title: Quantification of relative gene dosage by single-base extension and high-performance liquid chromatography: application to the SMN1/SMN2 gene.
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.
authors.affiliation: Institute of Biomedical Engineering, National Taiwan University, Taipei 100, Taiwan.
authors.forename: Chia-Cheng
authors.initials: CC
authors.lastname: Hung
authors.forename: Chien-Nan
authors.initials: CN
authors.lastname: Lee
authors.forename: Chih-Ping
authors.initials: CP
authors.lastname: Chen
authors.forename: Yuh-Jyh
authors.initials: YJ
authors.lastname: Jong
authors.forename: Chi-An
authors.initials: CA
authors.lastname: Chen
authors.forename: Wen-Fang
authors.initials: WF
authors.lastname: Cheng
authors.forename: Win-Li
authors.initials: WL
authors.lastname: Lin
authors.forename: Yi-Ning
authors.initials: YN
authors.lastname: Su
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D055532:SMN Complex Proteins; D014897:Spinal Muscular Atrophies of Childhood / Q000235:genetics; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein