MGC Home

Clone Info

• Where to Buy
• Vectors & Method Overviews

Sequencing Info

• MGC ESTs

Download Data

MGC Info

• Project Summary
• Project Teams
• NIH Institutes
• References

Other Species Collections

• Danio (ZGC)
• Xenopus (XGC)

Useful Links

• UCSC
• NCBI Home
• NCBI MGC Retrieval
• Full-length cDNA Projects
• CGAP
• OCG

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:

• 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.

References:

1. Siebert, P.D. and Larrick, J.W. (1998, eds.) Gene Cloning & Analysis by RT- PCR. BioTechniques Books (a Division of Eaton Publishing), Natick, MA.
2. 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.
3. 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.