Quantitative assessment of apolipoprotein E genotypes by image analysis of PCR-RFLP fragments

Yan Yun Wu, Rosario Delgado, Rene Costello, Trey Sunderland, Ruth Dukoff, Gyorgy Csako

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Apolipoprotein E (APOE) genotyping usually involves polymerase chain reaction (PCR) and assessment of restriction fragment length polymorphism (RFLP) by gel electrophoresis. We made determination of HhaI restriction endonuclease digestive patterns more objective and improved diagnostic accuracy with a quantitative approach using sensitive DNA stain (SYBR Green) and image analysis of gel patterns. For distinguishing true and partially-digested restriction fragments, band ratios were calculated for the staining intensity of gel patterns from 116 sample runs of 63 human blood specimens. Each of these specimens was independently genotyped for APOE by at least two (and most of them by three) different PCR-RFLP methods. Based on the distribution of band ratios, decision levels were established and used for developing a program for computer-aided interpretation of APOE genotypes (Microsoft Excel software). Appropriateness of the decision levels for band ratios was validated by APOE genotyping of additional 61 specimens. The approach described here is applicable to a variety of other molecular diagnostic techniques that are based on PCR-RFLP or sequence-specific signal amplifications. Copyright (C) 2000.

Original languageEnglish (US)
Pages (from-to)213-221
Number of pages9
JournalClinica Chimica Acta
Volume293
Issue number1-2
DOIs
StatePublished - Mar 2000
Externally publishedYes

Keywords

  • Alzheimer disease
  • Apolipoprotein E
  • Electrophoresis
  • Genotyping
  • Polymerase chain reaction (PCR)
  • Restriction fragment length polymorphism (RFLP)

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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