Unit

UNIT 6.4 Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR

  1. Hans Albertsen,
  2. Andrew Thliveris

Published Online: 1 MAY 2001

DOI: 10.1002/0471142905.hg0604s00

Current Protocols in Human Genetics

Current Protocols in Human Genetics

How to Cite

Albertsen, H. and Thliveris, A. 2001. Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR. Current Protocols in Human Genetics. 00:6.4:6.4.1–6.4.6.

Author Information

  1. University of Utah, Salt Lake City, Utah

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: FEB 1994

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.