Wieland I, Bolger G, Asouline G, Wigler M
Cold Spring Harbor Laboratory, NY 11724.
Proc Natl Acad Sci U S A. 1990 Apr;87(7):2720-4. doi: 10.1073/pnas.87.7.2720.
We describe a procedure for genomic difference cloning, a method for isolating sequences present in one genomic DNA population ("tester") that is absent in another ("driver"). By subtractive hybridization, a large excess of driver is used to remove sequences common to a biotinylated tester, enriching the "target" sequences that are unique to the tester. After repeated subtractive hybridization cycles, tester is separated from driver by avidin/biotin affinity chromatography, and single-stranded target is amplified by the polymerase chain reaction, rendering it double-stranded and clonable. We model two situations: the gain of sequences that result from infection with a pathogen and the loss of sequences that result from a large hemizygous deletion. We obtain 100- to 700-fold enrichment of target sequences.
我们描述了一种基因组差异克隆程序,这是一种分离存在于一个基因组DNA群体(“测试者”)中而在另一个群体(“驱动者”)中不存在的序列的方法。通过消减杂交,使用大量过量的驱动者来去除生物素化测试者中常见的序列,从而富集测试者特有的“靶标”序列。经过反复的消减杂交循环后,通过抗生物素蛋白/生物素亲和色谱法将测试者与驱动者分离,然后通过聚合酶链反应扩增单链靶标,使其成为双链且可克隆。我们模拟了两种情况:病原体感染导致的序列获得和大片段半合子缺失导致的序列丢失。我们获得了靶标序列100至700倍的富集。