Fluorescent sequencing from PCR templates

Bruce Roe BROE at AARDVARK.UCS.UOKNOR.EDU
Thu Dec 12 06:56:00 EST 1991


I received the following from domi at genethon.genethon.fr

 Date: Thu, 12 Dec 91 10:46:10 +0100
 From: domi at genethon.genethon.fr
 Subject: Re:Fluorescent sequencing
 To: BROE at aardvark.ucs.uoknor.edu, domi at genethon.genethon.fr
 Message-Id: <9112120946.AA16284 at genethon.genethon.fr>
 
---------------------------- 
 hi,
 your responses to these questions:
 
  Topic 1: Sequencing with PCR products as templates.
 > 
 > 1. What kind of success have you had with sequencing PCR products?
 > 2. If you have had "good" success with PCR product sequencing, how's about
 >    passing on the protocol for PCR product isolation and the sequencing
 >    conditions?
 > 
 > Topic 2:
 > 
 > 1. What kind of success have you had with sequencing with dye-terminators?
 > 2. Same as above.. could you share your protocol?
 
 1- Sequencing PCR. 

As a rule we have no trouble sequencing PCR products so long as the original
PCR  gave a relatively clean Product (ie, NO products with electrophoretic
mobilities  lower than the product to be sequenced, and low background fog). In
this case we  do not isolate the PCR product but sequence it directly from the
PCR filtered reaction. If the PCR is messy, we do a short (15 cycles)
re-amplification of the band to be sequenced, ie the band is cut out from a low
melting gel, the gel slice  is molten at 65 *C and an aliquot (10-15*l) used
directly for re-amplif. If the  PCR is relatively clean then we proceed
directly as above. We sequence using fluorescence labelled ddX and have no
trouble reaching 400-500 bases with a good accuracy (over 98%). However, some
PCR products, particularly those carried out from  cloned DNA (YACs, cosmids,
lamda libraries) often give very messy products with  very high background. In
these cases even what looks like a single band on gel very often contains a
variety of PCR products with homogenous sizes giving useless sequences. We deal
with those by isolating the band from a low melting gel, diluting the molten
slice by a factor of 5 or 6 and carrying out serial re-amlpifs. That seem to 
work quite well.

2- sequencing with labelled ddX: we use these on DS DNA or SS DNA when we do
reverse sequences. In general, if it was'nt for the unreadable first 20 or so
bases typical of DS sequencing, we would not be abble to tell the difference
between these and  standard SS m13 sequences using labelled primers. In terms
of read-lengths there  is only a small difference (500-600 on SS DNA by
standard methods versus 450-550  on DS DNA by labelled ddX).

 With respect to protocols: no problems sharing them with others. we are in the
 process of publishing the lot.
 
 bye
 dominique (communication officier for genethon-ceph)

-----------------------
Cheers.........Bruce



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