title: Individual single tube genotyping and DNA pooling by allele-specific PCR to uncover associations of polymorphisms with complex diseases.
abstract: BACKGROUND
  The genotyping for the study of the SNPs in different complex diseases require a great number of patients. In this sense, the determination of allele frequencies and genotypes requires a rapid and economical procedure.
  
  
  METHODS
  The genotype has been carried out by allele-specific PCR in single tube with the discrimination of the products of PCR by its T(m). For this purpose a GC tail was added to 5' extreme of the specific primer. The allele frequencies were also calculated by DNA pooling and QRT-PCR using allele-specific primers.
  
  
  RESULTS
  The use of the genotyping in single tube through allele-specific PCR and melting curves has led us to the accurate genotype of three polymorphisms of vdr (cdx-2), osteoprotegerin (A-163G) and ppar-gamma (C-681G) genes in 225 postmenopausal women to be associated to osteoporosis. Only the cdx-2 polymorphism was associated with a reduced bone mineral density (BMD). These data were similar to those obtained when the allele frequencies were calculated using QRT-PCR in DNA pools.
  
  
  CONCLUSIONS
  Individual genotyping with allele-specific PCR in single tube and melting curve analysis is a fast, trustworthy and economic method to study any SNP. We propose the following approach to determine the possible association of SNPs with complex and multifactorial diseases like osteoporosis, in which hundreds of individuals should be analyzed: construct control and problem groups, make DNA pools, and calculate pooled allelic frequencies. Genotyping each individual further permits to determine the genotypic distribution when differences in allelic frequencies are observed, thus allowing more complex statistical analyses (including other variables like age, weight, etc.).
authors.affiliation: SANYRES XXI (Grupo PRASA), Laboratorio Biología Molecular, Córdoba, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Casado-Díaz
authors.forename: Rafael
authors.initials: R
authors.lastname: Cuenca-Acevedo
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Quesada
authors.forename: Gabriel
authors.initials: G
authors.lastname: Dorado
mesh_terms: D000071616:CDX2 Transcription Factor; D015331:Cohort Studies; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D020412:Multifactorial Inheritance; D010024:Osteoporosis / Q000235:genetics*; D053244:Osteoprotegerin / Q000235:genetics; D047495:PPAR gamma / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D017698:Postmenopause; D015534:Trans-Activators / Q000235:genetics
chemical_list: D000071616:CDX2 Transcription Factor; D018398:Homeodomain Proteins; D053244:Osteoprotegerin; D047495:PPAR gamma; D015534:Trans-Activators; D004247:DNA