Login

Join for Free!
111310 members


PCR

Genetics as it applies to evolution, molecular biology, and medical aspects.

Moderator: BioTeam

PCR

Postby Julian92 » Mon Jan 28, 2013 12:23 pm

Can someone tell me why a mixture of 12 specific primers produces 12 bands from a single persons DNA. And why is the pattern of bands produced by multiplex PCR with the same 12 primers highly variable between individuals? What are the most likely explanations for individual D having less than 12 bands?

Yours,
Julian
User avatar
Julian92
Garter
Garter
 
Posts: 2
Joined: Mon Jan 28, 2013 12:08 pm
Location: Glasgow

Postby Julian92 » Tue Jan 29, 2013 3:10 pm

I realise that each of the 12 primer pairs bind to a different region of human genome; the VNTR's within each region, and that is how 12 distinct bands can be detected after PCR. But what i don't know is why the exact same primers that target the same location end up a different areas.

Yours,
Julian
User avatar
Julian92
Garter
Garter
 
Posts: 2
Joined: Mon Jan 28, 2013 12:08 pm
Location: Glasgow

Re: PCR

Postby jonmoulton » Tue Jan 29, 2013 4:16 pm

"what i don't know is why the exact same primers that target the same location end up a different areas."

I don't think that the same primers that target the same location do end up in different areas. The difference in the masses of the bands produced is due to the difference in the number of repeats between individuals.

Just looking at a single pair of primers, you target the same two loci in any human (unless the primers are poorly chosen and so there is off-target binding due to allelic variation). So, you prime at the locus of the forward primer target and prime at the locus of the reverse primer target, but the distance between these two targets varies with the individual's number of intervening repeats; that's the source of the band mass variation.

When you are using 12 pairs of primers, they are targeting 24 different binding loci. Each forward and reverse pair spans a region that has a variable number of base pairs between individuals, but a given individual will have a definite base distance between each pair of primers (barring complications like chromosomal rearrangements from cancer, etc.). This means that an individual whose chromosomes are tested against the 12 primer pairs will produce 12 bands, one for each primer pair, but the mass of the RT-PCR products will vary when compared between different individuals. This is not because each pair or primers is targeting different areas, but rather it is because the distance between the forward and reverse primer binding sites varies between individuals.

"What are the most likely explanations for individual D having less than 12 bands?"

If an individual has less than 12 bands, it could be because two of the PCR products have masses so similar that they cannot be distinguished. Another possibility is that there has been one or more mutation(s) in one of the primer binding sites so that the primer has some mispairs with its putative target and the affinity of the primer for the altered target is below the affinity needed for efficient PCR.
User avatar
jonmoulton
Viper
Viper
 
Posts: 415
Joined: Fri Feb 15, 2008 5:38 pm
Location: Philomath, Oregon, USA


Return to Genetics

Who is online

Users browsing this forum: No registered users and 0 guests

cron