Casado-Díaz Antonio, Cuenca-Acevedo Rafael, Quesada José Manuel, Dorado Gabriel
SANYRES XXI (Grupo PRASA), Laboratorio Biología Molecular, Córdoba, Spain.
Clin Chim Acta. 2007 Feb;376(1-2):155-62. doi: 10.1016/j.cca.2006.08.014. Epub 2006 Aug 17.
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.
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.
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.
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.).
对不同复杂疾病中的单核苷酸多态性（SNP）进行基因分型研究需要大量患者。从这个意义上讲，确定等位基因频率和基因型需要一种快速且经济的方法。
通过单管等位基因特异性PCR进行基因分型，并根据PCR产物的熔解温度（T(m)）来区分产物。为此，在特异性引物的5'端添加了一个GC尾。还通过使用等位基因特异性引物的DNA池化和定量逆转录PCR（QRT-PCR）计算等位基因频率。
通过等位基因特异性PCR和熔解曲线在单管中进行基因分型，使我们能够准确确定225名绝经后女性中维生素D受体（cdx-2）、骨保护素（A-163G）和过氧化物酶体增殖物激活受体γ（C-681G）基因的三种多态性与骨质疏松症的相关性。只有cdx-2多态性与骨密度（BMD）降低有关。这些数据与使用DNA池中的QRT-PCR计算等位基因频率时获得的数据相似。
单管等位基因特异性PCR和熔解曲线分析的个体基因分型是研究任何SNP的快速、可靠且经济的方法。我们提出以下方法来确定SNP与骨质疏松症等复杂多因素疾病的可能关联，其中应分析数百名个体：构建对照组和问题组，制作DNA池，并计算池化等位基因频率。当观察到等位基因频率差异时，对每个个体进行基因分型进一步允许确定基因型分布，从而允许进行更复杂的统计分析（包括年龄、体重等其他变量）。
