Polymerase Chain Reaction(PCR)

BY RAJU

What is the PCR?

PCR is the technique that take specific sequence of DNA and amplifies it to be used for further testing.

Purpose of the PCR is to amplify the lot of double stranded DNA molecules(fragments) with same size and sequence by enzymatic method and cycling condition.

PCR reaction :-

Essential components required: DNA template

DNA polymerase

Primer

dNTPs

Buffer to maintain PH

Divalent cations (Mg+2)

Monovalent cation Potassium ions

principle :-

PCR contain three process

1. Denaturation

2. Annealing

3. Extension

cycle repeated according to requirement

Principle :-

Denaturation

Denaturation is the first step of PCR , in which the DNA strands are separated by heating to 94–98 °C for 20–30 seconds.

It causes DNA melting of the DNA template by breaking the hydrogen bonds between two strands and resulting single-stranded DNA molecules.

Principle :-

AnnealingThe reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template.

This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should only bind to a perfectly complementary part of the template. If the temperature is too low, the primer could bind imperfectly. If it is too high, the primer might not bind.

Principle :-

AnnealingTypically the annealing temperature is about 3–5 °C below the melting temperature of the primers used.

Stable DNA–DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence.

The polymerase binds to the primer-template hybrid and begins DNA formation.

Principle :-

ExtensionThe temperature at this step depends on the DNA polymerase used. Taq DNA polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme in this step.

The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to amplify.

Principle :-

Extension At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction.

Primer are extended by adding nucleotides (dNTPs) complementary to the DNA template strand.

Now the first cycle over and second cycle continued, as PCR machine is automated thermocycler the same cycle repeated up to 30-40 times.

PCR reaction steps :-

Step-1

Denaturation

Step-2

Annealing

Step-3

Extension

PCR (contd…)

Primer in PCRPrimer are usually short, chemically synthesized oligonucleotides, with a length of about 18-30 bases, Ideal G-C content 40-60% but play around the length.

Melting temperature should be between 42-65°C

Primers serve as a starting point for DNA synthesis and they are hybridized to the target DNA, which is then copied by DNA polymerase.

Avoid primer-primer hybridization and pick the primer that do not form hairpins.

PCR (contd…)

Taq DNA polymerase in PCRTaq polymerase is a thermostable DNA polymerase named after thermophilic bacterium Thermus Aquaticus from which it was isolated.

Taq’s optimum activity between 75-80°C and in extension step applied temperature is 72°C in PCR.

Drawbacks of Taq is relatively low replication fidelity, it is because lacks of exonuclease proofreading activity.

Extension rate - 50 nucleotides/second.It has error rate measured at about 1 in 9,000 nucleotides.

PCR (contd…)

Pfu DNA polymerase in PCRPfu polymerase is a thermostable DNA polymerase named after hyperthermophilic archaeon pyrococcus furiosus from which it was isolated.

Optimum activity of Pfu is between 72°C .

It has 3’ to 5’ exonuclease proofreading activity, this means Pfu polymerase generate few errors than Taq polymerase.

Extension rate - 1000 nucleotides/minute.It has error rate measured at about 1 in 1.3 million nucleotides.

PCR (contd…) DNA meltingThe melting temperature defined as the temperature at which half of the DNA strands are in the single-stranded (ssDNA) state.

A=T less energy required to break AT bonds

G=C more energy required to break GC bonds

APPLICATIONS OF PCR

1. Diagnosis of genetic diseasesThe use of PCR in diagnosing genetic disease, whether inherited genetic changes or as a result of a spontaneous genetic mutations, is becoming more common. Diseases can be diagnosed even before birth. Examples include:

Genetic counseling-screening the parents for genetic diseases before deciding on having children.

APPLICATIONS OF PCR (contd…)

2. Genetic fingerprints

One of the most famous uses for PCR is in the creation of a genetic fingerprint (also known as DNA profiling) from a sample of blood or semen, or from a hair root. Much beloved by writers of detective fiction, genetic fingerprints are profiles of specific stretches of DNA that vary from person to person. PCR also plays a role in mitochondrial DNA analysis, used for samples from hair shafts and bones when other samples are not available.

Genetic analysis based on PCR is also used in paternity testing, and in tissue typing for organ transplantation.

APPLICATIONS OF PCR (contd…)

3. Detection and diagnosis of infectious diseases

PCR can detect infectious disease before standard serological laboratory tests (tests to detect the presence of antibodies), so allowing treatment to start much earlier. Because of this, PCR is also useful for screening donated blood for infections, and is especially useful for infections that are difficult to culture in the laboratory, such as tuberculosis.

APPLICATIONS OF PCR (contd…)

4. Detection of infection in the environment

PCR is used to monitor and track the spread of infectious disease within an animal or human population. PCR can also be used to detect bacterial and viral DNA in the environment, for example looking at pathogens in water supplies.

5. Personalised medicine

PCR is used in personalised medicine to select patients for certain treatments, for example in cancer when patients have a genetic change that makes a patient more or less likely to respond to a certain treatment.

APPLICATIONS OF PCR (contd…)

6. PCR in researchEscherichia coli in genetic engineering, and to amplify stretches of genetic material for Sanger sequencing – the Human Genome Project used PCR.

PCR can be used in analysis of gene expression, for example looking at levels of expression and when genes are switched on and off in physiological processes, including in health and disease.