An ancient Ta subclass L1 insertion results in an intragenic polymorphism in an intron of the NF1 gene.
An ancient Ta subclass L1 insertion results in an intragenic polymorphism in an intron of the NF1 gene.
复制标题
古代 Ta 亚类 L1 插入导致 NF1 基因内含子的基因内多态性。
DOI：
10.1093/hmg/3.3.517
复制
发表时间：
1994
影响因子：
3.5
通讯作者：
Ward,K
中科院分区：
文献类型：
--
作者：
Bleyl,S;Ainsworth,P;Nelson,L;Viskochil,D;Ward,K
1 型神经纤维瘤病基因外显子 30 和 31 之间的内含子包含 350 bp 插入多态性，对应于先前报道的 Taql 多态性 (1)。该多态性存在于 4.2 kb EcoKI 限制性片段中。我们克隆了该片段并命名其构建体pDV1。 9. 我们的实验室使用了pDV1。 9广泛地作为探针来检测这种基因内多态性作为诊断性NF1连锁测试中的两个等位基因系统。为了创建一个基于 PCR 的系统来检测这种多态性，我们通过连续 pDV1 的 PCR 扩增来定位多态性位点。 9 段。通过DNA测序，我们发现多态性是由两种形态引起的；一种插入了 5' 截短的 LI (LJNE-1) 片段，另一种则没有插入。 LI单元属于Ta子集(2)，并且包含LI单元共有的3'开放阅读框的一部分。该插入是 NF1 基因中六个古老多态性之一，其处于完全连锁不平衡和等位基因关联 (3)。为了确认多态性是由于 LI 元件的存在或缺失引起的，引物 DV1。 9 S2（5'-CCT TAA TCA TGA AGG GTG CTG ACC-3'）和引物 DV1。从L1片段侧翼的Genbank NF1序列(4)中选择9个T2(5'-GCA TGT GCC ATG GTG CCT GGC T-3')。在含有 150 ng 基因组 DNA、25 pmol 每种引物、200 fiM dNTP、1.5 mM MgCl 2 、10 mM Tris-HCl (pH 8.3)、40 mM NaCl、0.25 mM 亚精胺和 1.25 U Taq 聚合酶 (Perkin-Elmer Cetus) 的 50/tl 反应中进行 PCR。在94℃初始5分钟变性后，反应经历33个循环：94℃变性20秒，66℃退火20秒，72℃延伸40秒。 PCR产物在2%琼脂糖凝胶上进行尺寸分级，观察到与LI插入相关的472bp条带和与野生型相关的122bp条带（图1a.）。通过 PCR 获得的基因型与之前通过 RFLP 分析获得的基因型相同。共显性遗传已在 5 个 CEPH 家族中得到证实，86 个 CEPH、10 个亚洲个体和 10 个非洲个体 (3) 的等位基因频率为：
The intron between exons 30 and 31 of the neurofibromatosis type 1 gene contains a 350 bp insertion polymorphism which corresponds to a previously reported Taql polymorphism (1). This polymorphism lies in a 4.2 kb EcoKI restriction fragment. We have cloned this fragment and designated its construct pDVl. 9. Our laboratories have used pDV1. 9 extensively as a probe to detect this intragenic polymorphism as a two allele system in diagnostic NFl linkage testing. In an effort to create a PCR based system for the detection of this polymorphism, we localized the polymorphic site by PCR amplification of serial pDV1. 9 segments. By DNA sequencing, we discovered that the polymorphism is due to two morphs; one with, and one without the insertion of a 5'truncated LI (LJNE-1) fragment. The LI unit is of the Ta subset (2), and contains a portion of the 3'open reading frame common to LI units. The insertion is one of six ancient polymorphisms in the NFl gene which are in complete linkage disequilibrium and allelic association (3). To confirm that the polymorphism is due to the presence or absence of the LI element, primer DV1. 9 S2 (5'-CCT TAA TCA TGA AGG GTG CTG ACC-3'), and primer DV1. 9 T2 (5'-GCA TGT GCC ATG GTG CCT GGC T-3') were chosen from the Genbank NFl sequence (4) flanking the LI fragment. PCR was performed in 50/tl reactions containing 150 ng of genomic DNA, 25 pmol of each primer, 200 fiM dNTPs, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 40 mM NaCl, 0.25 mM Spermidine and 1.25 U Taq Polymerase (Perkin-Elmer Cetus). After an initial 5 min denaturation at 94 C, reactions were subjected to 33 cycles of denaturation at 94 C for 20 sec, annealing at 66 C for 20 sec, and extension at 72 C for 40 sec. PCR products were sizefractionated on a 2% agarose gel, and bands of 472 bp, correlating to the LI insertion, and 122 bp, for the wild type, were observed (Figure la.). Genotypes obtained by PCR were identical to those obtained previously by RFLP analysis. Codominant inheritance has been demonstrated in 5 CEPH families, and allele frequencies in 86 CEPH, 10 Asian, and 10 African individuals (3) were: