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PCR question

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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PCR question

Postby bizzaroSquirrel » Thu Apr 07, 2005 2:03 am

Hi guys,
I have a question from a prac in molecular biology and it has me stumped. Either i dont understand the question or i dont understand the theory. Hope you don't mind me posting the entire question, i have an exam in 8 weeks time for this subject so if you know the answer could you also explain it? That would help me out heaps more than just the answer ;)

Argh, i jsut typed out the biggest question, but i understood it as soon as i finished, oh well, i have another one :D

Q1) Imagine that a pair of primers amplify more fragments than the desired one. Which program parameters woudl you modify to suppress this? Would you increase or decrease thier values?

I have absolutely no idea. I searched for ages through the textbook and online.
I'm assumming prgram parameters that can be increased or decreased are either times or temperatures of the denature, renature and expansion phases. Is that right?
Only information i got was to raise annealing temperature to prevent low-temp annealing and synthesis, but i don't understand this.

I would ahve said design better primers, or purify the DNA, but these arent values that can be increased or decreased :?

Any ideas?
Thanks
james
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Postby canalon » Thu Apr 07, 2005 1:12 pm

In your question you have a specificity problem in your PCR. The obvious factors that can be adjusted in this case are:

- Annealing temperature, what you call "renature", if this temperature is increased, the primer-DNA structure are less stable, then hybridation between primers and would be stable only with a greater homology. At lower temp, a few base pair difference still allow hybridation and amplification.
- MgCl2 concentration: Taq polymerase need Mg++ nions to perform its elongation. Reduced concentration result in lower activity and higher specificity.

All details and more on this website at
http://www.protocol-online.org/prot/%20Molecular_Biology/PCR/Multiplex_PCR/

HTH

Patrick
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