PCR sequencing

Bill Melchior, NCTR/FDA WMELCHIOR at NTET.NCTR.FDA.GOV
Thu Jul 16 09:12:04 EST 1992


>what I want to find out are the
>optimum d:dd ratios for the 4 nucleotides.
>
>I would also appreciate any data on thermal stability of ddNTPs. Is it
>because they get degraded that one has to use such high concs ? Or is it
>simply because the traditional "labeling reaction" is running in parallel
>with the "termination reaction"?

I've not yet tried cycle sequencing, though I hope to start soon, but my
experience with Klenow probably applies.  I found that I had to adjust the
dNTP/ddNTP ratios (yes, by trial-and-error) from those given in protocols
in the literature to optimize things for my sequence.  (One of the reasons
I went to Sequenase when it was available.)

You might want to look at BRL's FOCUS vol 5 #1 (March 1983) for trouble-
shooting regular dideoxy sequencing.

(ddNTPs should be stable.)

________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||    to US regulators



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