonding
between complementary sequences of DNA & RNA.

•Arthur Kornberg has identified the first DNA polymerase and He was
awarded Nobel Prize in 1959.

•In 1976 a DNA polymerase is isolated from T. aquaticus. It is found to

retain its activity at temperatures above 75°C.

•In 1977 Frederick Sanger reports a method for determining the

sequence of DNA.
-- The technique involves an oligonucleotide primer, DNA
polymerase and modified nucleotide precursors that block further
extension of the primer in sequence-dependent manner. He is awarded
the Nobel Prize in 1980.
 PCR

The Polymerase Chain Reaction (PCR) provides an extremely

sensitive means of amplifying relatively large quantities of DNA

The technique was made possible by the discovery of Taq

polymerase, the DNA polymerase that is used by the bacterium
Thermus aquaticus that was discovered in hot springs

The primary materials, or reagents, used in PCR are:

- DNA nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want to amplify
- Primers, single-stranded DNAs between 20 and 50 nucleotides
long (oligonucleotides) that are complementary to a short
region on either side of the template DNA
- DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA
 PCR

The cycling reactions :

There are three major steps in a PCR, which are repeated for 20 to 40
cycles. This is done on an automated Thermo Cycler, which can heat
and cool the reaction tubes in a very short time.
Denaturation at around 94°C :
During the denaturation, the double strand melts open to single stranded
DNA, all enzymatic reactions stop (for example the extension from a
previous cycle).
Annealing at around 54°C :
Hydrogen bonds are constantly formed and broken between the single
stranded primer and the single stranded template. If the primers exactly
fit the template, the hydrogen bonds are so strong that the primer stays
attached
Extension at around 72°C :
The bases (complementary to the template) are coupled to the primer
on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the
template from 3' to 5' side, bases are added complementary to the
template)
PCR

The different steps of PCR

 PCR

Exponential increase of the number of copies during PCR

PCR

Every cycle results in a doubling of the number of strands DNA present

After the first few cycles, most of the product DNA strands made are
the same length as the distance between the primers

The result is a dramatic amplification of a the DNA that exists between

the primers. The amount of amplification is 2 raised to the n power; n
represents the number of cycles that are performed. After 20 cycles,
this would give approximately 1 million fold amplification. After 40
cycles the amplification would be 1 x 1012
Verification of PCR product
PCR and Contamination

The most important consideration in PCR is contamination

Even the smallest contamination with DNA could affect

amplification

For example, if a technician in a crime lab set up a test reaction

(with blood from the crime scene) after setting up a positive
control reaction (with blood from the suspect) cross
contamination between the samples could result in an
erroneous incrimination, even if the technician changed pipette
tips between samples. A few blood cells could volitilize in the
pipette, stick to the plastic of the pipette, and then get ejected
into the test sample

Modern labs take account of this fact and devote tremendous

effort to avoiding cross-contamination
Optimizing PCR protocols

PCR can be very tricky

While PCR is a very powerful technique, often enough it is not

possible to achieve optimum results without optimizing the
protocol

Critical PCR parameters:

- Concentration of DNA template, nucleotides, divalent cations

(especially Mg2+ ) and polymerase

- Error rate of the polymerase (Taq, Vent exo, Pfu)

- Primer design
Primer design

Primer design in PCR

Perhaps the most critical parameter for successful PCR is the design of primers

Primer selection
Critical variables are:
- primer length
- melting temperature (Tm)
- specificity
- complementary primer sequences
- G/C content
- 3’-end sequence
Primer length

- primer length is proportional to annealing efficiency: in general, the

longer the primer, the more inefficient the annealing

- the primers should not be too short as specificity decreases

 Primer design

G/C content
- ideally a primer should have a near random mix of nucleotides, a 50% GC content
- there should be no PolyG or PolyC stretches that can promote non-specific annealing
 Primer design

Melting temperature (Tm)

- the goal should be to design a primer with an annealing temperature of at least 50°C
- the relationship between annealing temperature and melting
temperature is one of the “Black Boxes” of PCR
- a general rule-of-thumb is to use an annealing temperature that is 5°C lower
than the melting temperature

- the melting temperatures of oligos are most accurately calculated using nearest
neighbor thermodynamic calculations with the formula:
Tm = H [S+ R ln (c/4)] –273.15 °C + 16.6 log 10 [K+]
(H is the enthalpy, S is the entropy for helix formation, R is the molar gas
constant and c is the concentration of primer)

- a good working approximation of this value can be calculated using the Wallace formula:
Tm = 4x (#C+#G) + 2x (#A+#T) °C
- both of the primers should be designed such that they have similar melting temperatures.
If primers are mismatched in terms of Tm, amplification will be less efficient or may not
work: the primer with the higher Tm will mis-prime at lower temperatures; the primer with
the lower Tm may not work at higher temperatures.
 Taq polymerase

•Taq polymerase is a
thermostable DNA polymerase
named after the thermophilic
bacterium Thermus aquaticus
from which it was originally
isolated

