disappearing sequences : any ideas

David C.Fritzinger dfritzin at nospam.hotmail.nospam.com
Sat Dec 29 15:50:02 EST 2001


In article <a0l54b$ee1$1 at mercury.hgmp.mrc.ac.uk>, "Morey, Roger"
<zbf0 at cdc.gov> wrote:

> We have also experienced a multiple T problem.  We were sequencing PCR
> products on a beckman 2000xl, and kept getting unclear reads after the
> multi-T site.  We then  cloned the product site on this and related species.
> For the few we were successful with actually had a distribution of 
> Ts ranging from say 13-19 in the population taken from cryostorage.  Those
> with even longer multi-T reads were not resolvable.    We also tried
> resolution with a gene scanned based approach.  I belive this could either
> be a hypervariable site, or just a problem with t ended priming, such as
> slippage.  I have recently read that primers with a T end are worse.  I
> wonder if there is a connection.   
> 
> Roger E Morey
> CDC NCID/DBMD/MSPB
> MS G34 Bldg17 # 2205
> 1600 Clifton rd
> Atlanta, GA  30333
> 

I believe the problem lies with the PCR, and not so much with the
sequencing. On long tracts of Ts or As, Taq tends to "stutter", either
adding an extra base, or removing one. This causes the poor, mixed
sequences past the  polyT or polyA stretch. If anyone knows a solution
for this, let me know.

David C. Fritzinger, Ph.D.
Associate Professor
Cancer Research  Center of Hawaii

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