I have a handful of primers, of which the details are unknown. In particular, I want to find the exact target sequence bases of the primers in their respective genes. So, I tried to find out, but I couldn't get it. Could anyone help me step by step to resolve this?
I uses NCBI nucleotide BLAST.
2- first concatenate a pair of PCR primers by inserting string of 20 or more N's in between the two primers, and then search the concatenated pair as one sequence. Since BLAST looks for local alignments and automatically searches both strands, there is no need to reverse complement the reverse primer before doing the concatenation or the search.
3-Searching against nr/nt database, by this way you can identitfy both the genes sequences and primers sites.
OR
Use the primer BLAST programme of NCBI .
If still have problem send me one of the gene sequences and all primer pairs sequences and I will do it as example
Zuhal A. Hamid
Yeah thanks for your valuable suggestion. I would refine my question further. Target primers and target genes are known. I would like to find out at where exactly Forward primer and reverse primer would bind in the target gene? Primer's binding sequence in the target gene I am asking how to find out?
You could try the Primer Blast software available at the NCBI site. It'ls quite helpful.
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
I uses NCBI nucleotide BLAST.
2- first concatenate a pair of PCR primers by inserting string of 20 or more N's in between the two primers, and then search the concatenated pair as one sequence. Since BLAST looks for local alignments and automatically searches both strands, there is no need to reverse complement the reverse primer before doing the concatenation or the search.
3-Searching against nr/nt database, by this way you can identitfy both the genes sequences and primers sites.
OR
Use the primer BLAST programme of NCBI .
If still have problem send me one of the gene sequences and all primer pairs sequences and I will do it as example
Zuhal A. Hamid
I use CLC Workbench (free trial) for these kind of questions. You can do "in silico" PCR on your target gene. If the 3'-ends of your primers align to your target gene, the software gives you a nice visual representation of where your primers bind and even the expected fragment size and sequence.
http://www.clcbio.com/products/clc-main-workbench/
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