lecture 5: PCR

what is PCR

method for amplification of specific dna sequences

what region is amplified

region between two known dna sequences

binding sites for two primers

known sequence sites

forward primer

binds to the bottom DNA strand

5’ → 3’

reverse primer

binds to the top DNA strand

3’ → 5’

denaturation

94C

separates two strands

each strand is a template for new strands

primer annealing

60C

forward and reverse primers bind complementary sites on opposite strands

dna polymerase binds to the 3’ end of the primer

extension

72C

polymerase extends from 3’ end of primer making complementary dna in 5’ → 3’ direction

polymerase synthesises through opposing primer sequence

first cycle products

polymerase may continue past the opposite site

some products may be longer than the finals

second cycle products

some new strands have a length defined by two primer positions

third cycle products

exact length product becomes the main product as cycling continues

describe features of Taq polymerase

robust

isolated from bacterium thermus aquaticus

thermostable

opt temp for activity 75-80C

low fidelity (error prone), no proofreading

products have an extra adenine at the 3’ end

components for Taq polymerase PCR

dna template

oligonucleotide primers

dNTPs

reaction buffer

Taq polymerase

describe features of Pfu polymerase

isolated from pyrococcus furiosus (100C optimum growth)

thermostable

slower tan Taq

less robust (more sensitive)

high fidelity, proofreading

blunt-ended

what is accuracy

average number of correct nucleotides a given polymerase incorporates before making an error

what are primer design considerations

1. avoid secondary structures (hairpins) or repeated sequences

2. ensure primers are non self-complementary (avoid primer-dimer formation)

3. ensure 3’ end base is correctly base paired otherwise they wont act as polymerase primers

addition of dna sequence

add sequence to primers 5’ end

primers 3’ end binds target dna

pcr copies added sequence into the final product

real time PCR - qPCR

measures product formation during cycles

results can be used to estimate how much target material was present at the start