cycle sequencing

Arun Prakash a.prakash at mailbox.uq.edu.au
Fri Oct 20 02:15:39 EST 2000


Thanks for the very useful comments. Our sscp results did not pick up this
particular mutation. We are still in the process of doing the reverse
sequencing. At the moment we are having problem with the primers.

What I'm trying to figure out about the BAFL is that is there any reason why
mutation cannot lead to more than one base change. In other words, what's the
chance of  say G mutating to C, A and T (thus giving rise to a BAFL-type
mutation)?

Arun

Justin Hopkins wrote:

> >
> > We are carrying out mutation analysis on carcinogen-induced p53
> > mutations using SSCP and cycle sequencing techniques. We observe sample
> > specific bands-on-all-four-lanes type mutations. The results are
> > reproducible and specific.
>
> Do you mean that you observe BAFLs only in samples with an abnormal SSCP
> pattern?
>
> BAFLs are usually caused by poor sequencing template, and my be carry
> through from a poor PCR template.
>
> > But because there are bands on all 4 lanes (
> > not necessarily of equal intensities)
>
> If one of the four has a clearly stronger signal, this is most likely the
> true base. If two bases have equal intensities stronger than the others,
> you may have a mutation.
>
> > we are not sure if they are
> > genuine mutations or just pcr related artefact. Is it safe to assume
> > that if one sample shows it and another doesn't then these bands are
> > indeed mutations?
>
> No.
>
> Have you observed these BAFLs in sequences from both directions? If not,
> then that will probably clear them up.
>
> BAFLs tend to come up at the same place in certain DNA sequences, perhaps
> due to 2° structure or bound protein. BAFLs that reoccur in the sample
> place in different sample are very likely to be artefacts.
>
> If you've sequenced in each direction, you still get bands across all four
> lanes, at a unique position, for a sample with a unique SSCP pattern, then
> I am afraid that is particularly baffling!
>
> Otherwise hope this helps.
>
> > Thanks for any helpful comments.
> >
> > Arun Prakash
> > Senior Research Fellow
> > Univeresity of Queensland
>
> --
> Justin Hopkins
> Cancer Genetics Laboratory
> Guy's Hospital
> London
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