Molecular Tools - Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is a laboratory technique for generating large quantities of DNA. Its is a cell free amplification technique for synthesize identical multiple copies of DNA of interest. Developed by Karry Mullis in 1984.

Principle of PCR

The double stranded DNA is denatured to separate into two individual strands and step is called as denaturation. Each strand is  allowed to hybridize with a primer and this step is called as renaturation, after that this duplex is used for DNA synthesis.

Requirement for PCR

1. A target DNA ( 100-30,000 bp in length).
2. Two primers which are complementary to regions flanking the target DNA.
3. Four deoxyribonucleotide (dATP, dTTP, dGTP, dCTP )
4. A thermostable DNA polymerase - Taq polymerase ( isolated from Thermus aquaticus).

Stages of PCR

1. Denaturation : When the temperature rises to 95 degree Celsius for one minute, Hydogen bond between DNA strands get break down and both strands are separated.
2. Renaturation or Annealing : As the temperature of mixture is cooled down to 55 degree Celsius, there will be complementary base pairing between primer and target flanking DNA strands. This process is called as renaturation or Annealing.
3. Synthesis : The initiation of DNA synthesis occurs at 3'-OH end of each primer. Synthesis is carried out using DNA polymerase ( Taq polymerase). Always synthesis worked out at 5' to 3' direction. The temperature is maintained by Taq polymerase and optimum temperature is 75 degree Celsius and reaction can be stop if temperature is raised around 95 degree Celsius.

Each cycle of PCR takes about 3-5 minutes. The ideal annealing temperature must be low enough to enable hybridization between primer and template, but high enough to prevent mismatched hybrids from forming. This temperature can be estimated by determining the melting temperature or Tm of the primer–template hybrid. The Tm is the temperature at which the correctly base-paired hybrid dissociates. 

A temperature 1–2 ◦C below this should be low enough to allow the correct primer–template hybrid to form, but too high for a hybrid with a single mismatch to be stable. The Tm can be determined experimentally, but is more usually calculated from the simple formula-

 

Tm = (4 × [G + C]) + (2 × [A + T])◦C

The annealing temperature for a PCR experiment is therefore determined by calculating the Tm for each primer and using a temperature of 1–2 ◦C below this figure. Note that this means the two primers should be designed so that they have identical Tm. If this is not the case, the appropriate annealing temperature for one primer may be too high or too low for the other member of the pair.

Technique to Study PCR products

There are wide range of procedures have been devised for studying PCR products, three techniques are particularly important:

• Gel electrophoresis of PCR products: The results of most PCR experiments are checked by running a portion of the amplified reaction mixture in an agarose gel. A band representing the amplified DNA may be visible after staining, or if the DNA yield is low the product can be detected using Southern hybridization. If the expected band is absent, or if additional bands are present, then something has gone wrong and the experiment must be repeated.
• Cloning of PCR products: Some applications require that after a PCR the resulting products are ligated into a vector and examined by any of the standard methods used for studying cloned DNA. This may sound easy, but there are complications
• Sequencing of PCR products

Variations of PCR Technique are;

Nested PCR- Primer binds to target DNA and sometime also to an undesired DNA by which both get amplified. To increase the specificity of PCR, nested primers are used.

Inverse PCR- Amplification of DNA of the unknown sequences is carried out from the known sequences. Restriction endonuclease is used in this process. 

Anchored PCR- Small sequence of nucleotide can be tagged to the target DNA

Reverse Transcription PCR - RNA molecule is converted to complementary DNA or cDNA by using the enzyme reverse transcriptase. Then cDNA serves as template to perform PCR.

Asymmetric PCR - Used to synthesis single-stranded DNA molecules which is mainly used for DNA sequencing.

Real-time Quantitative PCR - Double stranded DNA binds to ethidium bromide and this complex emit fluorescence that can be detected and Quantified.

 

Random Amplified Polymorphic DNA (RAPD) - In RAPD, short oligonucleotide primers are arbitrarily selected to amplify a set of DNA fragments randomly distributed throughout the genome.

 

Amplified Fragment Length Polymorphism (AFLP)-  It is based on the restriction fragment  length  polymorphism. AFLP is a very sensitive method for detecting polymorphism in the genomes.

Rapid Amplification Of cDNA Ends (RACE) : Reverse transcription, followed by PCR (RT-PCR)  results in the amplification of RNA sequence in cDNA form.

 

Question asked in GATE BT from this topics are:

• To determine required set of primer from the sequence of Target DNA.
• To calculate molecules of amplicon generated.
• To determine the amplified product present at any specified concentration.
• DNA polymerase used in PCR.
• To calculate melting temperature of given DNA sequence.
• RAPD and AFLP.