Screening Poly [dA/dT(−)] cDNA for Gene Identification

  • San Ming Wang
  • Scott C. Fears
  • Lin Zhang
  • Jian-Jun Chen
  • Janet D. Rowley
Part of the Methods in Molecular Biology™ book series (MIMB, volume 221)

Abstract

The goal for developing the SPGI (screening poly [da/dT(−)] cDNA for gene identification technique was to generate an efficient tool for maximal identification of the expressed genes from eukaryotic genomes. The impetus for developing the SPGI method was triggered by the observation that a large number of human novel transcripts have not been identified in spite of intensive efforts in the past decades using the expressed sequence tag (EST) approach (1, 2, 3, 4, 5, 6, 7, 8, 9). Based on our analysis, we believe that the use of complementary DNA (cDNA) libraries generated by regular oligo-(dT) primers contributes significantly to this problem (10,11). The cDNAs generated by oligo-(dT) priming contain various lengths of poly(dA/dT) tail sequences at the 3′ end. Most of the cDNA libraries are processed through normalization/subtraction before being used for sequencing analysis (7). During the process of normalization/subtraction, poly(dA)–poly(dT) hybrids will form randomly between unrelated cDNA templates. The removal of these hybrids causes the loss of cDNA templates. This phenomenon affects especially the low-copy cDNA templates, which represent most of the genes. To overcome this problem, we developed the SPGI method. In this method, a set of anchored oligo-(dT) primers is used for mRNA priming to prevent the inclusion of the poly(dA/dT) sequences from the 3′ ends of cDNAs. Using the poly [dA/dT(−)] cDNA for subtraction/normalization prevents the formation of poly(dA)–poly(dT) hybrids, therefore preventing the nonspecific loss of cDNAs because of the poly(dA)–poly(dT) hybrids.

Keywords

Vortex Phenol EDTA Agarose Hydroxyapatite 

References

  1. 1.
    Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J. M., Utterback, T. R., et al. (1992) Sequence identification of 2,375 human brain genes. Nature 355, 632–634.PubMedCrossRefGoogle Scholar
  2. 2.
    Boguski, M. S. (1995) The turning point in genome research. Trends Biochem. Sci. 20, 295–296.PubMedCrossRefGoogle Scholar
  3. 3.
    Fields, C., Adams, M. D., White, O., and Venter, J. C. (1994) How many genes in the human genome? Nat. Genet. 7, 345–346.PubMedCrossRefGoogle Scholar
  4. 4.
    Gerhold, D. and Caskey, C. T. (1996) It’s the genes! EST access to human genome content. BioEssay 18, 973–981.CrossRefGoogle Scholar
  5. 5.
    Mao, M., Fu, G., Wu, J. S., et al. (1998) Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning. Proc. Natl. Acad. Sci. USA 95, 8175–8180.PubMedCrossRefGoogle Scholar
  6. 6.
    Strausberg, R. L., Dahl, C. A., and Klausner, R. D. (1997) New opportunities for uncovering the molecular basis of cancer. Nat. Genet. 15, 415–416.PubMedCrossRefGoogle Scholar
  7. 7.
    Bonaldo, M. F., Lennon, G., and Soares, M. B. (1996) Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res. 6, 791–806.PubMedCrossRefGoogle Scholar
  8. 8.
    Wang, S. M. and Rowley, J. D. (1998) A strategy for genome-wide gene analysis: integrated procedure for gene identification. Proc. Natl. Acad. Sci. USA 95, 11,909–11,914.PubMedCrossRefGoogle Scholar
  9. 9.
    Velculescu, V. E., Madden, S. L., Zhang, L., et al. (1999) Analysis of human transcriptomes. Nat. Genet. 23, 387–388.PubMedCrossRefGoogle Scholar
  10. 10.
    Wang, S. M., Fears, S. C., Zhang, L., Chen, J.-J., and Rowley, J. D. (2000) Screening poly(dA/dT)− cDNAs for gene identification. Proc. Natl. Acad. Sci. USA 97, 4162–4167.PubMedCrossRefGoogle Scholar
  11. 11.
    Martin, K. J. and Pardee, A. B. (2000) Identifying expressed genes. Proc. Natl. Acad. Sci. USA 97, 3789–3791.PubMedCrossRefGoogle Scholar
  12. 12.
    Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) Serial analysis of gene expression. Science 270, 484–487.PubMedCrossRefGoogle Scholar
  13. 13.
    Zhou, G., Chen, J., Lee, S., Clark, T., Rowley, J. D., and Wang, S. M. (2001) The pattern of gene expression in human CD34+ hematopoietic stem/progenitor cells. Proc. Natl. Acad. Sci. USA 98, 13,966–13,971.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • San Ming Wang
    • 1
  • Scott C. Fears
    • 1
  • Lin Zhang
    • 2
  • Jian-Jun Chen
    • 3
  • Janet D. Rowley
    • 3
  1. 1.Section of Hematology and OncologyUniversity of Chicago Medical CenterChicago
  2. 2.Oncology CenterJohns Hopkins University School of MedicineBaltimore
  3. 3.Section of Hematology and OncologyUniversity of Chicago Medical CenterChicago

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