Or, should I ask, how to identify those intergenic spacer sequences that will be variable enough to allow for species-specific primer design?
I know that one needs to blast a few (better many) sequences on NCBI and identify those spaces that way, but does anyone has a specific instruction for that? I have spent two days looking for these and either I am tired out or I can't make a hit. Mostly I get partial sequences for 16 ribosomal RNA and I think this is not a good target, because it is highly conserved. I need something in between 16 and 23 to grab on to and I don't know how to make that search.
Thank you !
Dr. Ara Kooser answered similar question on this platform, which I found it helpful:
The first thing would be to gather up as many high quality 16S sequences from NCBI:
http://www.ncbi.nlm.nih.gov/
On the drop down menu select nucleotide and then in the search box add:
pectolytic 16S
Press search.
Grab as many full length sequences as you can. You will want to align then using ssu-align from the Eddy lab:
http://selab.janelia.org/software.html
You can look at the alignment of the 16S gene to find conserved areas for your primers to set. The big thing here is to compare your pectolytic 16S the non-pectolytic 16S sequences. I am going to guess you might not find a significant difference. If there is a difference it might be in the hypervariable regions of your pectolytic 16S, but I doubt it.
non-16S (pectic acid lyase?, I don't know a lot about the type of bacteria you are interested in)
If that is the case you can then search NCBI for genes that are unique to pectolytic bacteria. I would guess the genes that express the pectic enzyme might be unique to your buggies. I would design primers based on the alignment of one of the domains (the most conserved domain) of the pectic enzyme gene.
Whatever conserved domain of the gene you are interested in, find a sequence and then run a BLASTN on it. Gather all those hits. Align them. And look for a good area to sit you primers in.
UGENE is a nice piece of opensource software for looking at alignments.
http://ugene.unipro.ru/
You may also try this link:
Article PCR amplification of species specific sequences of 16S rDNA ...
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