Hello Dr. Kraev,
You can download the various SMART-cDNA protocol manuals
directly from the Clontech website <http://www.clontech.com>
or you can have them send them to you from.
Clontech
1020 East Meadow Circle
Palo Alto, CA 94303-4230, USA
800-662-2566 phone
650-424-8222 phone
650-424-1064
tech at clontech.com
Some of the references that I have found that relate to the
technique are:
Suppression subtractive hybridization: A versitle mehtod
for identifying differentially expressed genes
L. Diatchenko, S. Lukyanov, Y-F. C. Lau, P.D. Siebert
Methods in Enzymology 303:349-380, 1999
Construction of cDNA libraries from small amounts of
total RNA using the Suppression PCR effect
K. Lukyanov, L. Diatchenko, A. Chenchik, A Nanisetti,
P. Siebert, N. Usman, M. Matz, S. Lukyanov
Biochem. Biophys. Res. Comm 230:285-88, 1997
Amplification of cDNA ends based on template-switching
effect and step-out PCR
M. Matz, D. Shagin, E. Bogdanova, O. Britanova, S. Lukyanova
L. Diatchenko, A. Chenchik
Nucl. Acids Res. 27(6):1558-60, 1999
An improved PCR method for walking in uncloned genomic DNA
PD Seibert, A Chenchik, DE Kellogg, KA Lukyanov, SA Lukyanov
Nucl. Acids Res. 23(6):1087-88, 1995
Methods and compositions for full-length cDNA cloning using
a template-switching oligonucleotide.
A Chenchik, Y Zhu, P Siebert (clontech labs)
US Patent 5,962,272 Oct. 5, 1999
[you can locate and printout patents at <http://patents.ibm.com> ]
Generation and use of high-quality cDNA from small amounts
of total RNA by SMART PCR.
A Chenchik, Y Zhu, L Diatchenko, R Li, J Hill, PD Siebert
In: RT-PCR methods for gene cloning and analysis
eds. P Siebert, J Larrick (BioTechniques Books, Natick, MA)
1998, pp.305-319
[!P.S. if you happen to find this source I'd also be interested
in a copy of the article!]
[disclaimer: no connection with or endorsement of the above
companies]
I hope this helps,
Brad Turner
****************************************************************
Bradley Turner
Beth Israel Deaconess
Medical Center
Harvard Medical School 617-667-1215 phone
Division of Gastroenterology 617-667-2767 fax
Room Dana 536 bsturner at biosun.harvard.edu
330 Brookline Avenue bturner at caregroup.harvard.edu
Boston, MA 02215 turner at sprcore.caregroup.harvard.edu
****************************************************************
On Fri, 28 Jan 2000, Alexander S Kraev wrote:
> Dear netters,
> I have recently come across a particularly difficult case of locating a
> 5'-end of an RNA,
> which prompted to search for alternatives for doing the RACE experiments. I
> have been
> using a modification of the original procedure, that is tailing with deoxy-A
> and TdT, then
> making second strand with an anchor primer, ending in oligoT, and finally
> amplifying the
> product using PCR. In most of the resultant products sequenced with dye
> terminator
> chemistry directly the dT tail is not attached right to the presumable cDNA
> end but
> via three non-complementary G (on the coding strand) residues. Now, I have
> come across a Clontech
> kit which claims that (citation from the page 50 of the year 2000 catalog)
> "when reverse
> transcriptase reaches the end of the mRNA template, it adds a few (dC)
> residues...". Could
> anybody, i.e. from Clontech or other please provide a reference to the paper
> describing the
> primary data behind the (apparently) patented SMART technology? Are we led
> to believe that
> the three C residues I am seeing with Expand RT added to the cDNA end are
> the basis of SMART?
>> This question is so vital to me because I believe to be isolating a product
> of a cDNA synthesis block rather than a true capped 5'-end.
>> Alex
>
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