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cDNA Synthesis and Cloning

  • Alangar Ishwara Bhat
  • Govind Pratap Rao
Protocol
  • 44 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Majority of plant viruses contain single-stranded RNA as their genome. When genome sequence information of the virus is lacking, RT-PCR cannot be employed to amplify the viral genome. In such cases in order to characterize viral genomes, they have to be first converted into complementary DNA (cDNA) using reverse transcriptase enzyme and oligo d(T) or random primers. The cDNA is then made double stranded and ligated to a vector (usually a plasmid). The ligated vector is used to transform bacteria so that recombinant vector multiplies in the bacterium. The positive recombinant vector carrying viral insert is then identified and confirmed by different methods including sequencing. The identified recombinant clones can be stored in glycerol or long-term storage can be done through lyophilization.

Key words

RNA virus First strand cDNA synthesis Second strand synthesis Plasmid vector Ligation Transformation Recombinant clones 

References

  1. Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263–269CrossRefGoogle Scholar
  2. Okayama H, Berg P (1982) High-efficiency cloning of full length cDNA. Mol Cell Biol 2:161–170CrossRefGoogle Scholar
  3. Sambrook J, Russel DW (2001) Molecular cloning, vol I–III, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Alangar Ishwara Bhat
    • 1
  • Govind Pratap Rao
    • 2
  1. 1.Division of Crop ProtectionICAR-Indian Institute of Spices ResearchKozhikodeIndia
  2. 2.Division of Plant PathologyICAR-Indian Agricultural Research InstituteNew DelhiIndia

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