title: Rapid genotyping of the osteoporosis-associated polymorphic transcription factor Sp1 binding site in the COL1A1 gene by pyrosequencing.
abstract: We describe a novel polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencingbased assay for rapid genotyping of the polymorphic Sp1 binding site in the COL1A1 gene (1). A single nucleotide G-->T substitution polymorphism at this GC-rich site has recently been reported to be a predictive genetic marker for low bone mineral density (BMD). To simplify screening for this marker, we optimized PCR conditions and subjected the amplicons to pyrosequencing, which is a convenient high-throughput sequence analysis technique, readily amenable to automation. The analysis of 200 deidentified convenience DNA samples extracted from blood revealed genotype frequences in Hardy-Weinberg equilibrium (SS 68.0%, Ss 28.5%, and ss 3.5%) in agreement with other studies of European populations. This study demonstrates for the first time that pyrosequencing can be used for rapid identification of the osteoporosis-associated single nucleotide polymorphism (SNP) in the COL1A1 gene.
authors.affiliation: Strategic Development, Laboratoriemedicin Ostergötland, Linköping University Hospital, SE-581 85 Linköping, Sweden. katarina.ellnebo-svedlund@lio.se
authors.forename: Katarina
authors.initials: K
authors.lastname: Ellnebo-Svedlund
authors.forename: Lasse
authors.initials: L
authors.lastname: Larsson
authors.forename: Jon
authors.initials: J
authors.lastname: Jonasson
authors.forename: Per
authors.initials: P
authors.lastname: Magnusson
mesh_terms: D000483:Alleles; D001665:Binding Sites; D015519:Bone Density; D024042:Collagen Type I / Q000378:metabolism; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D010024:Osteoporosis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000235:genetics*
chemical_list: D024042:Collagen Type I; D016329:Sp1 Transcription Factor