Jian Yu ( Email: jianyu@jhmi.edu)
Last edited on May 1, 2000
Overview
This protocol was developed in Kinzler/Vogelstein laboratories to isolate cDNA fragments corresponding to novel SAGE tags that do not match EST databases using a PCR based method. This technique was employed in publications by Polyak et al.Nature.1997 18;389(6648):300-5 and by Yu et al. PNAS 1999 96(25):14517-14522.Using the polyA+ RNA prepared for the SAGE Library that has the most tags of interest,double stranded cDNA is synthesized with a specifically designed primer. After several enzymatic manipulations much like SAGE, the 3’ends enriched cDNA fragments (primary rSAGE library) is used as template for PCR reactions to obtain sufficient quantity of DNA, so called amplified rSAGE library. Second PCR amplification is carried out with SAGE tag specific primer and a common primer M13F to obtain specific product. Sequence of PCR product is used to verify the authenticity of a given SAGE tag and to obtain additional nucleotide sequence that can be used in database search or designing primers for 5’ RACE. The purified PCR produce can be used in a variety of hybridization based applications.
You will need the SAGE protocol as reference. Many steps of rSAGE are similar, even identical to that of SAGE and many reagents are shared by these two protocols. The same careful practice should be exercised to avoid PCR product contamination as much as possible. Close attention paid to the protocol during experiments will facilitate trouble-shooting should problems arise.
rSAGE Diagram
