12 - PCR efficiency and primer design

PCR efficiency formula

• A = A0 x P^n

• A = molar yield of amplicon after PCR

• A0 = original molar amount of target sequence

• P = efficiency of amplicon per cycle (2 = 100% efficiency as it is doubling per cycle)

• n = number of cycles

Example calculation

• Get weight of human genome (bp x 660)

• Get moles of starting amplicon (divide by moles of genome, include units)

• Get moles of target amplicon size (bp x 660)

• Get moles of amplicon yield (divide by moles of target)

• Plug into formula, take log of both sides for efficiency

Requirements for primer design

• Specificity - unique to target sequence, poly A/T or GC rich have less specificity, underrepresented nucleotides

• Efficiency - allow polymerase to extend

• Avoid secondary structures like primer dimers or hairpin loops

Primer binding rules

• Direction - always ordered 5’ to 3’

• Forward binds to lower (antisense) and reverse to sense

• The last 3-5 3’ nucleotides must match perfectly

• Mismatches tolerated in 5’ end, used for adding restriction sites, mutations

Primer dimers

• Caused by primers self annealing or cross annealing

• Homodimers - same primer binds to itself

• Heterodimers - forwards and reverse primers binding

• Reduces yield, visible as extra bands

• Avoid complementary sequences at 3’ end