An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues
An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues
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DOI：
10.1006/jmbi.1996.0049
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发表时间：
1996-02-02
影响因子：
5.6
通讯作者：
Gherardi, E
中科院分区：
文献类型：
--
作者：
Zaccolo, M;Williams, DM;Gherardi, E
我们描述了一种基于使用核苷类似物的三磷酸盐混合物的DNA随机诱变的新方法。该方法依赖于用标记聚合酶体外扩增DNA，并在6-（2-脱氧- β -d -核呋喃基）-3,4-二氢- 8h -嘧啶-[4,5- c][1,2]恶嗪-7-one（dP）和8-氧-2‘脱氧鸟苷（8-oxodG）的5’-三磷酸存在下进行扩增。新合成的dP （dPTP）的三磷酸衍生物是标签聚合酶的优良底物（K-m = 22 μ M，而TTP的K-m = 9.5 μ M）；它取代了TTP，效率降低了四倍，取代了dCTP。经过30个DNA扩增循环后，四种正常dNTPs和dPTP的等摩尔混合物产生以下四种过渡突变的频率：A—>G (4.4 × 10(-2)), T—>C (4.3 × 10(-2)), G—>A （1.1 × 10(-2)）和C—>T （1.0 × 10(-2)）。8-oxodG的三磷酸衍生物（8-oxodGTP）与相反的模板腺嘌呤结合，产生两个过渡突变（A- >C和T- >G），频率分别为0.8 × 10（-2）和1.2 × 10（-2）。含有dPTP和8-oxodGTP的反应混合物会导致dP和8- oxodg诱导的突变以及在没有插入和缺失的情况下广泛的密码子变化。所描述的方法与以前的诱变程序在三个方面不同：(1)它允许非常高的碱基替换频率（高达1.9 x 10(-1)），(2)它允许通过DNA扩增周期的数量来控制突变负荷，(3)它产生过渡和翻转突变。该程序可用于DNA和蛋白质突变体文库的生成，从中可以选择具有改进或新活性的物种。(C) 1996学术出版社有限公司
We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitro with Tag polymerase and in the presence of the 5'-triphosphates of 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2'deoxyguanosine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Tag polymerase (K-m = 22 mu M versus K-m = 9.5 mu M for TTP); it is incorporated in place of TTP and, with a similar to fourfold lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, equimolar mixtures of the four normal dNTPs and dPTP yield the following frequencies of the four transition mutations: A-->G (4.4 x 10(-2)), T-->C (4.3 x 10(-2)), G-->A (1.1 x 10(-2)) and C-->T (1.0 x 10(-2)). The triphosphate derivative of 8-oxodG (8-oxodGTP) is incorporated opposite template adenine and yields two transition mutations (A-->C and T-->G) at frequencies of 0.8 x 10(-2) and 1.2 x 10(-2) respectively. Reaction mixtures containing dPTP and 8-oxodGTP results in both dP and 8-oxodG-induced mutations and an extensive array of codon changes in the absence of insertions and deletions. The method described differs from previous mutagenesis procedures in three respects: (1) it enables very high frequencies of base substitutions (up to 1.9 x 10(-1)), (2) it allows control of the mutational load via the number of DNA amplification cycles and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected. (C) 1996 Academic Press Limited