SPECIFIC ENZYMATIC AMPLIFICATION OF DNA INVITRO - THE POLYMERASE CHAIN-REACTION
SPECIFIC ENZYMATIC AMPLIFICATION OF DNA INVITRO - THE POLYMERASE CHAIN-REACTION
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DOI：
10.1101/sqb.1986.051.01.032
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发表时间：
1986-01-01
期刊：
影响因子：
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通讯作者：
ERLICH, H
中科院分区：
文献类型：
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作者：
MULLIS, K;FALOONA, F;ERLICH, H
将 DNA (1# g) 溶解在 100# 1 含有 50 mM NaCl、10 mM Tris-Cl (pH 7.6) 和 10 mM MgCl2 的聚合酶缓冲液中。将反应混合物中四种脱氧核苷三磷酸中的每一种调节至1.5mM，并且将两种寡核苷酸引物中的每一种中调节至1/zM。聚合酶链式反应的单循环如下进行：将反应加热至95℃，持续2分钟，冷却至30℃，持续2分钟，并在2μl含有约0.1#1甘油的上述缓冲液中添加1单位的大肠杆菌DNA聚合酶I的Klenow片段(Klenow从US Biochemicals获得，在含有5U/#的50%甘油溶液中)。 我）。使延伸反应在30℃进行2分钟。循环终止并通过使反应返回到95℃持续2分钟来开始新的循环。在此报告的人类 DNA 扩增中，执行的循环数为 20 至 27 次。
DNA (1# g) was dissolved in 100# 1 of a polymerase buffer containing 50 mM NaC1, 10 mM Tris-Cl (pH 7.6), and 10 mM MgC12. The reaction mixture was adjusted to 1.5 mM in each of the four deoxynucleoside triphosphates and 1/zM in each of two oligonucleotide primers. A single cycle of the polymerase chain reaction was performed by heating the reaction to 95~ for 2 minutes, cooling to 30~ for 2 minutes, and adding 1 unit of the Klenow fragment of Escherichia coli DNA polymerase I in 2 gl of the buffer described above containing about 0.1# 1 of glycerol (Klenow was obtained from US Biochemicals in a 50% glycerol solution containing 5 U/# I). The extension reaction was allowed to proceed for 2 minutes at 30~ The cycle was terminated and a new cycle was initiated by returning the reaction to 95~ for 2 minutes. In the amplifications of human DNA reported here, the number of cycles performed ranged from 20 to 27.