homozygous primer for PCR

Question

[J.F.Jiang]

Hi all,

To design the primer for MiSeq, I found that it might be hard to design a perfect primer for those homozygous region.

say, for a 22 bp primer, if I use blast program to seek its homo condition, I found 17 bp could be matched in other chrom, some of this 5 bp are in the 5' of the primers, while others are randomly distributed.

So is there any guidance (ref?) for this development?

[Devon Ryan]

Why exactly are you trying to design primers for a MiSeq? Are you instead trying to design PCR primers to amplify a genomic region that you then want to sequence (presumably looking for look frequency changes, since otherwise a MiSeq is overkill)?