BIONET DNA Sequencing Survey

[Bruce Roe]

In article <9306202330.AA18198 at cscgpo.anu.edu.au>, Klaus.Matthaei at anu.edu.au writes...
>Stuff deleted.
> 
>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

Thanks Klaus, we too have observed that low enzyme gives more crossbands.
	
I also got a private reply which points to a couple of references:

"What about the recent reports of TdT chasing to get rid of these terminations?
 The one I've got beside me is from Ming Li in Calgary (Focus 15, 19-20 - ok, so
 its not a refereed journal, but the results look pretty convincing)!  Other
 reports (Biotechniques 9, 46; Ibid 12, 228) back this up. If this method
 really works, then that tells us the terminations mostly contain 3' dNTPs."

If these references are correct, and I have no reason to question them, it
looks like the enzyme actually does "fall off" and the cross bands are
terminated with 3' deoxynucleotides rather than 3' dideoxy's.

Thanks one and all....I'm off to find that issue of BioTechniques.
Cheers.....bruce