Three new point mutations in type II procollagen (COL2A1) and identification of a fourth family with the COL2A1 Arg519-->Cys base substitution using conformation sensitive gel electrophoresis.
Three new point mutations in type II procollagen (COL2A1) and identification of a fourth family with the COL2A1 Arg519-->Cys base substitution using conformation sensitive gel electrophoresis.
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II 型原胶原 (COL2A1) 中的三个新点突变，并使用构象敏感凝胶电泳鉴定了具有 COL2A1 Arg519-->Cys 碱基取代的第四个家族。
DOI：
10.1093/hmg/4.2.309
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发表时间：
1995
影响因子：
3.5
通讯作者：
Prockop,DJ
中科院分区：
文献类型：
--
作者：
Williams,CJ;Rock,M;Considine,E;McCarron,S;Gow,P;Ladda,R;McLain,D;Michels,VM;Murphy,W;Prockop,DJ
II型前胶原基因（COL2A1）因其在骨骺和关节软骨组织中丰富而成为广泛分析关节退行性疾病的目标。现在有相当多的证据支持它参与了至少两种骨发育不良家族的几种遗传性骨骼疾病：脊柱骨骺发育不良和Stickler/Kniest发育不良。为了建立COL2A1基因突变与各种骨发育不良亚群之间的关系，我们利用构象敏感凝胶电泳（CSGE; 1）在正常对照和生长板疾病和/或骨关节炎患者的混合人群中筛选潜在的突变。为了进行分析，我们使用了PCR引物来扩增基因，这通常是我们实验室用于序列研究的引物(2)。COL2A1基因的PCR产物大小为291 ~ 811bp，包括该基因的全部54个外显子和所有内含子/外显子边界区域。PCR扩增后，每个样品的20^ 1加热到98℃5分钟，然后在68℃孵育1小时，产生异源双链。运行缓冲液和电泳条件的细节已在其他地方描述(1)。然而，凝胶组成已被修改如下：将39.6 g丙烯酰胺和0.4 g双丙烯基哌嗪（BAP; Fluka）的原液在100 ml无菌蒸馏水中配制成含有10%（vol/vol）乙二醇（Sigma）和15%（wt/vol）甲酰胺（GIBCO/BRL）的10%聚丙烯酰胺凝胶。值得注意的是，1XTTE的组成为89 mM Tris/29 mM牛磺酸缓冲液/0.5 mM EDTA, pH 9.0（美国生化公司）。常规的CSGE分析一次对8例患者样本进行；36孔凝胶格式允许对8个样品中的每个样品分析4个PCR产物，加上额外的分子量标准通道。然而，为了充分利用大凝胶格式的分离能力，将每个样品的两个不同大小的异源双链PCR产物分别装在同一车道上。因此，对来自8个样品的32个COL2A1 PCR产物的完整分析可以通过在两天内运行四种凝胶来完成。
The type II procollagen gene (COL2A1) has been targeted for extensive analysis in degenerative diseases of the joint because of its abundance in epiphyseal and articular cartilage tissue. There is now considerable evidence to support its involvement in several types of heritable skeletal disorders in at least two bone dysplasia families: spondyloepiphyseal dysplasia and Stickler/Kniest dysplasia. In order to establish the relationship between COL2A1 gene mutations and various subsets of bone dysplasias, we have utilized Conformation Sensitive Gel Electrophoresis (CSGE; 1) to screen for potential mutations in a mixed population of normal controls and patients with growth plate disorders and/or osteoarthritis. For the analyses, we have used PCR primers for the amplification of the gene which are normally used in our laboratory for sequence studies (2). The PCR products from the COL2A1 gene ranged in size from 291 to 811 bp and included all 54 exons of the gene plus all intron/exon boundary regions. Following amplification by the PCR, 20^ 1 of each sample was heated to 98 C for 5 min followed by incubation at 68 C for 1 h to generate heteroduplexes. Details of the running buffer, and electrophoretic conditions have been described elsewhere (1). However, the gel composition has been modified as follows: a stock solution of 39.6 g of acrylamide and 0.4 g of bis-acrolylpiperazine (BAP; Fluka) is made in 100 ml of sterile, distilled water and used to prepare a 10% polyacrylamide gel containing 10%(vol/vol) ethylene glycol (Sigma), and 15%(wt/vol) formamide (GIBCO/BRL). It should also be noted that the composition of 1XTTE is 89 mM Tris/29 mM taurine buffer/0.5 mM EDTA, pH 9.0 (United States Biochemical Co.). CSGE analyses were routinely performed on eight patient samples at once; the 36-well gel format permitted the analysis of four PCR products for each of the eight samples, plus extra lanes for molecular weight standards. However, to make full use of the separating capacity of the large gel format, two separate heteroduplexed PCR products of different sizes from each sample were loaded in the same lane. Thus, analysis of a complete panel of 32 COL2A1 PCR products from eight samples could be accomplished by running four gels over the course of two days.