DIRECT CLONING AND SEQUENCE-ANALYSIS OF ENZYMATICALLY AMPLIFIED GENOMIC SEQUENCES
DIRECT CLONING AND SEQUENCE-ANALYSIS OF ENZYMATICALLY AMPLIFIED GENOMIC SEQUENCES
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DOI：
10.1126/science.3461561
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发表时间：
1986-09-05
期刊：
影响因子：
56.9
通讯作者：
ERLICH, HA
中科院分区：
文献类型：
--
作者：
SCHARF, SJ;HORN, GT;ERLICH, HA
描述了一种将基因组DNA的酶扩增片段直接克隆到M13载体用于序列分析的方法。人类β-珠蛋白基因的110碱基对片段和人类白细胞抗原DQ.Alpha的242碱基对片段。用聚合酶链式反应方法扩增基因座，这是一种基于DNA聚合酶I反复变性、引物退火和延伸的循环过程。通过对克隆产物的分析，可以对S扩增方法的特异性和保真度进行定量评估。鉴于观察到的序列错误频率较低，该方法有望成为从纳克量的DNA中快速获得可靠基因组序列的方法。
A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human .beta.-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ.alpha. locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5'' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method''s specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.