cycle sequencing

Justin Hopkins zmga2150 at kcl.ac.uk
Mon Oct 16 04:55:29 EST 2000


>
> 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






More information about the Methods mailing list