I try to obtain sequence of highly variable part noncoding element by sanger sequencing. After polyA/polyT repeat (in direct and reverse sequencing) signal are decreased greatly of interrupted. Because of expected variability of this region and not good quality of sequence after repeat it seems be difficult to create internal primers. Maybe other solutions of such problem are known?
Hi Andrii,
this could be a solution if I understand you right http://www.agctsequencing.com/TroubleshootingGuideF.asp
Dear Tetiana,
Thank you for your answer and proposedsolution of this problem. I know this way, but it's looks like unsuitable for me in 2 reasons:
1) I expect polyA/polyT repeats at least in 2 regions of my product, so in such way primers will be unspecific
2) I have less lenth of repeats than 25 b.p. and their lenth is variable
This is a common problem with all repeat regions. Unfortunately there aren't many good solutions. In my experience, sometimes reducing the template concentration in the cycle sequencing reaction (I assume you're doing cycle sequencing) to the bare minimum sometimes allows sequencing though repeat regions, although my experience was working with triplet repeats, not mono-base strings. There are also formulations of the reaction mix which use alternative nucleotides, but that probably wouldn't help with a mono-base string.
Sorry I don't have any better suggestions - good luck.
Dear Laura B Geyer,
what concentration of template you use for your reasons? Yes, dinucleotide and trinucleotide repeats in general are quite easier to fix than mononucleotide.
Andriii,
It will depend somewhat on the locus and the length of the sequence before the repeat region so it takes some optimization. To be honest I don't usually bother quantifing PCR products when sequencing so I generally use a relative concentration. Essentially when I run into this problem I try repeating the reaction with 1/2 to 1/4 the amount of starting template. You can probably go quite a bit lower than that, but at some point you'll simply get no signal. If you want to quantify, I recommend checking out what the recommended concentration is for the kit you're using and go with the lower limit (or 10-20% less than that- their recommendations tend to be conservative).
Good Luck.
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