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Related Experiment Videos

Fluorescence-based DHPLC for allelic quantification by single-nucleotide primer extension.

K Kosaki1, H Yoshihashi, Y Ohashi

  • 1Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan. kkoskai@med.keio.ac.jp

Journal of Biochemical and Biophysical Methods
|February 17, 2001
PubMed
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This study shows that single-nucleotide primer extension (SNuPE) with fluorescence-based denaturing HPLC (DHPLC) can accurately quantify DNA alleles. This method precisely determines the relative amount of a specific allele in a DNA sample.

Area of Science:

  • Molecular Biology
  • Genetics
  • Analytical Chemistry

Background:

  • Accurate quantification of DNA alleles is crucial for genetic studies.
  • Existing methods may have limitations in precision or throughput.

Purpose of the Study:

  • To evaluate the quantitative accuracy of single-nucleotide primer extension (SNuPE) combined with fluorescence-based denaturing HPLC (DHPLC) for determining DNA allele proportions.

Main Methods:

  • Utilized SNuPE incorporating a ROX-labeled dideoxy CTP (ROX-ddCTP) at the polymorphic site.
  • Separated the extended primer from unincorporated dye terminators using ion-pair reversed-phase liquid chromatography.
  • Measured the signal intensity of incorporated ROX-ddCTP.

Main Results:

Related Experiment Videos

  • The signal intensity of incorporated ROX-ddCTP showed a strong correlation with the relative amount of the C-allele.
  • Quantification was accurate over a range of one order of magnitude.
  • Demonstrated the ability to determine the molar proportion of a specific allele.

Conclusions:

  • SNuPE coupled with fluorescence-based DHPLC provides an accurate method for quantitative allele determination.
  • This technique is effective for analyzing the relative molar proportion of alleles in genomic DNA.
  • The combined method offers a precise approach for genetic polymorphism analysis.