title: Molecular haplotyping of tandem single nucleotide polymorphisms by allele-specific PCR.
abstract: BARD1 Val507Met (1592A>G) is an interesting marker for association studies on cancer risk. However, studies are scarce in the literature, probably reflecting the methodological problem imposed by the fact that next to the 1592A>G stands the 1591C>T single nucleotide polymorphism (SNP). We have designed an allele-specific PCR method capable of molecular haplotyping tandem SNPs. In the tandem SNPs haplotyping assay (tSNPh), four reverse primers are designed to be perfect matches of each potential haplotype. The forward primer is labeled with a fluorochrome. PCR products are analyzed by capillary electrophoresis. Haplotyping is performed by size calling. To ascertain the accuracy and reproducibility of the assay, we measured the level of concordance with sequencing data in 124 samples. In vitro-generated templates have been used for further testing. We developed a novel and reliable assay that permits typing two SNPs directly adjacent to each other, avoiding mutual interferences. The method is amenable to automation and high throughput. We expect that this assay will contribute to clarifying the role of BARD1 in cancer susceptibility. In addition, we suggest that tandem SNPs are potentially interesting polymorphic markers in which molecular haplotyping can be performed easily.
authors.affiliation: Laboratorio de Oncología Molecular y Servicio de Oncología Médica, Hospital Clínico San Carlos, 28040 Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Cañadas
authors.forename: Ana
authors.initials: A
authors.lastname: Sánchez-de-Abajo
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: Fernández
authors.forename: Miguel
authors.initials: M
authors.lastname: Martín
authors.forename: Eduardo
authors.initials: E
authors.lastname: Diaz-Rubio
authors.forename: Trinidad
authors.initials: T
authors.lastname: Caldés
authors.forename: Miguel
authors.initials: M
authors.lastname: de la Hoya
mesh_terms: D000483:Alleles; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods; D005819:Genetic Markers; D015894:Genome, Human; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D025202:Molecular Diagnostic Techniques; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D025521:Tumor Suppressor Proteins; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases