How can I calculate the target pcr product size from the primer position in source sequence , cleavage fragment?
primer sequence
F: 5′-ATCTTCCGAAGAATACAAGA-3′
R: 5′-CTCAACCCCTATTTAACCTTT-3′
Gene - AGAP2 , Restriction enzymes (HinfI) - SNP rs12368653
F: 5′-AGC TGG CCC TGG CAC TGA CTC TGC TCT-3′
R:5′-ATG GAA ACA CCT TGC TTC TTC TCC CTC-3′
Gene - CYP2R1, Restriction enzymes (FokI) - SNP rs2228570
F:5’-GGAAGCTTTGGAGAGCTGAA-3’
R:5’-GCCATAAGTCCAACCAGGAA-3’
Gene - CYP2R1, Restriction enzymes (FokI) - SNP rs12794714
F:5′-AAATAATGAGCAAATGAAAGAAGAC-3’
R:5′-CAATAACAGCAAAGAAATGAGTAGA-3’
Gene - DPB, Restriction enzymes (HaeIII)) - SNP rs7041 and rs4588
BLAST your primers to get the exact genomic sequence. Then find the cut sites for the SNP variants. Either use a cut site finding program (NEB has one for their enzymes) or just search by hand.
Good luck!
Hi Asaad,
NEB is a good site to get the restriction map of your amplicons (for NebCutter, http://nc2.neb.com/NEBcutter2/) as Katie advised you, but you'll need the amplicon sequence and the easiest way is to make an in-silico PCR at the UCSC site (http://genome.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=hg19&org=Human&wp_f=&wp_r=&wp_size=4000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=771722473_3TCAAC836k7Bzgk7Zh0s7twcC54p)...you'll get Tm, positions and sequences.
fred
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