3.9 PCR

What is Polymerase Chain Reaction (PCR)? Give me some background information about it!

technique that allows scientists to make millions of copies of desired gene fragment in hours

• PCR allows scientists to perform genetic testing on incredibly tiny amounts of tissue - even a single cell

• PCR can even be used on tissue that has been degraded by environment

• Used in medicine, genetics, biotechnology and forensics

  • HIV research

  • Mummy of Pharaoh Ramses III

  • The Human Genome Project

  • Crime Scene Investigation

What is Step 1?

Denaturation

DNA sample is heated to 94°C - 96°C.

Heating breaks hydrogen bonds that hold double stranded DNA together - result is complimentary single strands

What is Step 2?

Annealing

DNA primers added. Cooled to 50 ̊C - 65 ̊C

2 different primers (forward and reverse) - chosen to be complimentary to opposite ends of target region being copied

What is Step 3?

Elongation

Taq DNA Polymerase added. Heated to 72 ̊C

Taq DNA polymerase comes from bacterium Thermus aquaticus, which is found in hot springs - enzyme does not denature at 72 ̊ C

Taq DNA polymerase joins at primers and moves along strands adding complimentary base pairs as it goes - result is two incomplete copies of DNA, each containing target region

Cycle 1 Complete

What is Step 4?

Repeat steps 1 - 3

• After cycle 2, the result is four incomplete copies of DNA, each containing target region

• Taq DNA polymerase added; heat to 72 ̊C

Cycle 2

What is Step 5?

Repeat Steps 1-4

PCR vs. DNA Replication

Table 1: DNA Replication and PCR	DNA Replication	PCR
Double-stranded DNA Separates	Topoisomerase DNA helicase	High Temperature (94 ̊C - 96 ̊C)
Primers Added to Single Strands	RNA Primers	DNA Primers (forward and reverse)
Complimentary Strand Synthesized	DNA polymerase III copies entire DNA strand	Taq DNA polymerase copies DNA fragments
Result	Copy of entire DNA molecule	After 30 cycles, over a billion identical DNA fragments