D.1.1.4 - Polymerase Chain Reactions + Gel Electrophoresis - Biology

PCR

Polymerase chain reaction

PCR Definition

A polymerase chain reaction is an artificial method of replicating DNA under laboratory conditions → it is used to amplify quantities of a specific sequence of DNA

In PCR each reaction

double the amount of DNA → standard sequence of 30 cycles → over 1 billion copies

PCR reaction occurs in

a thermal cycler

Phases in thermal cycler

Denaturation, annealing, elongation

denaturation

DNA sample is heated (approx. 90°C) to separate two strands

annealing

sample is cooled (approx. 55°c) to allow primers to anneal (primers then copy the sequence)

elongation

sample is heated to the optimal temperature for a heat-tolerant polymerase (tag) to function (approx. 75°c) → heat tolerant DNA polymerase Taq copies strand and replicates it

Taq polymerase

Enzyme from the thermus aquaticus, which is able to function at high temperatures and is thus used in PCR without denaturing

Taq polymerase function

extends the nucleotide chain from the primers

Gel electrophoresis

laboratory technique used to separate and isolate DNA fragments based on mass

restriction endonuclease

an enzyme acting on DNA/RNA fragments within the sequence restricting them and breaking the phospho di ester bond to create specific DNA fragments by cutting sequences.

applications

dna profiling