ENGINEERING HYBRID GENES WITHOUT THE USE OF RESTRICTION ENZYMES - GENE-SPLICING BY OVERLAP EXTENSION
ENGINEERING HYBRID GENES WITHOUT THE USE OF RESTRICTION ENZYMES - GENE-SPLICING BY OVERLAP EXTENSION
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DOI：
10.1016/0378-1119(89)90359-4
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发表时间：
1989-04-15
期刊：
影响因子：
3.5
通讯作者：
PEASE, LR
中科院分区：
文献类型：
--
作者：
HORTON, RM;HUNT, HD;PEASE, LR
重叠延伸基因剪接是一种不考虑重组位点核苷酸序列，不使用限制性内切酶或连接酶，在精确连接处重组DNA分子的新方法。要重组的基因片段在单独的聚合酶链反应（pcr）中产生。引物的设计使产物的末端包含互补序列。当这些PCR产物混合、变性和再退火时，具有匹配序列的3'端链重叠，并作为彼此的引物。DNA聚合酶对这种重叠的延伸产生了一种分子，在这种分子中，原始序列被“拼接”在一起。该技术用于构建一个编码由两种不同的小鼠i类主要组织相容性基因组成的马赛克融合蛋白的基因。与标准的重组DNA技术相比，这种简单而广泛适用的方法具有显著的优势。
Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3'' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are ''spliced'' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different mouse class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.