Inverse PCR - Can I use ipcr to determine full-length sequence of unknown ORF? (Oct/05/2007 )
I was wondering if anyone as used inverse pcr to try and amplify and sequence an unknown ORF? I have a gene that I am working with that has not been fully sequenced in baboon. I have several exons sequenced but I still need to fill in the gaps. Standard pcr has not worked probably because I only have human known sequence for the gene and baboon seems to be different in these areas so I can't get the primers to work. I have also tried RACE and been unsuccessful. I thought I could use inverse pcr because I have known internal sequence but I don't want to sequence the entire gene just the ORF. Any suggestions?
This should be very straightforward. Find an enzyme that cuts your genomic DNA about every 1Kb or so (but fails to cut at the ends of the sequence you already have). Make outward directed primers pointing away from each other on the known sequence, about 30-50 bp from each end. Cut the genomic DNA. Heat kill the enzyme, then dilute to about 20 ng/ul in a ligation reaction. Ligate the cut DNA fragments (T4 DNA ligase, buffer, room temperature for 30 minutes will probably work). Use the ligation product as a PCR template with your primers. Depending on the genome size, you will probably need many cycles. You may need to do nested PCR, in which case you'll need to amplify first with a set of inner (more centrally located) primers, then amplify with the pair on the outside. Gel purify the band (expect 2kb) and sequence from each end with the PCR primer. You should see the end of the known fragment followed by the genomic DNA of the flanking regions. Rinse, lather, repeat to find larger regions. You'll likely need to switch enzymes.
Thanks for your suggestions Phage. I will definitely give then a try.