Probes or primers that span introns - (Apr/27/2005 )

Why should i select probes or primers that span introns when I design for cDNA sequences in RT-qPCR?

Thanks for any ideas.

-justwonder-

By spanning an intron with your primers/probes you avoid amplifying any contaminating genomic DNA (which contains introns).

-Steph1-

QUOTE (Steph1 @ Apr 28 2005, 01:15 AM)

By spanning an intron with your primers/probes you avoid amplifying any contaminating genomic DNA (which contains introns).

Should i span the whole intron or only the junction between exon and intron? How can i prevent amplification of genomic DNA since it has alot of introns and not only one intron?

-justwonder-

Hi Justwonder,

I have not done any realtime RT-PCR and don't know whether primers spanning introns will help because you are not going to see the bands.

The purpose of having primers span introns is to differentiate whether a band is amplified from genomic DNA or cDNA. If your RNA is contaminated with DNA and primers span one or more introns, you will get two bands with one from the cDNA and the other from DNA. It is better to let the primers span introns that is not so big so that you can get the extra band if there is contamination. Another strategy is to let at least one of the primers bridge an exon-exon junction. But I prefer the former. To know more about RT-PCR primer design, check this http://www.protocol-online.org/forums/inde...?showtopic=2706

-pcrman-

Hi pcrman,

Thanks.

I have only heard about exon-intron junctions, but i have noticed you used exon-exon junctions. Does this junction exist?

Thanks again.

-justwonder-

Exon-exon junctions exist in cDNA

-methylnick-

QUOTE

Exon-exon junctions exist in cDNA

Exact what I meant.

-pcrman-