Primer design for HLA locus.
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6.9 years ago

Hey every 1, i have designed primers for HLA locus DPA1(exon 2 region) based on Real-Time PCR (qPCR) Primer Design guidelines. primer will start from intron regions to cover full exonic region.

F-CAGCAACAGAGAATGTCAGC

R-CCCTGAAGCAGCAATTGATG

to check for amplification of only a single region  i have used in silico PCR UCSC but it shows multiple region from chr6. 

pls kindly help me with solving this.

thanks

 

 

 

HLA primer design • 3.3k views
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1
Entering edit mode
6.9 years ago

I checked your case, the 3 sequences found are identical. If you do a BLAT (fast BLAST) search of the 351bp sequence against the last version of the human genome in Ensembl you will obtain only one region. I suppose PCR UCSC uses an old version of the genome with redundant annotations of MHC loci as their are a complex family of paralogues.

Anyway be careful designing primers for this family of genes, you are in the risk of not covering all the human allele repertoire (even if you design primers in constant regions), and also you'll find non-specific PCR products because the primers will amplify paralogue loci regions even if they don't match 100% perfectly.

Check your primers in wet-lab, and tell us the results ;)

Here is my Ensembl search:

http://www.ensembl.org/Homo_sapiens/Tools/Blast/Results?db=core;tl=hloRm1DxKtyA1Cw9-648559

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