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PCR with Degenerate primers

PCR with Degenerate primers - PCR - Polymerase Chain Reaction Forum

PCR with Degenerate primers - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 11-11-2009, 07:13 PM
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Smile PCR with Degenerate primers



Hi all, I amplified my target gene using degenerate primers. I used several sets of degenerate primers reported so far for that particular gene in different bacteria none of them worked except for one set. The amplicon was sequenced and it didn't show any match with blast search but when same sequence was searched with blastx, it matched (81% identity) with the protein we are looking for. Is it ok to use this amplicon for my further studies which are gene expression studies for this particular gene under different conditions etc. The problem is there is not even a single report on genetic studies on this particular gene in this bacteria. My question is: Can I use this for futher studies and how I'll justify when I publish my work? Waiting for expert views and suggestions.
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Old 11-12-2009, 05:17 PM
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Default Re: PCR with Degenerate primers

Is the set of degenerate primers tested on a group of your bacteria or just on type/reference strain(s)? I actually mean that the justification for using degenerate primers is some polymorphism within the particular gene so that standard primers won't cover all possible versions of the gene. But maybe there is some kind of possibility to design standard primers on the basis of several sequences of the gene from several diferent strains? This will require some sequencing though.

This is not an easy issue to solve. If you have no other options and there is some polymorphism in the sequence you study then you'll need to be sure that your degenerate primers are specific enough. I would recommend thorough specificity test on a number of different templates (not only on DNA from closely related bacteria). There is GenBank and blast, but some genomes are not fully known yet...
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Old 11-13-2009, 05:19 PM
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Smile Re: PCR with Degenerate primers

Hi Aga,

Thank you very much for the reply. The degenerate primers I used were designed by multiple sequence alignment of the target gene in different gram positive bacteria. The primers have been used in other gram positive bacteria. Actually, I used several sets of degenerate primers earlier but nothing worked. The only difference in those primers was that they were designed by mutilple sequence alignment of protein sequences ( target gene)of gram positive as well as gram negative bacteria and the working primers are designed only by protein sequence alignment of gram positive bacteria. I am working with gram positive bacteria ( Arthrobacter) which has been reported to be involved in degradation of Hydrocarbons but there are hardly any genetic studies done so far and there is no reference sequence for this particular organism in the Genbank. What should I do further so that I can find some solid answer for sequence of my target gene. Waiting for your expert views and suggestions.
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Old 11-16-2009, 03:49 PM
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Smile Re: PCR with Degenerate primers

Quote:
Originally Posted by Aga View Post
Is the set of degenerate primers tested on a group of your bacteria or just on type/reference strain(s)? I actually mean that the justification for using degenerate primers is some polymorphism within the particular gene so that standard primers won't cover all possible versions of the gene. But maybe there is some kind of possibility to design standard primers on the basis of several sequences of the gene from several diferent strains? This will require some sequencing though.

This is not an easy issue to solve. If you have no other options and there is some polymorphism in the sequence you study then you'll need to be sure that your degenerate primers are specific enough. I would recommend thorough specificity test on a number of different templates (not only on DNA from closely related bacteria). There is GenBank and blast, but some genomes are not fully known yet...
Hi Aga:

Thank you very much for the prompt reply. The gene I am targeting is highly diverse among different bacterial strains although the protein sequence has certain conserved regions. The degenerate primers were designed by multiple sequence alignment of protein sequences of the target gene in differnt bacteria. The primers have been tested for specificity in different bacteria but the bacterial culture I am working with has been studied only as hydrocarbon degrader but there are no genetic studies done on this particular genus. I need some guidance on what should I do next and how should I proceed with my further studies. Waiting for expert suggestions. Thanks once again.
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