Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR

Hans Albertsen1, Andrew Thliveris1

1 University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 6.4
DOI:  10.1002/0471142905.hg0604s00
Online Posting Date:  May, 2001
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Abstract

This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template.This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA.

     
 
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Table of Contents

  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  MaterialsFor common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • 2 µg total yeast DNA from a yeast artificial chromosome (YAC‐bearing) clone containing cDNA of interest in a 50‐µl agarose plug (unit 5.1)
  • Restriction endonucleases and 1× buffers
  • TE buffer, pH 7.8
  • 1 Weiss U/µl T4 DNA ligase and 10× buffer ( appendix 2D)
  • 95% and 70% (v/v) ethanol
  • 10× PCR amplification buffer ( appendix 2D; determine appropriate MgCl 2 concentration empirically)
  • 5 U/µl Taq DNA polymerase
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • Mineral oil
  • SeaKem GTG agarose (FMC Bioproducts)
  • 1× TBE buffer ( appendix 2D)
  • 10 mg/ml ethidium bromide ( appendix 2D; store at 4°C in the dark)
  • 40 pmol/µl M13 universal primer: 5′‐TGTAAAACGACGGCCAGT‐3′
  • 40 pmol/µl M13 reverse primer: 5′‐CAGGAAACAGCTATGACC‐3′
  • 15‐ml tube (e.g., Falcon 2006 or equivalent)
  • 67° and 100°C water baths
  • Centricon‐100 concentrator (Amicon)
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA ( appendix 3C), agarose gel electrophoresis (unit 2.7), isolation of DNA fragments from agarose gels (CPMB UNIT ), PCR amplification (CPMB UNIT ), and direct DNA sequencing of PCR products (CPMB UNIT )
CAUTION: Ethidium bromide is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
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Figures

Videos

Literature Cited

Literature Cited
   Crick, F. 1979. Split genes and RNA splicing. Science 204:264‐271.
   Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., Robertson, M., Sargent, L., Krapcho, K., Wolff, E., Burt, R., Hughes, J.P., Warrington, J., McPherson, J., Wasmuth, J., Le Paslier, D., Abderrahim, H., Cohen, D., Leppert, M., and White, R. 1991. Identification and characterization of the familial adenomatous coli gene. Cell 66:589‐600.
   Ochman, H., Gerler, A.S., and Hartl, D.L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621‐623.
Key Reference
   Groden, et al., 1991. See above.
  First description of the concept of using inverse PCR to identify intron/exon boundaries.
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