Do you have to sequence both strands? (anecdotes)
jwoodget at ocicl.oci.utoronto.ca
Thu Apr 14 18:20:55 EST 1994
Over the past five years my lab has sequenced at least twenty new cDNAs in a
competitive area. We've taken a variety of approaches such as subcloning
fragments, nested deletion series (the best in my view), custom oligos;
mainly using double-stranded DNA, occasionally using an ABI sequenator.
While I wholeheartedly agree in the need to sequence both strands, there are
several anecdotal observations I'd like to add.
One project involved a particularly GC-rich, novel gene (about 3.5 kb). The
student cloned both bovine and human cDNAs and bombarded them with all of the
above strategies. Over twenty specific oligos were used to read across
compressions, etc. Various denaturation techniques were employed. Both
strands in both genes were sequenced yet we still made two separate errors
which shifted and then restored the correct ORF over a 40 base pair stretch.
Moral: doing everthing "right" doesn't guarentee accuracy.
Another project involved a gene from slime mould which has an AT-rich genome.
We used both radioactive and automated sequencing and the latter was abysmal.
ABI used this aprticular gene to specifically improve the 373s performance in
dealing with AT repeats, using the radioactively-determined sequence as the
benchmark (which resulted, in part, to the introduction of the 5-filter
wheel). Moral: hi-tec isn't always hi-acc.
This week I downloaded a sequence from NCBI via its accession number.
Translation of the cDNA did not yield the same ORF as in the GenBank entry
due to a single base deletion. Although programs like Authorin check for
such errors, many submissions are made without these tests. Moral: some
mistakes are easily found.
Finally, in a competitive area, the degree of redundancy of sequencing has to
be kept to a minimum for reasons of both efficiency and effectiveness (i.e.
each strand should be sequenced only once). This is perhaps becoming more of
a problem. Short cuts are bound to be taken. How many people sequence both
strands (or even entirely one strand) of a site-directed mutant? There are
controls (protein expression, etc) but I've certainly chanced upon unintended
errors which would have been undetectable other than by sequencing. Moral:
Cell/Nature/Science don't wait for ever.
Enough moralising, got to get this paper off.......
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