PCR Flashcards
What is initially needed for PCR?
Template DNA, the four DNA nucleotides, thermostable Taq polymerase, buffer and an automated reaction vessel known as a thermal cycler.
What is PCR used for?
For the amplification of DNA fragments. Amplification refers to the generation of many identical copies of a particular DNA fragment. PCR involves a cycle of reactions at three different temperatures. By the end of each cycle, the number of copies of the template DNA fragment has doubled.
What is needed for PCR to happen?
A sample of DNA is needed to act as a template for amplification . A small sample of DNA isolated from a trace of blood, skin, hair, semen or cheek cells is sufficient. Two primers target the section of DNA to be replicated. Each one is carefully selected to be complementary to a short stretch of bases on one of the two DNA strands. The primers bind to the template and allow DNA replication to occur in the 5’ to 3’ direction. As each cycle progresses, the DNA between the two primer binding sites will be replicated.
What is a thermal cycler?
Is a machine the automatically controls the thirty to sixty cycles of the following three different temperatures. As each thermal cycle doubles the number of copies of the target DNA, the number of copies of DNA increases exponentially. After 30 cycles, it is possible to achieve amplification to a billion copies.
What are the three stages in the thermal cycler?
Stage 1- denaturation at 95’C
Stage 2- annealing of primers at 55’C
Stage 3- DNA extension at 72’C
What happens in stage 1?
The mixture is heated to 95’C to denature the DNA by breaking the hydrogen bonds between bases. Taq polymerase is thermostable and does not denature.
What happens in stage 2?
Cooling allows the primers to anneal (form hydrogen bonds) to their complementary base sequences on the single stranded DNA .
What happens in stage 3?
The optimum temperature for the Taq polymerase to extend the complementary strands in a 5’ to 3’ direction is 72’C.
