Deshler J O
Department of Molecular Genetics, City of Hope, Duarte, California 91010.
Genet Anal Tech Appl. 1992 Aug;9(4):103-6. doi: 10.1016/1050-3862(92)90048-a.
Random mutagenesis of cloned DNA fragments can be an important tool for understanding genes and/or gene products. I report here a simple procedure for generating random mutations in any cloned DNA fragment in vitro. This method utilizes the polymerase chain reaction (PCR) to amplify chemically damaged single-stranded DNA containing the DNA fragment of interest. A mutagenized pool of plasmid DNA is produced by cloning the PCR product (containing chemically induced sequence heterogeneity) into a new vector. Therefore, extreme chemical treatments can be used that biologically inactivate the original vector. In this report, 10% of the plasmids contained a mutation in a 97-nucleotide DNA insert, giving a mutagenesis frequency of 10(-3)/base pair. Of 3000 yeast transformants screened, seven intron mutations were isolated that activate a cryptic 3' splice site, suggesting that 2% of the mutations could give rise to the desired phenotype. The seven mutations included transitions, transversions and single nucleotide deletions, demonstrating the well-balanced repertoire of nucleotide changes derived from this procedure.
克隆DNA片段的随机诱变可以成为理解基因和/或基因产物的重要工具。我在此报告一种在体外任何克隆DNA片段中产生随机突变的简单方法。该方法利用聚合酶链反应（PCR）扩增含有感兴趣DNA片段的化学损伤单链DNA。通过将PCR产物（含有化学诱导的序列异质性）克隆到新载体中产生诱变的质粒DNA库。因此，可以使用极端化学处理使原始载体在生物学上失活。在本报告中，10%的质粒在一个97个核苷酸的DNA插入片段中含有一个突变，诱变频率为10^(-3)/碱基对。在筛选的3000个酵母转化体中，分离出7个内含子突变，这些突变激活了一个隐蔽的3'剪接位点，表明2%的突变可产生所需表型。这7个突变包括转换、颠换和单核苷酸缺失，证明了该方法产生的核苷酸变化种类均衡。
