Molecular Biotechnology

, Volume 22, Issue 3, pp 223–230 | Cite as

Global amplification of cDNA from limiting amounts of tissue

An improved method for gene cloning and analysis
  • M. K. Reddy
  • Suresh Nair
  • S. K. Sopory


In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.

Index Entries

RACE-PCR reverse transcription pea streptavidin-coated magnetic particles 


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  1. 1.
    Akowittz, A., and Manuelidis, L. (1989) A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA. Gene 81, 296–306.CrossRefGoogle Scholar
  2. 2.
    Belyavsky, A., Vinogradova, T. and Rajewsky, K. (1989) PCR-based cDNA library construction: general cDNA libraries at the level of a few cells. Nucleic Acids Res. 17, 2919–2932.PubMedCrossRefGoogle Scholar
  3. 3.
    Domec, C., Garbay, B., Fournier, M., and Bonnet, J. (1990) cDNA library construction from small amounts of unfractionated RNA: association of cDNA synthesises with polymerase chain reaction amplification. Anal. Biochem. 188, 422–426.PubMedCrossRefGoogle Scholar
  4. 4.
    Kulpa, D., Topping, R., and Telesnitsky, A. (1997) Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer associated reverse transcriptase errors. EMBO J. 16, 856–865.PubMedCrossRefGoogle Scholar
  5. 5.
    Chenchik, A., Zhu, Y.Y., Diatchenko, L., Li, R., Hill, J., and Siebert P.D. (1998) Generation and use of high-quality cDNA from small amounts of RNA by SMART PCR. in: Gene Cloning and Analysis by RTPCR, (Siebert, P., and Larrick, J. W., eds.), BioTechniques Books, Natick, MA.Google Scholar
  6. 6.
    Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002.PubMedCrossRefGoogle Scholar
  7. 7.
    Kato, S., Sekine, S., Oh, S. W., Kim, N. S., Umezawa, Y., Abe, N., Yokoyama-Kobayashi, M., and Aoki, T. (1994) Construction of a human full-length cDNA bank. Gene 150, 243–250.PubMedCrossRefGoogle Scholar
  8. 8.
    Maruyama, K., and Sugano, S. (1994) Oligo-capping: A simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotide. Gene 138, 171–174.PubMedCrossRefGoogle Scholar
  9. 9.
    Chomezynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol chloroform extraction. Anal. Biochem. 162, 156–159.Google Scholar
  10. 10.
    Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA.Google Scholar
  11. 11.
    Reddy, M. K., Nair, S. and Tewari, K. K. (1998) Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea. Plant Mol. Biol. 37, 773–784.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  1. 1.International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali MargNew DelhiIndia

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