THE POLYMERASE CHAIN REACTION

4. THE POLYMERASE CHAIN REACTION

Basic PCR technique is important and indispensable in molecular biology, as the DNA fragment can be amplified. Useful in amplifying a DNA sequence whose nucleotide sequence is not known.

Principles of PCR are in 3 steps namely:

i) Denaturation at 94⁰ C (10-30 seconds) where one double-stranded DNA molecule is denatured to 2 molecules of single-stranded DNA.

ii) Annealing at 45-65⁰ C (30 seconds onwards depending on the size of the sequence) where specific primers can hybridize to the single strand in the opposite direction. In this step, if lower temperatures are set, the primer can hybridize many sites, while at 50-60⁰ C the primer can hybridize the target sequence only. Normally, amplifies 1kb/minute or 1 kb/30 seconds in modern PCR machines.

iii) Extension at 72⁰ C, the primer initiates to extend by use of DNA polymerase.

The above-mentioned 3 steps complete one cycle. In the second cycle, there are 2 double-stranded DNA that are denatured to single-stranded DNA to form 4 molecules of single-stranded DNA.

CASSETTE-LIGATION MEDIATED PCR

5 steps are involved in this kind of PCR namely:

1. Digestion of DNA with restriction enzymes by using Hind III.
2. Ligation of cleavage product to synthetic double-stranded DNA with Hind III site.
3. The first PCR using ligated DNA and template and 2 primers. One primer hybridizes to the target sequences and the other primer hybridizes to the cassette.
4. The second round PCR gives more specificity to amplification. The template is used from the first PCR product plus 2 primers. These 2 are engineered to be hybridized into the primer sites of the previous first PCR primers.

3’ – RACE ( 3 prime rapid amplification cDNA end)

This is used when cloning cDNA from eukaryotic cells such as fungi, we use the 3’-race method. In eukaryotic cells, mRNA are added to the polyA tail at the 3’ end. cDNA can then be synthesized using reverse transcriptase. Oligo dT primers are used in many cases to synthesis cDNA. Some sequences are also added at the end of the 5’ of the oligo dT primer. Then, the specific primer for the target sequence can be constructed, then a PCR can be done using the cDNA as the template. After the first round of PCR, a second round of PCR is done again; hence another primer must be constructed. Using this method, cDNA can be cloned rapidly. It is noteworthy that bacteria mRNA does not contain poly A tail.