designing primers for genes not sequenced yet - (Dec/04/2009 )
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?
Clone the fragment into a vector of known sequence, then sequence the unknown insert using primers designed to the vector sequence.
The cloning approach works well if you can select the gene somehow, or sequence enough to find the gene. If you have a specific gene in mind, then the best approach is to design gene-specific primers for (typically) a portion of the gene. This can be done by finding the gene in other related species in Genbank, then aligning the genes to find highly conserved regions. You then design primers facing each other within these conserved regions. Usually, this requires the use of degenerate primers, since the codon table has degeneracies. Try to find a highly conserved region with either Met or Trp codons, and use the unique codons for those amino acids at the 3' end of primers. Avoid using Leu, Arg, Ser with six codons possible. The remainder can be coded for with degenerate bases in the third position of the codon.
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.