bbraun at ucla.edu
bbraun at ucla.edu
Thu Feb 29 00:45:29 EST 1996
In <lamphier-270296013135 at pl-ueno122.infosphere.or.jp>, lamphier at po.infosphere.or.jp (Marc Lamphier) writes:
>In article <voeller-2402962201480001 at voeller.his.com>, voeller at his.com (Jim
>> In article <4gl3qu$8bd at mark.ucdavis.edu>, ez022056 at dale.ucdavis.edu
>> (Edward Wang) wrote:
>> > We dumped the project after 1.5 years and 70+ false positives...
>> > Good luck
>> The differential display method has became very popular. I know of many at
>> my institution who have used this method for various projects and I
>> suspect likewise at others. I have attended a number of seminars where
>> researchers have presented preliminary results from differential display.
>> I have seen a handful of published articles regarding genes cloned using
>> the technique. I have not really seen anything substantial come out as a
>> result. Dr. Wang's comment seems much more typical. Anyone want to
>> Jim Voeller
>> Lombardi Cancer Center
>> Georgetown University
>> Washington DC, USA
>We have used the method extensively in our own laboratory and come up with
>a number of bona fide positives -- genes that were clearly induced in one
>cell type and not another. But there are a lot of bugs with the method.
>Reproducibility is one -- and the suggestion to use duplicate lanes of the
>same sample is a good one. Secondly a lot of junk appears at the bottom of
>the gel and we learned to ignore anything below a certain length. Thirdly,
>the DNAs amplified are from the 3' end of the message and tend to be AT
>rich, and this caused two kinds of problems: a) as probes they often
>hybridized poorly in Northerns, b) the sequence information was often
>useless because it was so far 3' to the coding region that it didn't show
>up in databases and often provided no amino acid sequence to look for
>motifs, etc. We often had to re-probe a lambda cDNA library and re-sequence
>before we could determine if we had anything of potential interest.
>After much work we got several genuine positives (although the clones
>themselves turned out not to be terribly interesting)
>Dept. of Immunology
>University of Tokyo
These problems also reflect what I have heard from many people.
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