PCR amplification of extracted DNA fragment
The technique involves repeated rounds of in-vitro DNA synthesis by a thermo stable enzyme, such as Taq DNA polymerase which is isolated from bacteria called Thermus aquaticus. To perform a PCR reaction, PCR reaction mixture is prepared for which a small qwuatity of the target DNA ia added to an eppendorf with a buffer containing Taq DNA polymerase, oligonucteoide primers, the four deoxyribonucloetide of DNA (dNTPs), and the cofactor Mgcl2.
The PCR mixture is subjected to PCR cucles which consiste of following three steps
- Denaturation: During this step, DNA is denatured into single stranded DNA.DNA denaturation is the critical step in the DNA amplification reaction. The time specified for DNA depends on variuos factors like, nature of template, GC Content, secondary structure etc. For most amplifications, incubation time for DNA denaturation is 30 sec at 92o C to 95o C, with 94o C being standard choice.
- Primer Annealing: During this step, forward and reverse primers are annealed to their complementary sequences on either side of target sequence. The specified temperature is calculated empirically and is usually 5o C less than the Tm of the primers used. Extreme annealing temperature may result in non specific amplifications.
- Extension: During this step, annealed primers are extended by DNA polymerase to synthesize the compllementary DNA strands. Primer extension is usually carried out at a temperature of 72o C. Time used foir extension depends on thew length of target sequence.
Usually PCR is subjected to 30-40 cycles. As the amplification proceeds, the DNA sequences between the primers doubles after each cycle.
|1OX assay Taq polymerase buffer||5 µL|
|dNTPs mix (2.5 mM each)||2 µL|
|Template DNA (100 ng/ µL)||2 µL|
|Forward primer (10 pM)||2 µL|
|Reverse primer (10 pM)||2 µL|
|Taq DNA polymerase (5U/µL)||1.25U (0.25 µL)|
|Sterile water||36.75 µL|
|Total reaction volume||50 µL|
|Step||Temperature C||Time||Number of cycles|
|Initial denaturation||94||1 – 3 min||1|
|Denaturation||94||30 sec||30 – 40|
|Annealing||Tm – 5||30 sec||–|
|Final extension||72||5-10 min||1|