UV mutation of PCR products?

Rafael Maldonado rafael at howard.genetics.utah.edu
Thu Oct 31 14:57:36 EST 1996


On Tue, 29 Oct 1996, Rasmus J. wrote:

> In an attempt to sequence gel-cleaned PCR fragments via cyclesequencing
> we discovered, that apparantly a short exposure (ca. 5 min) to
> short-waved UV light (355nm) was enough to make a sequencing impossible.
> But anyway, all PCR fragments could be cloned in a T-overhang vector
> (selfmade) and white transformands were obtained.

Probably, the UV is breaking your DNA. Is it in good conditions after UV 
treatment? Have you re-run the DNA after the UV treatment? Can you clone 
the UV trated fragments? Has the insert in those clonings the proper size?

Have you made controls of DNA with not treatment to UV?

Have you tried treating other, non-PCR fragment with UV?

Have our tried long-wave, less mutagenic UV? Or less intensity? Have you 
tried other UV sources?

> Now my questions:
> 1. If such a mutated fragment is cloned, will it be replicated normal

Yes. The problem is that very energetic UV can break DNA.

> (high copy) and could sequencing of these clones lead to the right
> DNA-sequence? 

I do not understand this question.

> 2. Could it be possible, that the cause of the problems is a radical
> T-dimer mutation and crosslinking of the different fragments with each
> other?

I do not think so. You will need far more energy than UV for making 
intramolecular links in DNA.


Rafa

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