Optimisation of dCAPS markers (degenerate primers) - problems with detection of heterozygotes (Mar/06/2007 )
I designed dCAPS marker (degenerated primer with 1 missmatch + restriction cutting) and "nearly" everything works well. I am able to distinguish wild type (digested PCR product: 225 bp and 17 bp) and mutant genotype (PCR product 242 bp). However, I have tested a detection sensitivity of heterozygotes (DNA mix of W and M genotypes: 10:90 , 20:80 , 30:70 , 40:60 , 50:50) and thats a problem. I can differentiate W genotype in DNA mix 50:50 %, but the second band (225 bp - digested PCR product) isnt visible if DNA concentration of W genotype is lower than 50 %. Does anybody know, how to solve this problem? Why am I able to separate homozygotes not heterozygotes?
Thanks for your suggestions.
PCR mix [20 µl]: 1 µl DNA (30 ng), 1 x Taq buf, 0,1 µM (tested 0,1 a 0,2 µM) primers, 200 µM dNTPs, Mg2+ 1,5 mM (tested 1-3 mM); Ta = 55 - 60 C (Tm of shorter = reverse primer is 60 C).
RE (BstXI): 55 C, 3 h, 4 U enzyme + 5 µl PCR mix + 1x buffer (final volume 10 µl)
a general scheme:
FORWARD PRIMER 5’...CCCAAAATGG
WILD TYPE 5’ ...CACAAAATGGTGG
MUTANT TYPE 5’ ...CACAAAATGGAGG
C ... missmatch
Ok, I try to ask in a different way:
Does anybody optimized PCR marker which perfectly amplified single homozygous templates, but problem appeared in heterozygous plants (true heterozygotes or DNA mix of different genotypes (alleles)) - primers preferentially amplify only one of the two templates?