PCR question

[Guest lordSquirrel]

Hi guys

I feel pretty dodgy having my first post in the homework forum, but i've done my share of lurking dammit, i've lurked my little heart out, and now i need some help

 

The question is for a prac in molecular biology.

 

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?

Only information i found was to raise annealing temperature to prevent low-temp annealing and synthesis, but i don't understand this.

 

I would have said design better primers, or to purify the DNA, but these aren't values that can be increased or decreased.

 

Any ideas?

Thanks

james

[Dak]

primers anneal to sequenses with a strength thats proportional to the amount of conformation between the primer and the sequence. the amount of heat nessesary to split the primer and the dna apart is proportional to the strength of annealment, so if the primer is completely complementary to the desired sequence, and partially complementary to other sequenses, then whacking the temperature up untill its almost hot enough to unanneal the primer and the dna-that-you-want-pcr'd should unanneal the primer from the other sequenses, as if they are only partially complementary they will be annealed weaker, and so will require less heat to unanneal them.

 

i think. its been a while since iv done this, and its a little fuzzy in my memory. ie you should chek this