Rapid Amplification of cDNA Ends question
Posted by:
hianghao (IP Logged)
Date: January 05, 2010 11:33AM
Hi, i am new in molecular biology and i need some help in RACE...
In 5' RACE, after the production of 1st strand cDNA using gene-specific-primer 1 and addition of polyT tail, another gsp 2 is used together with anchor primer to amplify that cDNA. My question is, why don't we use gsp1 for amplification instead of gsp2?
2nd is, if gsp1 & 2 are parts of known sequence (designated as X) which i obtain from 3'RACE, will X have overlapping sequence with cDNA obtain from 5'RACE? If yes, can i assume that the overlapping sequence is the sequence of gsp2+a few nucleotide, since i get gsp1 from X?
3rd, can i directly use cDNA generated from 3'RACE as primer for 5'RACE, instead of using some part of it to design gsp1? If not, y?