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question about sequncing


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#1 chicken

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Posted 18 February 2010 - 10:09 PM

in automated sequencing we get a chromatogram in which peaks show which nucleotide is present (A. T .G .C).

my question is

what is the meaning of hieght of peak, sometime it is short and sometime it is long for same nucleotide.

plz answer any one

#2 mdfenko

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Posted 19 February 2010 - 09:11 AM

the simplest explanation (and there may be more than one reason) is that the strands formed are terminated randomly. this means that you may have more copies of one size than another of nearly the same size.
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#3 jpd

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Posted 24 February 2010 - 01:10 PM

What we usually infer from peak heights here is the dosage. Sure, the sequencing process is random; but if you start from roughly the same amounts of each strand, half of the dosage of the peak should be coming from one allele and half from the other. Yes the peaks all vary in height; but that pattern in peak height should be pretty similar across samples for the same sequence*. If I remember correctly, the difference in peak heights is also affected by the sequence pattern because of the way the instrument detects each base. So for a string of A's you'll probably notice that the first peak is a little higher but then drops off.

*When the sequence is not very clean but we suspect that there is a real base pair change, we compare the dosage amount of the suspicious peak to that of the WT or control. If the dosage drops by half, the change is probably real. There's a program called Sequence Pilot that saves the peak height information and you can compare the normal peak heights to the one you're looking at.




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