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pDNR-LIB Library Method Overview
BD Biosciences Clontech, Mountain View, CA, constructed libraries in the pDNR-LIB vector using the 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:
- 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 occured only when RT reached the 5'-end of mRNA template.
- The 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.
References:
- 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.
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