Entering edit mode
11.0 years ago
r.a.konrad
•
0
Hello,
After I obtained the results from sequencing, I proceeded to curate the sequences and identifying (with Vector NTI) the primers I used in the sequence. The primers, although are degenerated, were found with no mismatches.
The problem is that when I blast the squence using BLASTn or BLASTX, the results are malate synthase, a gene/protein that I totally didn't expected and totally unrelated to the gene I'm interesed. These results were like 13 of the 20 sequences while 5 were of the gene I was expecting.
Please tell me what went wrong. I'm a bit desperate. I used a clone library with pGEM and chemocompetent DH5-alpha.
This is more a molecular biology protocol question than a bioinformatics question. From the point of view of BLAST, nothing has "gone wrong". You just have unexpected sequences.
I have to agree with Neil. Either something went wrong during the actual cloning or your expectations of what you were going to clone are wrong. And as such this indeed is not really a bioinformatics question.
I'd disagree - depending on the source of the annotation, incorrect functional labels can easily propagate through the sequence databases from just one contaminant or incorrect initial annotation. I'd be interested in knowing the provenance of the "malate synthase" annotation before jumping to conclusions.
This is true, and I'd be interested in hearing some more details of this particular case. Although I strongly suspect this is a biology issue and not bioinformatics per se. But either way there is likely a useful primer in here (I suspect) on how to properly analyze the data and use bioinformatics to figure out the solution.
Did you conduct a primer blast when you designed the primers? This should always be done when designing primers.
Josh I assume you are referring to NCBI's Primer BLAST service (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), and not some other Primer BLAST?
There may be a few iterations of it elsewhere but they should be the same. You want to make sure your primers are specific for your site of interest and only your site of interest.
What gene were you expecting to amplify and what organism are you looking in? Also were you amplifying a specific exon/domain region of your gene of interest?