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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Comparative Analysis of Human DNA Variations by Fluorescence-based Sequencing of PCR Products
Pui-yan Kwok,[1,2] Christopher Carlson, Thomas D. Yager, Wendy Ankener, and Deborah Nickerson
 Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195  Division of Dermatology, Washington University School of Medicine, St. Louis, Missouri 63110  Division of Nephrology, Hospital for Sick Children, Toronto, Canada M56 1 X8
Automated, direct cycle sequencing of purified double-stranded PCR products using Taq polymerase and fluorescently-labeled dideoxynucleotide terminators provides a robust and highly reproducible method to identify DNA sequence variations in sequence-tagged sites (STSs). We have developed a simple and sensitive approach that is capable of identifying a single heterozygote among homozygotes even when the standard base-calling program (ABI 373 analysis program) is unable to distinguish between these individuals. Using this approach, we have scanned for the presence of common DNA sequence polymorphisms in more than 200 STSs from the human genome. We have found that three criteria are important in accurately calling heterozygous bases: 1) a greater than 40% decrease in normalized peak height of the heterozygous nucleotide compared to the homozygous nucleotide, 2) the appearance of a new and significant peak at the location of the heterozygous nucleotide, and 3) a significant change in the normalized peak height of the nucleotide immediately adjacent to the polymorphic site. With these criteria, we now routinely detect heterozygous bases with negligible false-positives. We have also extended the use of quantitative peak height analysis by comparing sequencing traces from a heterozygous individual to that obtained from a pooled DNA sample. This comparison provides a system for rapidly determining allele frequencies for single nucleotide polymorphisms in a population of interest, and also provides a method for rapidly comparing allele frequencies in regions of genetic or evolutionary interest among different human populations.