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pDNR-LIB Library Method Overview
BD Biosciences Clontech, Mountain View, CA, constructed libraries in the pDNR-LIB vector
SMART (Switching Mechanism at the 5'-end of RNA Transcript) proprietary method. For details of the protocol,
please read the Creator SMART cDNA Library Construction Kit User Manual. Briefly,
the method is as follows:
- BD PowerScript Reverse transcriptase (RT) was used to add
a stretch of cytosine residues on the end of newly synthesized cDNA. This terminal
transferase activity of the RT is only significant when RT reaches the 5'-end of mRNA template.
- The BD SMART Oligo, containing an adaptor sequence and dG, specifically bound to the dC stretch
generating the extended template for RT. Second strand synthesis was easily accomplished using linear
or exponential amplification with the adaptor primers.
- Adaptors containing the rare
asymmetrical restriction sites for Sfi I enabled directional cloning of the cDNA into the
Creator pDNR-LIB Library Vector.
Siebert, P.D. and Larrick, J.W. (1998, eds.) Gene Cloning & Analysis by RT- PCR. BioTechniques Books (a Division of Eaton Publishing), Natick, MA.
Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD. Reverse transcriptase template switching: a
SMART approach for full-length cDNA library construction.
Biotechniques 2001 Apr;30(4):892-7.
Zhumabayeva B, Diatchenko L, Chenchik A, Siebert PD. Use of SMART- generated cDNA for gene expression
studies in multiple human tumors.
Biotechniques 2001 Jan;30(1):158-63.