Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences.
Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences.
复制标题
通过聚合酶链式反应 (gd-PCR) 的定量适应确定基因剂量：快速检测基因序列的缺失和重复。
DOI：
10.1006/geno.1994.1270
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发表时间：
1994
期刊：
影响因子：
4.4
通讯作者：
Shuldiner,AR
中科院分区：
文献类型：
--
作者：
Celi,FS;Cohen,MM;Antonarakis,SE;Wertheimer,E;Roth,J;Shuldiner,AR
基于聚合酶链反应（PCR）的筛选方法，如变性梯度凝胶电泳、单链构象多态性和异源双链分析，是检测点突变以及小缺失和插入的有力工具，但无法检测外显子、基因或染色体的杂合缺失或重复。我们现在报告一种基于PCR的方法，指定基因剂量PCR（gd-PCR），允许快速筛选杂合缺失和重复的基因或外显子。基因剂量PCR是一种定量方法，其中两个体外合成的DNA内标物与基因组DNA样品共扩增，一个对应于感兴趣的基因（测试序列），另一个对应于参考（二体）基因（参考序列）。两种内标物均设计为使用相同的引物对进行扩增，其效率与基因组DNA对应物的效率相似，产生的PCR产物略小于基因组DNA的产物。在[32 P]dCTP存在下，扩增约等摩尔量的两种内标物和基因组DNA，得到4种放射性标记的PCR产物;在电泳和产物定量后，很容易计算基因剂量。为了验证，分析了56名受试者的基因组DNA，其中28名患有细胞遗传学证实的唐氏综合征（21三体），28名对照为21号染色体二体。使用β-淀粉样前体蛋白基因（APP：染色体21 q21）作为测试序列，对照受试者的校正平均基因剂量为2.00 ± 0.29，而唐氏综合征受试者的平均基因剂量为3.05 ± 0.27。两组之间的所有样本都有明显的分离。我们还成功地使用gd-PCR检测等位基因缺失，通过筛选胰岛素受体基因（IR;染色体19p13.2-p13.3）的相关区域，在三个无关的极端胰岛素抵抗的遗传综合征的患者，已知的外显子3，外显子14，或外显子17-22的缺失杂合子。基因剂量PCR是一种通用、快速、灵敏的方法，用于筛查外显子、基因或染色体的重复和缺失，在临床和研究环境中具有广泛的应用。
Screening methods based on the polymerase chain reaction (PCR), such as denaturing gradient gel electrophoresis, single-stranded conformational polymorphism, and heteroduplex analysis, are powerful tools for the detection of point mutations as well as small deletions and insertions, but are unable to detect heterozygous deletions or duplications of exons, genes, or chromosomes. We now report a PCR-based approach, designated gene dosage-PCR (gd-PCR), that allows rapid screening for heterozygous deletions and duplications of genes or exons. Gene dosage-PCR is a quantitative method in which twoin vitrosynthesized DNA internal standards are coamplified with the genomic DNA sample, one corresponding to the gene of interest (test sequence) and the other to a reference (disomic) gene (reference sequence). Both internal standards are designed to be amplified with the same primer pairs and with efficiencies similar to those of their genomic DNA counterparts, yielding PCR products slightly smaller than those derived from genomic DNA. Amplification of approximately equimolar amounts of the two internal standards and genomic DNA, in the presence of [32P]dCTP, results in four radiolabeled PCR products; after electrophoresis and quantification of the products, gene dosage is easily calculated. For validation, genomic DNA from 56 subjects, 28 with cytogenetically documented Down syndrome (trisomy 21) and 28 controls that were disomic for chromosome 21, was assayed. Using the β-amyloid precursor protein gene (APP: chromosome 21q21) as the test sequence, control subjects had an adjusted mean gene dose of 2.00 ± 0.29, while subjects with Down syndrome had a mean gene dose of 3.05 ± 0.27. There was a clear separation of all of the samples between the two groups. We also successfully used gd-PCR to detect allelic deletions by screening pertinent regions of the insulin receptor gene (IR; chromosome 19p13.2-p13.3) in three unrelated patients with genetic syndromes of extreme insulin resistance—known heterozygotes for deletions of either exon 3, exon 14, or exons 17-22. Gene dosage-PCR is a versatile, rapid, and sensitive method for screening for duplications and deletions of exons, genes, or chromosomes, with broad application in both clinical and research settings.