Polymerase Chain Reaction

PCR

• Repetitive amplification of a piece or region of DNA• Numerous uses

– Straightforward amplification & cloning of DNA

– RT-PCR – reverse transcription coupled with PCR to amplify mRNAs (cDNAs actually are template)

– Production of cDNA libraries

– Mutagenesis

– Sequencing

PCR Requirements

• DNA template– DNA that will be amplified (copied)

• Oligodeoxynucleotide primers– anneal to template to allow DNA replication

• thermostable DNA polymerase– DNA polymerase extends the primers to synthesize a

copy of the template DNA – thermostable polymerases allow automation and

repeated rounds of DNA denaturation

• deoxynucleotides and appriate reaction conditions– dNTPs are incorporated into synthesized DNA,

buffered pH, & Mg2+ to allow enzyme activity of DNA pol

PCR: The Process

1. Begin with a DNA template• Insert in vector• 1st strand cDNA• Genomic DNA

AAAAAAA

TTTTTTT

PCR: The Process

AAAAAAATTTTTTT

2. Denature template 3. Anneal primers

AAAAAAA

TTTTTTT

PCR: The Process

3. Extend primers with thermostable DNA polymerase• Taq• Pfu

This ends a PCR cycleAdditional cycles will repeat

these three steps

PCR: The Process

Beginning of 2nd cycle1. Melt newly synthesized

DNA from template• New strands of DNA are

now also available as templates

2. Anneal primers 3. Extend primers

1 & 2

3

PCR: The Process

• Beginning of 3rd cycle• Melt newly synthesized

DNA from template– All new strands of DNA

are now also available as templates

• Anneal primers • Extend primers

PCR: Yields

• How much amplification can be achieved?– Each cycle of PCR theoretically doubles the number of

template molecules

– Therefore the rate of amplification is 2n

Where n is the number of amplification cycles

– This will reach a practical maximum yield due to reagent (primer & dNTPs) concentration limits and maximum rate due to limiting enzyme concentrations. This upper limit is about 1x106 X amplification.

PCR: YieldsExample: Starting with 2ng of 5kb DNA template to amplify a 1Kb

insert, what is the theoretical yield after 20 cycles? After 30?

1. How many template molecules are there?

= 5000bp X 660g bp/mol bp = 3.3x106 g template/mol template

= 2x10-9 g template 3.3x106 g temp/mol temp = 6x10-16 mol temp

= 6x10-16 mol temp X 6.02x1023 molec/mol = 3.64x108 molecules

2. How many molecules of insert can be made in 20 cycles? 30?

3.64x108 molecules x 220 = 3.8x1014 molecules – 106 X

3.64x108 molecules x 230 = 3.9x1017 molecules – 109 X