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Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension–application to the variants of mannose‐binding lectin gene
Author(s) -
Zerefos Panayotis G.,
Ioannou Penelope C.,
TraegerSynodinos Joanne,
Dimissianos Gerasimos,
Kanavakis Emmanuel,
Christopoulos Theodore K.
Publication year - 2006
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.20300
Subject(s) - biology , aequorin , mannan binding lectin , primer extension , genotyping , genetics , primer (cosmetics) , lectin , photoprotein , gene , snp , microbiology and biotechnology , computational biology , single nucleotide polymorphism , genotype , bioluminescence , base sequence , biochemistry , chemistry , organic chemistry , intracellular
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 Ca 2+ . 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. Hum Mutat 27(3), 279–285, 2006. © 2006 Wiley‐Liss, Inc.