Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg(201) mutations in G alpha(s) from patients with fibrous dysplasia of bone
Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg(201) mutations in G alpha(s) from patients with fibrous dysplasia of bone
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DOI：
10.1016/s8756-3282(97)00107-5
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发表时间：
1997-08-01
期刊：
影响因子：
4.1
通讯作者：
Glorieux, FH
中科院分区：
文献类型：
--
作者：
Candeliere, GA;Roughley, PJ;Glorieux, FH
α（s）G蛋白亚基的arg（201）密码子中的突变与多种疾病相关，但是这些突变的分析由于它们的镶嵌呈现而变得复杂。为了克服与可能以低和可变产量存在于整个身体中的基因组突变的分析相关的问题，已经开发了一种基于聚合酶链式反应（PCR）的技术，其允许选择性扩增来自突变等位基因的产物，该技术使用定点诱变从对限制性内切酶消化敏感的正常等位基因产生PCR产物，而从耐消化的突变等位基因产生PCR产物，扩增和消化的连续和重复循环允许选择性富集来自突变等位基因的产物。该技术已被应用于骨纤维异常增殖症患者的分析，其中Ga 1突变的结果可能从单骨性病变到多骨性病变而不同，并且已经用从骨活检标本或外周血白细胞分离的DNA进行了分析。除了先前描述的arg --> his和arg --> cys置换外，分析还在其中一名患者中检测到一种新的arg --> ser置换。该患者表现为全骨骨化病，可能代表了纤维性发育不良的一个独特亚组。(C)Elsevier Science Inc.出版。All rights reserved.
Mutations in the arg(201) codon of the alpha(s) G protein subunit have been associated with a variety of disorders, but analysis of such mutations has been complicated by their mosaic presentation, To overcome the problems associated with the analysis of genomic mutations that may be present in low and variable yield throughout the body, a polymerase chain reaction (PCR)-based technique has been developed that allows the selective amplification of products from the mutant allele, This technique uses site-directed mutagenesis to generate a PCR product from the normal allele that is susceptible to restriction endonuclease digestion, whereas that from the mutant allele is resistant to digestion, Consecutive and repeated cycles of amplification and digestion allow selective enrichment of the product from the mutant allele, The technique has been applied to the analysis of patients with fibrous dysplasia of bone, where the consequence of Ga, mutations may vary from monostotic to polyostotic lesions, and has been performed with DNA isolated from either bone biopsy specimens or peripheral blood leukocytes, In addition to the previously described arg --> his and arg --> cys substitutions, the analyses have detected a novel arg --> ser substitution in one of the patients, This patient presented with a panostotic disease and may represent a unique subgroup of fibrous dysplasia. (C) 1997 by Elsevier Science Inc. All rights reserved.