Fluorescent Sequencing for Heterozygote Mutation Detection

  • Colin A. Graham
  • Alison J. M. Hill
Part of the Methods in Molecular Biology™ book series (MIMB, volume 167)

Abstract

Direct sequencing of PCR products using the dideoxy chain termination procedure developed by Sanger et al. (1) is now the most commonly used method for defining specific mutations. The main benefits of this method lie in its ease of use and this has been enhanced in recent years by the introduction of fluorescent labels and automated detection systems which obviate the need for radioactivity. Although the initial purchase price for automated sequencers is high, this is compensated for by single-tube reaction chemistry and rapid analysis and base calling.

Keywords

HPLC Dust Urea EDTA DMSO 

References

  1. 1.
    Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.PubMedCrossRefGoogle Scholar
  2. 2.
    Hawkins, T. L., Du, Z., Halloran, N. D., and Wilson, R. K. (1992) Fluorescence chemistries for automated primer-directed DNA sequencing. Electrophoresis 13, 552–559.PubMedCrossRefGoogle Scholar
  3. 3.
    Bjournson, A. J. and Copper, J. E. (1992) Band-stab PCR: a simple technique for the purification of individual PCR products. Nucleic Acids Res. 20, 4675.CrossRefGoogle Scholar
  4. 4.
    Rao, V. B. (1994) Direct sequencing of polymerase chain reaction-amplified DNA. Anal. Biochem. 216, 1–14.PubMedCrossRefGoogle Scholar
  5. 5.
    Davies. J., Winchester, B. G., and Malcolm, S. (1993) Mutation analysis in patients with the typical form of Anderson-Fabry disease. Hum. Mol. Genet. 2, 1051–1053.PubMedCrossRefGoogle Scholar
  6. 6.
    Donis-Keller, H., Dou, S., Chi, D., et al. (1993) Mutations in the RET proto-oncogene are associated with MEN 2a and FMTC. Hum. Mol. Genet. 2, 851–856.PubMedCrossRefGoogle Scholar
  7. 7.
    Hobbs, H. H., Browm, M. S., and Goldstein, J. L. (1992) Molecular Genetics of the LDL receptor gene in familial hypercholesterolaemia. Hum. Mutat. 1, 445–466.PubMedCrossRefGoogle Scholar
  8. 8.
    Graham, C. A., McLean, E., Ward, A. J., et al. (1999) Mutation screening and genotype:phenotype correlation in familial hypercholesterolaemia. Atherosclerosis 147/2, 309–316.CrossRefGoogle Scholar
  9. 9.
    Hughes, D. J., Hill, A. J. M., Macek, M., et al. (1996) Mutation characterisation of CFTR gene 206 Northern Irish CF families: Thirty mutations, including two novel, account for 94% of CF chromosomes. Hum. Mutat. 8, 340–347.PubMedCrossRefGoogle Scholar
  10. 10.
    Chillon, M., Casals, T., Gimenez, J., et al. (1994) Analysis of the CFTR gene in the Spanish population: SSCP screening for 60 known mutations and identification of 4 new mutations (Q30X, A120T, 1812-1 G>A and 3667del4) Hum. Mutat. 3, 223–230.PubMedCrossRefGoogle Scholar
  11. 11.
    van der Luijt, R., Khan, P. M., Vasen, H., et al. (1994) Rapid detection of translation-terminating mutations at the Adenomatous Polyposis Coli (APC) gene by direct protein truncation test. Genomics 20, 1–4.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Colin A. Graham
    • 1
  • Alison J. M. Hill
    • 2
  1. 1.Northern Ireland Regional Genetics CentreBelfast City Hospital TrustNorthern Ireland, UK
  2. 2.Department of Anatomy and PhysiologyUniversity of DundeeScotland, UK

Personalised recommendations