UNIDIRECTIONAL DIGESTION WITH EXONUCLEASE-III CREATES TARGETED BREAKPOINTS FOR DNA SEQUENCING
UNIDIRECTIONAL DIGESTION WITH EXONUCLEASE-III CREATES TARGETED BREAKPOINTS FOR DNA SEQUENCING
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DOI：
10.1016/0378-1119(84)90153-7
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发表时间：
1984-01-01
期刊：
影响因子：
3.5
通讯作者：
HENIKOFF, S
中科院分区：
文献类型：
--
作者：
HENIKOFF, S
描述了一种非常适合长时间DNA测序的缺失衍生物的快速产生和克隆的方法。该方法基于外核酸酶III的2个有用特征：以非常均匀的速率和未能在DNA末端启动消化的过程消化，并以4碱基3'' - 突起。该方法应用于单链噬菌体M13MP18中克隆的4570 bp果蝇基因组DNA片段。通过在果蝇DNA和测序引物结合位点之间的矢量的polylinker区域中的第一个切割复制形式（RF）DNA进行了有序的缺失克隆。一种酶留下了一个4碱基3'' - 突出，保护载体的其余部分免受核酸外切酶III攻击，从而使插入序列的单向消化从5''' - 其他酶留下的突出端。以均匀的间隔去除等分试样，并用S1核酸酶，Klenow DNA聚合酶和T4 DNA连接酶处理，然后用于转染统一的细胞。从每个等分试样得出的大多数产生的克隆被预测的程度只有略微散射，即使对于超过4 kb的删除也是如此。该方法允许有效分离大型DNA片段的小预选区域内缺失断点的簇，从而允许非随机序列分析。
A method is described for the rapid generation and cloning of deletion derivatives well-suited for the sequencing of long stretches of DNA. This method is based on 2 useful features of exonuclease III: processive digestion at a very uniform rate and failure to initiate digestion at DNA ends with 4-base 3''-protrusions. The method was applied to a 4570-bp Drosophila genomic DNA fragment cloned in the single-stranded phage vector M13mp18. An ordered set of deletion clones was made by first cutting replicative form (RF) DNA with 2 restriction enzymes in the polylinker region of the vector between the Drosophila DNA and the sequencing primer binding site. One enzyme left a 4-base 3''-protrusion that protected the remainder of the vector from exonuclease III attack, allowing unidirectional digestion of the insert sequence from the 5''-protruding end left by the other enzyme. Aliquots were removed at uniform intervals, treated with S1 nuclease, Klenow DNA polymerase and T4 DNA ligase, and then used to transfect competent cells. Most of the resulting clones derived from each aliquot were deleted to a predicted extent with only slight scatter, even for deletions of more than 4 kb. The method permits efficient isolation of clusters of deletion breakpoints within small preselected regions of large DNA segments, allowing nonrandom sequence analysis.