A novel ochre mutation in the beta-thalassemia gene of a Thai. Identification by direct cloning of the entire beta-globin gene amplified using polymerase chain reactions.
A novel ochre mutation in the beta-thalassemia gene of a Thai. Identification by direct cloning of the entire beta-globin gene amplified using polymerase chain reactions.
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泰国β-地中海贫血基因中的一种新的赭色突变。
DOI：
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发表时间：
1989
影响因子：
4.8
通讯作者：
Y. Fukumaki
中科院分区：
文献类型：
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作者：
S. Fucharoen;G. Fucharoen;P. Fucharoen;Y. Fukumaki
研究了来自泰国患者β-地中海贫血和HbE疾病的复合杂合子的β-珠蛋白基因。使用Taq DNA聚合酶通过聚合酶链反应扩增含有整个β-珠蛋白基因的3.0-内切酶片段，随后将扩增产物直接克隆到质粒DNA中。地中海贫血基因的序列分析显示，只有一个碱基的变化，C-A颠换密码子内的氨基酸35。这种新的突变产生了一个过早的终止子，TAA，一个赭石密码子，并导致β 0-地中海贫血表型。当使用常规克隆技术、扩增产物的直接测序以及与等位基因特异性寡核苷酸探针的杂交来分析该突变时，获得了相同的结果。从正常个体的基因组DNA获得的扩增产物的5个克隆的3.0-内切酶插入物的序列分析中未检测到错误掺入。这种方法是一种快速和准确的方法，用于分子克隆β-珠蛋白基因和其他基因，其部分核苷酸序列是已知的。
The beta-globin genes from a Thai patient compound heterozygous for beta-thalassemia and HbE disease were investigated. The 3.0-kilobase fragment containing the entire beta-globin gene was amplified by polymerase chain reaction, using Taq DNA polymerase followed by direct cloning of the amplified product into plasmid DNA. Sequence analysis of the thalassemia gene revealed only one base change, a C-A transversion within codon for an amino acid 35. This new mutation creates a premature terminator, TAA, an ochre codon, and results in a beta 0-thalassemia phenotype. The same result was obtained when this mutation was analyzed using a conventional cloning technique, direct sequencing of the amplified product, and hybridization with allele-specific oligonucleotide probes. No misincorporation was detected in the sequence analysis of the 3.0-kilobase insert of five clones of the amplified products obtained from genomic DNA of a normal individual. This approach is a rapid and accurate method for molecular cloning of the beta-globin gene and also other genes, the partial nucleotide sequences of which are known.