Analytical and Bioanalytical Chemistry

, Volume 388, Issue 8, pp 1747–1754

Genotyping of single nucleotide polymorphisms by primer extension reaction and a dual-analyte bio/chemiluminometric assay

  • Jessica Konstantou
  • Penelope C. Ioannou
  • Theodore K. Christopoulos
Original Paper

DOI: 10.1007/s00216-007-1383-z

Cite this article as:
Konstantou, J., Ioannou, P.C. & Christopoulos, T.K. Anal Bioanal Chem (2007) 388: 1747. doi:10.1007/s00216-007-1383-z

Abstract

Primer extension reaction (PEXT) is the most widely used approach to genotyping of single nucleotide polymorphisms (SNP). It is based on the high accuracy of nucleotide incorporation by the DNA polymerase. We propose a dual-analyte bio/chemiluminometric method for the simultaneous detection of the PEXT reaction products of the normal and mutant allele in a high sample-throughput format. PCR-amplified DNA fragments that span the SNP of interest are subjected to two PEXT reactions using normal and mutant primers in the presence of digoxigenin-dUTP and biotin-dUTP. Both primers contain a d(A)30 segment at the 5′-end but differ in the final nucleotide at the 3′-end. Under optimized conditions only the primer that is perfectly complementary with the interrogated DNA will be extended by DNA polymerase and lead to a digoxigenin- or biotin-labeled product. The products of the PEXT reactions are mixed, denatured, and captured in microtiter wells through hybridization with immobilized oligo(dT) strands. Detection is performed by adding a mixture of antidigoxigenin–alkaline phosphatase (ALP) conjugate and a streptavidin–aequorin conjugate. The flash-type bioluminescent reaction of aequorin is triggered by the addition of Ca2+. ALP is then measured by adding the appropriate chemiluminogenic substrate. The method was evaluated by genotyping two SNPs of the human mannose-binding lectin gene (MBL2) and one SNP of the cytochrome P450 gene CYP2D6. Patient genotypes showed 100% concordance with direct DNA sequencing data.

Keywords

Single nucleotide polymorphisms Genotyping (Bio)Chemiluminescence High throughput Dual analyte assay 

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Jessica Konstantou
    • 1
  • Penelope C. Ioannou
    • 1
  • Theodore K. Christopoulos
    • 2
    • 3
  1. 1.Laboratory of Analytical Chemistry, Department of ChemistryAthens UniversityAthensGreece
  2. 2.Department of ChemistryUniversity of PatrasPatrasGreece
  3. 3.Foundation for Research and Technology HellasInstitute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT)PatrasGreece