Li J, Wang F, Kashuba V, Wahlestedt C, Zabarovsky E R
Karolinska Institute, Stockholm, Sweden.
Biotechniques. 2001 Oct;31(4):788, 790, 792-3. doi: 10.2144/01314st05.
The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of d7TP After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.
特定基因组序列的缺失被认为会影响某些疾病（如癌症）的发病机制。鉴定这些序列可为疾病治疗提供新的治疗途径。在此，我们描述了一种简单且可靠的方法，称为缺失序列克隆法（CODE），该方法可从复杂的人类基因组中选择性克隆缺失序列。简而言之，来自两个来源（人类正常样本和肿瘤样本）的基因组DNA用限制性内切酶（如BamHI、BglII和BclI）消化，然后与特殊接头连接，并通过PCR扩增。测试者（正常）DNA使用生物素化引物和dNTPs进行扩增。驱动者（肿瘤）DNA使用非生物素化引物进行扩增，但用dUTP代替d7TP。变性和杂交后，所有驱动者DNA用尿嘧啶-DNA糖基化酶（UDG）破坏，所有不完全杂交体用绿豆核酸酶消化。从驱动者DNA中缺失但存在于测试者DNA中的序列用链霉亲和素磁珠纯化，该循环再重复三次。此程序导致从驱动者DNA样本中纯合缺失但存在于测试者DNA部分中的基因组序列的快速分离和高效克隆。
