Template Re-annealing Problem

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Navin
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Template Re-annealing Problem

Post by Navin » Sat Nov 24, 2007 10:33 am

In PCR, after the denaturation step, and then the temaperature is lowered to around 60 degrees celcius for the annealing of the primers,

Why is it that the 2 template strands do not re-anneal?

This question came out in my exam and some of the available options were;
A. The primer is small
B. 60 degrees celcius is too high for the template to reanneal
C. The primer concentration is high

The thing is - all those options seem plausible to me. What do you reckon?
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Post by MrMistery » Sat Nov 24, 2007 2:02 pm

i would go with B, through elimination:
A is true. So? It does not actually offer an explanation
C is also true. Not only it does not offer an explanation, but also is very ambiguous: high compared to what?
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Post by mith » Sat Nov 24, 2007 5:04 pm

C is probably the right answer you only have 2 template strands while your primer concentration is much much higher throughout the process.
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Post by canalon » Sat Nov 24, 2007 7:39 pm

C is the answer. In fact the huge excess of primer compared o template is the limiting step of the PCR. When too many primers have been used the eficiency drops dramatically.
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Post by MrMistery » Sat Nov 24, 2007 8:37 pm

i was thinking C might be, but then I thought that the relative concentrations change through the process and it still works just as fine. But now i remember that i've read that the efficiency drops sharply after 30 cycles. My bad
But cut me some slack, as i said the answer choices are strangely presented
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Post by mith » Sat Nov 24, 2007 11:36 pm

Helps if you've worked in a lab :)
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Post by Navin » Sun Nov 25, 2007 3:36 am

Thanks!

Yep, i guess C is the limiting step.

In fact - I have done PCR many times - but i didnt question what limited the number of steps! Well, I guess it just pays to be extra inquisitive.
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Post by canalon » Mon Nov 26, 2007 12:00 am

MrMistery wrote: But now i remember that i've read that the efficiency drops sharply after 30 cycles. My bad
But cut me some slack, as i said the answer choices are strangely presented


Not necessarily true, it also depends on the amount of starting material. With low amounts you might need more han 30 cycles to enter the limiting step. But then the enzyme is also losing some efficiency... which can be a pain when trying to detect low amount of DNA in qPCR
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Post by MrMistery » Tue Nov 27, 2007 7:52 pm

obviously the book was listing an average case
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