So my question is base don the following. I have a protein I want from an unsequenced organism. So I went and got the same protein from organisms closelly related and aligned the protein from them (did not find well conserved DNA sequences to use these), I looked into the conserved protein regions, design degenerate primers. However I could not find conserved regions in the flanking of the genes (as I already refereed, the few DNA sequences I found did not share relevant conservation).
So this left me with degenerate primers on 2 regions of the protein around 100 a.a. away from each end. If I use regular PCR for amplification my sequence is going to be cut too short and I won't know my whole gene.
So my idea was, if I could use the fluorescence marked bases directly on the first PCR I make, like we do when we already have the amplified product and we want to sequence, but in this case I would be making the sequences for sequecing directly from the gDNA with one primer at a time. So that the sequencing wouldnt be restrain between the 2 regions I know, but instead would be left open until the polimerase had activity to polymerize. I know this may lead to very low signals or even no signals at all, or just a mess in the sequencing sheet. However assuming I could get a relatively high concentration of genomic DNA (more template to start with) would this be possible? I know it is kind of a dumb idea, but your input and other suggestions to resolve this problem will be must appreciated.
UPDATE:
My goal is to be able to get the full dna sequence to synthesize the protein from the specific species that is not sequenced yet, which should give me a slightly different protein than the ones already documented from other species. Therefore I've been thinking that the DNA sequence may be divided by several exons. This way my best chance would probably be to just extract total RNA and try to sequence the mRNA for my protein. Any ideas to do the same think but with Reverse transcriptase instead?
Thank you for your suggestions
Hi Miguel,
Very interesting idea! Doing the sequencing reaction with gDNA as template could theoretically work. Maybe it is possible for some bacteria. What is the organism you are working with?
The use of degenerated primers in the sequencing reaction could cause problems. The use of gDNA should work if only one primer is added and binds to exactly one site. There will be two or more products if the binding site is not unique. Even binding of some primer parts will lead to the formation of several products. Therefore, sequencing will show an overlay of the different signals. The binding to multiple location within an entire genome is very likely. Therefore, using gDNA as template in sequencing will probably not work. Nevertheless, you could try it.
Good luck!
if you have any good sequence then you could try inverse pcr where you restriction digest the dna and ligate the ends of the cut fragments to form a circle. In one sequence you have your known sequence and the sequence up to the unknown restriction sites in both directions so 2 pcr primers in the known sequence facing away from each other will produce a pcr product which can be sequenced normally to give the unknown part of the sequence
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