designing primers for genes not sequenced yet
Posted 04 December 2009 - 02:49 AM
I have always wondered how a new gene is sequenced. If the gene was never sequenced before then how do people design primers to amplify the gene?
if one has to start from the mRNA and reverse transcribe even then there is need for a primer to selectively amplify the target cDNA.. how do people circumvent this?
If primers are designed based on the already available sequences of closely related species.. how effective are they?
are there any basic rules to be followed in such primer designing?
Posted 04 December 2009 - 02:14 PM
Posted 04 December 2009 - 06:17 PM
If you manage to find the gene with this primer set, you can amplify and sequence this region. Then you can design outward facing primers from that (now known) sequence and do inverse pcr to find flanking sequence.
Another possible approach is to use a closely related species gene as a probe for finding your target gene. A library of genomic fragments in plasmids can be screened with this approach (colony blots and hybridization). Then you can sequence the targeted plasmid. You could also probe a southern blot of your target organism DNA to locate a band containing your gene, then cut it out and clone, again selecting for the correct plasmid insert.