Detection by PCR of a VNTR polymorphism at D4S43.
Detection by PCR of a VNTR polymorphism at D4S43.
复制标题
通过 PCR 检测 D4S43 处的 VNTR 多态性。
DOI：
10.1093/nar/19.17.4772
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发表时间：
1991
影响因子：
14.9
通讯作者：
Gusella,JF
中科院分区：
文献类型：
--
作者：
Horn,GT;McClatchey,AI;Richards,B;MacDonald,ME;Gusella,JF
来源和说明：匿名 DNA 探针 pKPI。图 65 揭示了人类 D4S43 基因座内的 VNTR 多态性，检测为 Stul RFLP (1)。我们对紧邻该探针的 1269 bp PstI 片段进行了测序，以便开发 VNTR 的 PCR 引物（EMBL 登录号 X60679）。发现一个 14 bp 富含 G/C 的核心 (GGGGAGGGGGAAGA) 在距该片段一端约 350 bp 的区域中不完美地重复 6 次。 PCR 扩增：最初尝试通过 PCR 扩增该区域，结果产生了意外大小的产物。我们推测该区域富含 G/C 的性质或 PCR 过程中产生的替代 DNA 结构干扰了扩增过程。然而，使用 7-deaza-dGTP (2) 可以有效获得预期大小范围内的多态性 PCR 产物。在每个引物 GH437 (5'-GACCACAGAGAGCTTAGTGGAGC-TT-3') 和 GH436 (5'-GACCACTTCACTGACATCCACATCT-3') 以及“标准”缓冲液和 dNTP 浓度 (3) 中使用 0.4 IzM 进行 DNA 扩增，但总 dGTP 的 60% 是 7-脱氮杂-dGTP (Pharmacia#) 27-2090-02）。基因组 DNA (1 Ag) 使用 94°C 20 秒、55°C 20 秒和 74°C 20 秒扩增 28 个循环，最终在 74°C 孵育 5 分钟。发现 PCR 产物的相对大小与 Southern 印迹 RFLP 分析中看到的片段类别相对应，并且在两代和三代家族中验证了等位基因的共显性分离（数据未显示）。
Source and Desciption: The anonymous DNA probe pKPI. 65 reveals a VNTR polymorphism, detected as a Stul RFLP, within the human D4S43 locus (1). We have sequenced a 1269 bp PstI fragment located immediately adjacent to this probe in order to develop PCR primers for the VNTR (EMBL accession number X60679). A 14 bp G/C-rich core (GGGGAGGGGGAAGA) was found to be imperfectly repeated 6 times in a region approximately 350 bp from one end of this fragment. PCRAmplification: Initial attempts to amplify this region by PCR led to products of an unexpected size. We speculate that either the G/C-rich nature of this region or alternate DNA structures produced during PCR were interfering with the amplification process. However, the use of 7-deaza-dGTP (2) led to an efficient yield of polymorphic PCR products in the expected size range. DNA amplification was performed with 0.4 IzM in eachof the primers GH437 (5'-GACCACAGAGAGCTTAGTGGAGC-TT-3') and GH436 (5'-GACCACTTCACTGACATCCACATCT-3'), and'standard'buffer and dNTP concentrations (3), except that 60% of the total dGTP was 7-deaza-dGTP (Pharmacia# 27-2090-02). GenomicDNA (1 Ag) was amplified for 28 cycles using 20 sec at 94 C, 20 sec at 55 C, and 20 sec at 74 C, with a final incubation of 5 min at 74'C. The relative sizes of the PCR products were found to correspond with the classes of fragments seen in Southern blot RFLP analysis, and co-dominant segregation of the alleles was verified in two-and three-generation families (data not shown).