PCR – Polymerase Chain reaction

– amplification of region between 2 specific primers

– multiple cycles of denaturation, annealing, and extension

denaturation

– melts double stranded template (before PCR)

– melts extension products off template

(at beginning of cycles)

annealing – primer anneals to template

extension – primer extended by polymerase

Components of standard PCR reactions

thermostable DNA polymerases

– from thermophilic eubacteria or archaea

– several available commercially

– all require primers

– so only amplify DNA between added primers

1. Taq polymerase – from Thermus aquaticus

– hot spring Archaea

– most commonly used enzyme for PCR

advantages:

high rate of activity (~2000 nucleotides per minute)

– extends primers efficiently

stable to 97.5°C – during denaturation of DNA

problems:

no 3'-5' exonuclease activity (no proofreading)

– high rate of mistakes (1 per 2 X 10⁴ bp)

– problem if PCR product cloned &/or sequenced

– may cause early termination of extension

– mismatched ends extended poorly

– PCR using Taq polymerase

– cannot amplify distances > 2 kb very well

– most extension products not full length

2. polymerases from Pyrococcus species

– deep sea vent extreme thermophiles

– several types available from different suppliers

advantages:

have proofreading activity – lower error rate

– will amplify long sequences successfully

– up to 25 kb

problems:

not as efficient as Taq – take longer to extend primers

– however, you can use both at once!

– combination of Taq and proofreading polymerase

– Taq produces most of sequence

– proofreading enzyme fixes mismatched ends

– get efficient amplification of long sequence

primers

– 2 oligonucleotide primers specific for ends of sequence

requirements:

18-25 nucleotides long

– shorter primers occur multiple times by random chance

40-60% GC – good range for optimal melting temperature

– homopolymers not good

– melting temperature for both primers should be similar

– so both anneal at same temperature

– annealing temperature usually 3-5°C less than oligo T_m

– get stringent conditions for specific hybridization

– What if T_m not known?

– e.g. mix of degenerate primers

(based on amino acid sequence)

– primer for specific gene in different organism

– some mismatches present

– can do “touchdown PCR”

– start annealing at temperatures ~ 3°C above ideal T_m

– decrease annealing temp. 1°C every couple of cycles

– eventually optimal temperature reached

– start at more stringent conditions

– so most specific products made first

– amplified more

– make up most of DNA produced

no complimentary sequences

– within one primer – forms secondary structure

– primer anneals to itself instead of template

– between 2 primers – especially at 3' end

– get “primer dimer”

– product of PCR is only primer sequence

– to repeated sequences in target genomic DNA

– to vector sequences (if vector DNA present)

Note

– check most of the above using DNA analysis programs

– usually have primer design tools

– check for sequence homology

– calculate T_m from GC content

– may check primers against sequence databases

– for repetitive DNA or vector sequences

reaction buffer – optimal conditions for enzyme activity

includes:

dNTPs – for DNA synthesis

buffer – optimal pH

salt concentration – proper concentration of KCl

Mg²⁺ – required for enzyme activity

– also affects primer hybridization

– may have to be adjusted to optimize results

DNA template – crude preps often okay – but not always

– phenol, detergents etc. may interfere with PCR

– have to clean up DNA

– do positive control to test if PCR conditions a problem

(if possible – may not have suitable DNA)

– contamination also problem

– 1 contaminating molecule may produce visible results

– always do negative control

– no DNA, band seen – problem

preventing contamination

– use dedicated equipment & solutions for PCR

– e.g. micropipettors contaminated with DNA aerosols – can prevent by using tips containing filters

– can destroy “leftover” DNA from previous reactions

– using UV radiation – damages DNA

– or by making PCR products using dUTP

– instead of dTTP

– added okay by polymerase

– when DNA treated with uracil N-glycosylase

– DNA depyrimidated & destroyed

total amount of DNA present also important

– too little, will need more cycles to produce enough

– more chances for errors

– too much encourages non-specific hybridization

– optimum amount is ~ 3 X 10⁵ copies of desired gene

– 1 ng bacterial DNA, 1 µg human DNA