引物设计原则 qUence. Length of PCR product should ideally be less than 150 bp Avoid complementary sequences within and between primers and probes 月L ■ Avoid mismatches Avoid a 3-end T as this has a greater tolerance of mismatch Length 18-30 nucleotides GC content 4060% ■-2°Cx(A+T)+4℃x(c+G (simplified) PRIMER3 PRIMERS PRIMER EXPRESS引物设计原则