sequencing not good after reading poly A tail..why? - (Nov/14/2007 )

Hi everyone,

I am trying to sequence a 600bp fragment with a gene specific reverse primer. The sequencing results were good for the first 120bp (last 16bp of the 120bp is a polyA tail). I blasted the first 120bp and confirmed that is is my gene of interest. However, after the poly A tail, the sequences just went "mad". There are sequences after the poly A tail that i need to know. Can anyone help explain that and how to solve it?

thanks

p/s I have part of the sequencing attached

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This is (sadly) very typical. It can come from many things. Poly A regions are unstable, because the polymerase slips during the extension. This can happen with the pcr enzyme, with the plasmid replication in the cell, or with the sequencing reaction, or all three. What you typically see is the pattern you have, of uncertainty in the number of copies of A that are present. If you look closely at the sequence following, you'll see that it is two copies (or more) of the correct sequence shifted by one or more bases and added together. Sometimes you can directly read that sequence, but often it is difficult. I once solved a difficult problem of this kind by making a primer AAAAAAAAAAAAAAAAAAG and using it as the sequencing primer. You won't get sequence close the the end of this primer (as usual) but you can get good sequence. You can then often make a primer (reverse) to that good sequence and resequence backward to fill in the gap. Your sequence, however, I note has a second place where the primer I suggested above will prime -- to the poly T region following (preceeded by a C). Ugh. The poly G region following might be a good anchor, but there is probably an identical issue with sequencing in reverse across the poly T region.

If your template is good, it might be worth trying a sequencing reaction with the extension temperature lowered from the normal (I think 60C) which will lower the possible slippling. This won't help if your plasmid has different length sequences.

How badly do you need to know this? Transposon insertion? exonuclease followed by subcloning?

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I looked very carefully at your electropherogram, and I believe there is enough information there to assign pretty good sequence to it. It appear that each base is spread across three locations. As I read the sequence it is:

.....aaaaaa ggt cca gct cga tgc gcg tgg cca c ttttt...

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how did you read the sequence? i tried but couldnt get it.

i am trying a "new" method to do 5' and 3' race using adapter primers and ligating the 5' and 3' end together. So, you read it like this...

-(-primer-->)---------------(5'UTR)(adapter)(adapter2)(3' UTR)--------------(<--primer-)--


p/s: i got the method from this paper: Huang JC, Chen F. 2006. Simultaneous amplification of 5' and 3' cDNA ends based on template-switching effect and inverse PCR. Biotechniques 40(2):187-9.

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