Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension–application to the variants of mannose-binding lectin gene

Abstract

Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.-619G>C and c.-290G>C (promoter region), and one SNP at position c.-66C>T of the 5' untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5' end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3 sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data.

Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

“…10) The chemical conjugate of aequorin with streptavidin has been reported and successfully used in various assays. [11][12][13] The fusion protein of streptavidin with beetle luciferases has previously been prepared and applied to detect biotinylated IgG; click beetle luciferase 14) and firefly luciferase 15) were respectively fused to the carboxyl and amino terminus of streptavidin. These streptavidin-fused luciferases retained both the binding ability of biotinylated IgG and luminescence activity.…”

Bioluminescent reporters for identification of gene allelic variants

“…The use of CBP instead of free coelenterazin to start luciferase bioluminescence significantly simplifies the analysis because this protein is stable when stored in solution and in frozen and dried form. Note that the effective ness of the bioluminescent reporters for the SNP iden tification was demonstrated by the successful use of another photoprotein, akvorin of the jellyfish Aequorea victoria, for genotyping polymorphisms of the gene encoding mannozose binding lectin (MBL) [10]. …”

Heterozygous promotor haplotype LXA/LYB in MBL-deficiency associated with myopathy and left ventricular hypertrabeculation/noncompaction

“…MBL opsonizes pathogens for phagocytosis directly or via complement activation [5]. Glu)], four promotor polymorphisms -619 g/c, -290 g/c [6], -550 g/c (alleles H/L), and -221c/g (alleles X/Y), forming the haplotypes HY, LX, and LY [5,7], and a polymorphism (G3130C) in exon 4 [8]. The wild-type allele is known as allele A.…”