GC-Rich Template Amplification Failure (End-point PCR Primers)

Question

Accepted Answer

PCR fails to amplify templates with high GC content or produces very low yields Common causes: 1) GC-rich regions form stable secondary structures preventing amplification 2) Standard MgCl2 insufficient for GC-rich templates 3) Annealing temperature not optimized for GC-rich sequences 4) Insufficient extension time for difficult templates 5) Potassium in reaction stabilizing GC structures Solutions: 1) Increase extension time from 1 min/kb to 1.5 min/kb 2) Vary PCR cycles from 20-40 (35 cycles typical) 3) Denature at 95°C for 45 seconds 4) Use MgSO4 instead of MgCl2 for GC-rich templates 5) Test higher annealing temperatures starting at primer Tm, increasing in 2°C increments up to six temperatures 6) Add Platinum GC Enhancer solution 7) Eliminate potassium from amplification reactions 8) Use combination of 1.0 M betaine with 6–8% DMSO or 5% DMSO with 1.2–1.8 M betaine