•T. aquaticus is a bacterium that lives in hot springs and hydrothermal

vents, and Taq polymerase was identified as an enzyme able to
withstand the protein-denaturing conditions (high temperature) required
during PCR. Therefore it replaced the DNA polymerase from E.coli
originally used in PCR. Taq's temperature optimum for activity is 75-
80°C, with a half-life of 9 minutes at 97.5°C, and can replicate a 1000
base pair strand of DNA in less than 10 seconds at 72°C
One of Taq's drawbacks

-is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease
proofreading activity, and has an error rate measured at about 1 in 9,000
nucleotides. Some thermostable DNA polymerases have been isolated
from other thermophilic bacteria and archaea, such as Pfu DNA
polymerase, possessing a proofreading activity, and are being used
instead of (or in combination with) Taq for high-fidelity amplification.
Other Enzymes

•Pfu DNA polymerase is an enzyme from hyperthermophilic archaeon

Pyrococcus furiosus. Pfu is used to quickly amplify DNA in polymerase chain
reaction (PCR) processes. 'proofreading' properties compared to other
thermostable polymerases.
Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity.

•Vent enzyme from thermococcus litoralis.

Application of PCR

• Isolation of genomic DNA

PCR allows isolation of DNA fragments from genomic DNA by selective
amplification of a specific region of DNA. This use of PCR augments many
methods, such as generating hybridization probes for Southern or northern
hybridization and DNA cloning, which require larger amounts of DNA,
representing a specific DNA region.

• DNA sequencing
to determine unknown PCR-amplified sequences in which one of the
amplification primers may be used in Sanger sequencing. Bacterial colonies
(E.coli) can be rapidly screened by PCR for correct DNA vector constructs.

• Genetic fingerprinting; a forensic technique used to identify a person or

organism by comparing experimental DNAs through different PCR-based
methods.
•Amplification and quantitation of DNA
Quantitative PCR methods allow the estimation of the amount of a
given sequence present in a sample – a technique often applied to
quantitatively determine levels of gene expression. Real-time PCR is
an established tool for DNA quantification that measures the
accumulation of DNA product after each round of PCR amplification.
•PCR in diagnosis of diseases
PCR permits identification of non-cultivatable or slow-growing
microorganisms such as mycobacteria, anaerobic bacteria, or viruses from
tissue culture assays and animal models. The basis for PCR diagnostic
applications in microbiology is the detection of infectious agents and the
discrimination of non-pathogenic from pathogenic strains by virtue of specific
genes.
•VNTR PCR
involves few modifications to the basic PCR process, but instead targets
areas of the genome that exhibit length variation. The analysis of the
genotypes of the sample usually involves simple sizing of the amplification
products by gel electrophoresis. Analysis of smaller VNTR segments known
as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting
databases such as CODIS.
Hot-start PCR

is a technique that modifies the way that a PCR mixture is initially heated.
During this step the polymerase is active, but the target has not yet been
denatured and the primers may be able to bind to non-specific locations (or
even to each other). The technique can be performed manually by heating
the reaction components to the melting temperature (e.g. 95°C) before
adding the polymerase. Alternatively, specialized systems have been
developed that inhibit the polymerase's activity at ambient temperature,
either by the binding of an antibody, or by the presence of covalently bound
inhibitors that only dissociate after a high-temperature activation step. 'Hot-
start/cold-finish PCR' is achieved with new hybrid polymerases that are
inactive at ambient temperature and are only activated at elevated
temperatures.
In Colony PCR

bacterial colonies are rapidly screened by PCR for correct DNA vector
constructs. Colonies are sampled with a sterile toothpick and dabbed into a
master mix. To free the DNA for amplification, PCR is either started with an
extended time at 95°C (when standard polymerase is used), or with a
shortened denaturation step at 100°C and special chimeric DNA
polymerase. Colonies from the master mix that shows the desired product
are then tested individually.
 Pretreatments and extensions
The basic PCR process can sometimes precede or follow another technique:

RT-PCR (or Reverse Transcription PCR) is a common method used to amplify, isolate,
or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a
reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two
reactions are compatible enough that they can be run in the same mixture tube, with the
initial heating step of PCR being used to inactivate the transcriptase. Also, the
polymerase described below exhibits RT activity, and can carry out the entire combined
reaction. RT-PCR is widely used in expression profiling, which determines the
expression of a gene or identifies the sequence of an RNA transcript (including
transcription start and termination sites and, if the genomic DNA sequence of a gene is
known, to map the location of exons and introns in the gene). The 5' end of a gene
(corresponding to the transcription start site) is typically identified by an RT-PCR method
named RACE-PCR, short for Rapid Amplification of cDNA Ends. (Note that the acronym
RT-PCR has more recently been applied to Real-Time PCR, a version of Quantitative
PCR described above.)
 Thank you

Research is what I'm doing when I don't know what I'm doing.
~Wernher Von Braun