Inverse PCR - Can I use cDNA to do ipcr? (Jan/04/2008 )
Hi everyone,
Does anybody know if I can use cDNA for inverse pcr? I have a know sequence from my ORF but need the surrounding sequence. I don't want to sequence the entire gene so I thought I might be able to use just cDNA as a template. Could I just circularize the cDNA using T4 ligase and the amplify with inverse primers? Any suggestions are appreciated. Thanks
Is this single stranded cDNA or already amplified?
This would be mRNA from cell culture converted to cDNA
Why not try the regular inverse PCR-technique? since you know the sequence of your ORF, just cut your genomic DNA with an enzyme (or 2 different enzymes with compatible ends) that cuts halfway in your ORF and "often enough" in the genomic DNA (an enzyme that has a recognition sequence of 6 bp cuts once every 4096bp in theory). Ligate this mixture in a large volume (you want to have as much self-ligation as possible) and then amplify with primers facing outwards from the recognition site. This should amplify your ORF + the surrounding part (untill the site where the restrictions took place) that you can then sequence.
I was just trying to avoid sequencing large pieces of DNA and then dealing with mapping intron/exon boundaries. Thought someone might know of a way to use cDNA instead. I am only interested in getting the coding region of my gene. Thanks for your input