BIONET DNA Sequencing Survey

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Sun Jun 20 18:35:35 EST 1993


Stuff deleted.

>>  Also, aren't bands in all
>>four lanes caused by enzyme "fall-off" (which does tend to be
>>caused by sequence secondary structure, just like compression)?
>> 
>
>There is no evidence that it's "fall-off" or the enzyme just
>mis-incorporates d/ddNTP's when it hits a stable sequence secondary
>structure.  But you are correct about secondary structure being the 
>culprit.
>
>I have a tendency to think about bands in all 4 lanes as being due 
>to "template compression" i.e.  secondary structure, since "product
>compression" also is due to secondary structure ( fold back )
>in the synthesized strand.
>
>Cheers......bruce

I am not so sure that secondary structure is entirely to blame.  If you are
too stingey with your sequenase you can generate cross bands every where. 
If you titrate the enzyme you get crossbands in indirect proportion to the
amount of enzyme until at the lowest concentration you get no sequence that
is readable.  Decreasing the enzyme should not increase the secondary
structure but the crossbands increase nevertheless.  Perhaps the
processivity of sequenase is affected at low protein concentration??

Cheers, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If you don't do: you rust"
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