what is the criterion for selection of primers while doing 16s rRNA sequencing?
The 16S gene has regions that are highly conserved across species as well as regions of variability that can be used to differentiate species. If you want to amplify and then sequence the entire 16S gene, there are common primers (8F and 1492R) that work well for all, or virtually all species. There are then other common primers that can be used to walk the gene if a full length sequence is required. A list of many of these primer sequences can be found at http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
or alternatively try http://www.lutzonilab.net/primers/page604.shtml
As always, I suggest taking the sequence there and comparing it to several primer sequences in the scientific literature to make sure that there are no mistakes.
The 16S gene has regions that are highly conserved across species as well as regions of variability that can be used to differentiate species. If you want to amplify and then sequence the entire 16S gene, there are common primers (8F and 1492R) that work well for all, or virtually all species. There are then other common primers that can be used to walk the gene if a full length sequence is required. A list of many of these primer sequences can be found at http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
or alternatively try http://www.lutzonilab.net/primers/page604.shtml
As always, I suggest taking the sequence there and comparing it to several primer sequences in the scientific literature to make sure that there are no mistakes.
Thanks for a comprehensive answer Merrell ! But if the strain is unknown then we go through universal primers....Right?
Universal primers should work with ALL species......so if you have an unknown, you should be able to amplify the gene using the universal primers.
now the picture is clear. ..............we can simply BLAST the sequence with already reported or any specific software for it?
Correct. That should help you identify the species. You can also look at the RDB at http://rdp.cme.msu.edu/
This is often more accurate since Genbank is full of a ton of sequences that have not been properly identified or confirmed.
Assuming that you are analyzing an unknown bacteria, you should first opt for the universal primers. They essentially cover up all possible bacteria. BLAST the sequence against the NCBI database.
Dear Irsa, Merrel is right, first you should amplify with universal primers then if you got good result. it is ok go for BLAST otherwise go with different set of primers reported for different variable regions in the 16S gene. you will get the identity.
What is the size of gene sequence will likely get if i use 8f and 1492r primer
@ diviana tan! Yeah but more authentic then biochemical characterizations.
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