Contracting Primer Extension

David L. Haviland, PhD dhavilan at IMM2.IMM.UTH.TMC.EDU
Sun Aug 30 14:20:43 EST 1998


On Fri, 28 Aug 1998, Sabah Hussain wrote:


> Dear netters. I have been trying primer extension experiments on a
> recently cloned genomic DNA promoter fragment without any sucess. I was
> wondering whether there are commerical sites or companies which provide
> primer extension services for a reasonable price.Any help in this matter
> is greately appreciated.

Ahhh...not quite...  you need a fair bit more background on what the assay
is for and how to do it.  I'd suggest the Harvard Manual as one place to
start and any of the gene structure manuscripts that  appear in JBC on a
near monthly basis.

You can not perform primer extension analysis from cloned material.
Primer extension is conducted using mRNA (or total RNA  as a last resort)
obtained from tissues or cell lines that express your favorite gene and an
anti-sense oligo (no less than a 40 mer, treated using RNAse-free
reagents) spaced about a 100-150bp off the end of where one expects to
see the end of the cDNA. Using AMV-RT one reverse transcribes a cDNA from
the oligo to the end of the mRNA.  You then digest away the single
stranded RNA and run the product out on a sequencing gel, if it all works,
you should see the DNA/RNA band that sould be witin 3-5% of the
transcription start site.  

THE only thing you could use your cloned genomic fragment for would be the
sequence ladder (using the *same* oligo used in the primer extension
assay) as a means of counting up the number of bases and determine where
the TIS site is.

A "service" for primer extension wouldn't be very profitable and I've yet
to hear of such a service offered.

David

==========================
David L. Haviland, PhD
Assistant Professor, Immunology
University of Texas, HSC - Houston
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX  77030
==========================






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