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SMART cDNA synthesis

SMART cDNA synthesis method utilizes specific feature of Moloney murine leukemia virus reverse transcriptase (RT) to add a few non-template deoxynucleotides (mostly C) to the 3' end of a newly synthesized cDNA strand upon reaching the 5' end of the RNA template (Schmidt and Mueller, 1999). When added to the RT reaction, oligonucleotides containing an oligo(riboG) sequence on the 3' end (TS-oligo) form pairs with the deoxycytidine stretch produced by RT. The reverse transcriptase continues replication using the TS-oligo as a template, leading to 3' addition of the complimentary TS-sequence to serve as a universal 5' terminal site for primer annealing during following cDNA amplification (Chenchik et al., 1998; Matz et al., 1999).

SMART-prepared cDNA is full-length-enriched and well suitable for generation of cDNA libraries for various applications (Zhu et al., 2001).

Schematic outline of SMART cDNA synthesis.

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References

  • Chenchik A, Zhu YY, Diatchenko L, Li R, Hill J and Siebert PD. (1998) Gene Cloning and Analysis by RT-PCR, ed. by P Siebert and J Larrick, BioTechniques Books, Natick, MA, 305-319.
  • Matz M, Shagin D, Bogdanova E, Britanova O, Lukyanov S, Diatchenko L, Chenchik A (1999) Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res. 27, 1558-1560.
  • Schmidt WM and Mueller MW (1999) CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full length cDNA in PCR-mediated analyses of mRNAs. Nucleic Acid Res. 27, e31.
  • Zhu Y.Y., Machleder E.M., Chenchik A., Li R., Siebert P.D. (2001) Reverse transcriptase template switching, a SMART approach for full-length cDNA library construction. Biotechniques 30, 892-897.
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