UNIT 7.9 Mutation Detection Using Automated Fluorescence-Based Sequencing

  1. Kate T. Montgomery1,
  2. Oleg Iartchouck1,
  3. Li Li2,
  4. Anoja Perera3,
  5. Yosuf Yassin4,
  6. Alex Tamburino5,
  7. Stephanie Loomis1,
  8. Raju Kucherlapati1

Published Online: 1 APR 2008

DOI: 10.1002/0471142905.hg0709s57

Current Protocols in Human Genetics

Current Protocols in Human Genetics

How to Cite

Montgomery, K. T., Iartchouck, O., Li, L., Perera, A., Yassin, Y., Tamburino, A., Loomis, S. and Kucherlapati, R. 2008. Mutation Detection Using Automated Fluorescence-Based Sequencing. Current Protocols in Human Genetics. 57:7.9:7.9.1–7.9.31.

Author Information

  1. 1

    Harvard Medical School – Partners Healthcare Center for Genetics and Genomics, Boston, Massachusetts

  2. 2

    Albany Medical College, Albany, New York

  3. 3

    Stowers Institute for Medical Research, Kansas City, Missouri

  4. 4

    Dana Farber Cancer Institute, Boston, Massachusetts

  5. 5

    GlaxoSmithKline MDR, Boston, Massachusetts

Publication History

  1. Published Online: 1 APR 2008
  2. Published Print: APR 2008


The development of high-throughput DNA sequencing techniques has made direct DNA sequencing of PCR-amplified genomic DNA a rapid and economical approach to the identification of polymorphisms that may play a role in disease. Point mutations as well as small insertions or deletions are readily identified by DNA sequencing. The mutations may be heterozygous (occurring in one allele while the other allele retains the normal sequence) or homozygous (occurring in both alleles). Sequencing alone cannot discriminate between true homozygosity and apparent homozygosity due to the loss of one allele due to a large deletion. In this unit, strategies are presented for using PCR amplification and automated fluorescence-based sequencing to identify sequence variation. The size of the project and laboratory preference and experience will dictate how the data is managed and which software tools are used for analysis. A high-throughput protocol is given that has been used to search for mutations in over 200 different genes at the Harvard Medical School – Partners Center for Genetics and Genomics (HPCGG, http://www.hpcgg.org/). Curr. Protoc. Hum. Genet. 57:7.9.1-7.9.31. © 2008 by John Wiley & Sons, Inc.


  • Genetic mutation;
  • DNA sequencing;
  • mutation identification;
  • PCR amplification