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

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.

Obelin mutants as reporters in bioluminescent dual-analyte binding assay

“…see ref. [8][9][10]. We see obvious reasons for applying this technique in any dual assay regardless of its type -immune, hybridization or both.…”

Gene Assays Based on Bio(Chemi)luminescence

“…47,48 A major step was the subsequent development of a dual-analyte bio(chemi)luminometric assay for SNP genotyping by PEXT. 49 The method consists of: (a) one PCR to amplify the genomic region of interest, (b) PEXT reactions in the presence of digoxigenin-dUTP (normal primer) and biotin-dUTP (mutant primer) and (c) a microtiter well-based assay of extension products with ALP conjugated to anti-digoxigenin antibody (for detection of the normal allele) and an aequorin-streptavidin conjugate (for detection of the mutant allele). For each SNP, two primers were designed for PEXT (normal and mutant primer) and they were extended in two separate reactions.…”

“…In the above papers, [47][48][49] all assays used PEXT reactions for allele discrimination and, in all cases, both extended and non-extended primers were captured to the solid phase either through their poly(dA) segment 47,49 or their biotin moiety. 48 The difference in a subsequent paper is that only the extended primers were captured to the solid phase through the incorporated biotins.…”

Engineering Bioluminescent Proteins: Expanding their Analytical Potential

“…Christopoulos and colleagues have employed photoprotein-based DNA hybridization methods for digoxigenin and prostate specific antigen mRNA assays, for a quantitative PCR technique, and for the detection of transgenes and single nucleotide polymorphisms (13). …”