Quantification of relative gene dosage by single-base extension and high-performance liquid chromatography: Application to the SMN1/SMN2 gene
Journal
Analytical Chemistry
Journal Volume
77
Journal Issue
21
Pages
6960-6968
Date Issued
2005
Author(s)
Abstract
One of the most commonly used techniques for genotyping of single-nucleotide polymorphism (SNP) is detection of single-base extensions (SBEs). We present a new, rapid, simple, and highly reliable method for accurate quantification of SNP variants in a single reaction. Our approach is based on SBE detection coupled with high-performance liquid chromatography (HPLC) quantification. To demonstrate the utility of our approach, we report data to determine the gene dosage for relative amounts of alleles in a homologous gene, allowing detection of mutation causing exon skipping in human SMN genes to determine the ratio between the copy numbers of the SMN1/SMN2 gene. We successfully determined the relative ratio of the SMN1 and SMN2 genes and showed assay characteristics using the SBE reaction coupled with HPLC. This assay approach readily scaled to high parallelization with multiplex SBE reactions in a single sample screened in one analysis. By screening for particular SNP genotypes, this assay can be used to determine the relative gene dosage that correlates highly with the patient's disease state. The next challenge is to apply this novel methodology in a clinical screening and quantification setting for special gene regions within highly homologous genes. ? 2005 American Chemical Society.
SDGs
Other Subjects
Gene dosage; Genotyping; Single-base extensions; Single-nucleotide polymorphism (SNP); Correlation methods; Diseases; High performance liquid chromatography; Mutagenesis; Reaction kinetics; Genes; allele; article; gene; gene dosage; genotype; high performance liquid chromatography; quantitative analysis; reliability; screening; sequence homology; single nucleotide polymorphism; SMN1 gene; SMN2 gene; spinal muscular atrophy; Base Sequence; Chromatography, High Pressure Liquid; Cyclic AMP Response Element-Binding Protein; Gene Dosage; Humans; Molecular Sequence Data; Nerve Tissue Proteins; Polymorphism, Single Nucleotide; RNA-Binding Proteins; SMN Complex Proteins; Spinal Muscular Atrophies of Childhood; Survival of Motor Neuron 1 Protein; Survival of Motor Neuron 2 Protein
Type
journal article