Polymerase Chain Reaction (PCR)
What is PCR?
Test used to make many copies of (amplify) small sections of DNA or genes to detect the presence or absence of a gene to help identify disease/infection
What are the steps of PCR?
1. Denaturation
2. Annealing
3. Extension
Can be used to amplify the genes associated with cancers from DNA
What is denaturation?
-First step of PCR
-DNA is heated to 90-100C
-H-bonds between the strands of DNA broken
-DNA strands are separated (denatured)
-Single-strand used as template in annealing
What are the different types of PCR?
1. End-point (regular PCR)
2. Allele-specific (ARMS)
3. Reverse transcription (RT)
4. Realtime/quantitative
What is annealing?
-Second step of PCR
-Temp of DNA lowered to 45-65C
-primers attach to the DNA template (act as starting point for DNA synthesis)
What are the characteristics of PCR?
-During early cycles of PCR, amplification is exponential (doubling)
-During later cycles of PCR amplification plateaus as components (primers or NTPs) are exhausted and become limiting
What is extension?
-Third step in PCR process
-heat increased to 72C (optimal temp for Taq DNA pol.)
-Taq DNA pol. attaches to primer and builds complementary DNA strand by adding new bases 5'-3'