Studies on clonality by PCR analysis of the PGK-1 gene.
Studies on clonality by PCR analysis of the PGK-1 gene.
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通过 PGK-1 基因的 PCR 分析研究克隆性。
DOI：
10.1093/nar/19.10.2794
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发表时间：
1991
影响因子：
14.9
通讯作者：
J. Landegent
中科院分区：
文献类型：
--
作者：
H. van Kamp;R. Jansen;R. Willemze;W. Fibbe;J. Landegent
分析多态X染色体连锁基因PGK-1和HPRT(1)的甲基化模式已被广泛用于评估这些标记之一杂合子的女性患者的人类肿瘤细胞的克隆性质。如果能用聚合酶链式反应(2)进行分析，将极大地促进这一程序的适用性。PGK-1基因似乎是一个很好的候选基因，因为甲基化位点(Hpafl位点)和多态的BstX1位点距离DNA不到1kb。大约33%的女性在这个位置上可能是杂合子(1)。为了进行PCR扩增，必须将已知的外显子1(3)的核苷酸序列延伸到包含该多态的区域。用A1正义和B1反义引物进行PCR扩增获得333bp的探针，筛选出X噬菌体EMBL3中的Sau3A部分基因组文库(图1)。对分离到的X克隆(X 7.4)进行限制性内切酶图谱分析和Southern杂交，确定合适的DNA片段，并将其亚克隆到pUC 13中。用双脱氧链终止法，利用通用的正反向测序引物，建立了含有0.8kb BamHI片段(PP3)的亚克隆序列。因为分析的DNA没有显示BstXI位点，所以又合成了2个引物，并对A2-B2PCR产生的片段进行了序列分析，这些片段来自一名确实显示BstXI位点的男性和一名杂合子女性。用位于BstXI酶切位点3‘的A1和新的引物B3分析克隆性。因此，扩增的片段
Analysis of the methylation pattern of the polymorphic X-chromosome linked genes PGK-1 and HPRT (1) have been widely used to assess the clonal nature of human tumour cells in female patients heterozygous for one of these markers. The applicability of this procedure would be greatly facilitated if it could be analysed by PCR (2). The PGK-1 gene seemed to be a good candidate since the methylation sites (Hpafl sites) and the polymorphic BstXl site are within 1 kb of DNA. About 33 % of females are expected to be heterozygous at this site (1). For PCR amplification, the known nucleotide sequence from exon 1 (3) had to be extended into the region containing the polymorphism. A 'Sau 3A partial' genomic library in the X-phage EMBL3 was screened with a 333 bp probe that was obtained by PCR amplification using the Al sense and Bl antisense primers (Fig. 1). One of the X clones isolated, (X 7.4), was subjected to restriction mapping and Southern hybridization to determine the appropriate DNA fragment for subcloning into pUC 13. The sequence of a subclone containing a 0.8 kb BamHI segment (pP3), was established by the dideoxy chain-termination method using the universal 'forward and reverse' sequencing primers. Because the DNA analysed did not reveal a BstXI site, 2 additional primers were synthesized, and sequence analysis was also performed on A2-B2 PCR-generated fragments from a male who did exhibit the BstXI site, and from a heterozygous female. Clonality was analysed using the Al primer and a new primer B3, located 3' of the BstXI site. The amplified fragment thus