Korean J Lab Med.  2010 Jun;30(3):325-328. 10.3343/kjlm.2010.30.3.325.

Discrepancy in Genotyping of Apolipoprotein E between Allele-Specific PCR and Fluorescence Resonance Energy Transfer or Sequencing

• Park CH
• Lee ST
• Ki CS
• Kim JW

• KMID: 1781635
• DOI: http://doi.org/10.3343/kjlm.2010.30.3.325

Abstract

The human apolipoprotein E (APOE) gene contains several single-nucleotide polymorphisms (SNPs) that are distributed across the gene. The genotype of the APOE gene has important implications as a risk factor for various diseases. We observed 2 cases in which the results of allele-specific PCR (AS-PCR) of the APOE gene were not consistent with those of fluorescence resonance energy transfer (FRET) or sequencing analysis. In these cases, genotyping by AS-PCR showed that patients were epsilon2 homozygotes, while sequencing analysis and FRET showed that they were epsilon2/epsilon3 heterozygotes. Herein, we describe the causes of the errors in genotyping and describe the significance of these errors.

Keyword

APOE; Allele-specific PCR; Fluorescence resonance energy transfer; Genotyping; Sequencing

MeSH Terms

Alleles
Apolipoprotein E2/genetics
Apolipoprotein E3/genetics
Apolipoproteins E/*genetics
*Fluorescence Resonance Energy Transfer
Genotype
Homozygote
Humans
*Polymerase Chain Reaction
Polymorphism, Single Nucleotide
Risk Factors
*Sequence Analysis, DNA

Figure

• 

  Fig. 1. A flowchart summarizing the analytic courses and results for the 673 cases.

• 

  Fig. 2. Agarose gel electrophoresis of AS-PCR products. Case 1 and case 2 were ε2/ε2 homozygous. Abbreviation: M, APOE marker.

• 

  Fig. 3. Detection of mutations in the APOE gene by PCR-direct sequencing analyses. Case 1 and case 2 showed 388TT (left), 526TC (middle), and 2 point hemizygous mutations (784A∗ and 787A∗) (right).

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  Fig. 4. PCR bands of each allele (ε2, ε3, ε4, and ε7) and their interpretations in AS-PCR. The ε7 alleles of genotypes ε3/ε7 and ε4/ε7 were interpreted as ε3, and the ε7 allele of genotype ε2/ε7 was interpreted as ε2 (dotted box).

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