Do you have to sequence both strands?

Martin Kennedy mkennedy at chmeds.ac.nz
Mon Apr 4 16:11:14 EST 1994


In article <gosink-020494174955 at microb3.biostat.washington.edu>, gosink at u.washington.edu (John Gosink) writes:
> Hi,
> 						I was having a discussion with a person down the hall.  They claim
> that there is _no_ regulation that says you have to sequence both strands
> of a length of DNA (they are working with cloned PCR products) for
> submission to Genbank and/or a referreed paper.  Do you have any references
> on this subject?
> 
> 			                              -John
> 
> P.S.  They read a single strand, but make it a point to read each gel on
> two different occasions and/or by two different people.
-- 
If they want the sequence to be correct, they absolutely MUST do both strands;
no matter how beautiful the gels look, sequencing the other strand just about
always identifies regions of conflict that will need to be resolved with dITP
or other compression-resolving tricks.  If they don't care whether the sequence
is right or wrong, and whether others try to make primers from their bum
sequence, or waste endless bench hours because a submitted sequence was wrong,
then tell them they must at the very least indicate in their submission that
the sequence was only determined on one strand.  One other point; if they are
working with cloned PCR products, they better sequence multiple isolates from
distinct PCR reactions to insure against PCR errors; I for one wouldn't believe
a sequence if it came from one PCR clone, let alone if it was only sequenced on
one strand.  Finally, any decent sequence, from an experienced lab, isn't going
to be just sequenced on both strands once.  For accurate sequence, I'd
recommend at least 3 reads of each strand, preferably from different priming
positions.  Sequencing might be relatively trivial these days, but it is still
a lot of work to get right.


Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
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