I have an environmental genomic DNA sample that includes three different newly-discovered 134 nt novel sequences. I want to do inverse PCR (using restriction enzymes and ligation) to begin sequencing the flanking regions of the three sequences. Since I have three different sequences, I don't want to use the reverse complement of my original primer as I might be sequencing flanking regions of different molecules. My idea is to move back into left and right ends and use inverse PCR primers about 15-40 nt from the left and right ends where there is some variability. The last 10 nt or so at each end also shows variability so sequencing would confirm that I am sequencing flanking regions of the same molecule. So, how do I find appropriate primers since primer selection software assumes I know something about what's between the primers? I can supply the three aligned sequences.
Do not worry too much about the quality of the primers as the whole possible target that you are amplifying from is very small. It is only important that the primers do not have a hairpin loop or homologous with the other primer so as to minimise primer dimer. If a pcr works then it will be present in huge amounts compared with any small amount of non specific amplification. The amount of target self ligation may be small so many cycles of pcr or nested primers may be needed
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