I am currently working on an RT-PCR experiment designing different set of genes for running on a 96 well plate. When contacted several companies, some said they adopt intron spanning primer assay for my set of specific genes to aviod gDNA contamination. Other companies were of the opinion of exon-exon assay and insisted me on using on-column DNase treatments at several steps of the process to successfully avoid gDNA.
I was just wondering how each method works to avoid gDNA contamination. Valuable inpts will be highly appreciated.
Thanks
in exon-exon primer we have to design the primers in such a way to make its annealing occur on the boundaries of two adjacent exon . by this design we obligate the amplification to occur only on the spliced mRNA , other wise the amplification will not occur for genome or non spliced mRNA due to the presence of intron between these two exons .
Both of the methods you mention will work; however i would suggest always designing primers that span 2 exons. this will keep you from having problems with splice site variation. i.e., a gDNA pcr product will be much longer than the cDNA product because it will include the intervening intron(s). simply stated:
primerF-exon1-intron-exon2-primerR (gDNA) vs primerF-exon1-exon2-primerR (cDNA)
hope this helps,
Best
James
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