sage的原理及其应用.pptxVIP

SAGE的原理及其应用;What is SAGE?;How does SAGE work?;3. Digest the double stranded cDNA with a sequence specific restriction enzyme (an anchoring enzyme) that cleaves most transcripts at least once. Nla III is used as an anchoring enzyme since Nla III sites are known to occur approximately every 250 bp. ;4. Divide the cDNA into two fractions and ligate with two adapters (A and B, ~40 bp each). The adapters contain cohesive 4-bp overhangs complementary to the Nla III digested cDNA, a Type IIS restriction enzyme (tagging enzyme) recognition site at the 3′ end, and priming sites for PCR amplification