Smith D I, Mangrulker R, Geist R, Gilbert J, Kinsman K, Drabkin H, Golembieski W
Department of Molecular Biology and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201.
Genomics. 1989 May;4(4):453-9. doi: 10.1016/0888-7543(89)90268-1.
We have generated chromosome 3-specific recombinant libraries in both lambda and cosmid cloning vectors starting with somatic cell hybrids (hamster/human) containing either an intact chromosome 3 or a chromosome 3 with an interstitial deletion removing 75% of long-arm sequences. The libraries contained between 2 X 10(5) and 5 X 10(6) independent recombinants. Approximately 2% of the recombinants in these libraries contain inserts of human DNA. These were identified by hybridizing the recombinants to radioactively labeled total human DNA. Over 2500 recombinants containing human DNA were isolated from these various libraries and DNA was prepared from each of them. This represents 80,000 kb of cloned chromosome 3 sequences. One-third of the DNAs were digested with EcoRI or HindIII, and fragments free of repetitive sequences were radioactively labeled using random hexanucleotide primers and tested as unique sequence hybridization probes. Over 6500 of the fragments were tested and of these 758 were unique sequence probes with minimal or no background hybridization. Their hybridization only to chromosome 3 was verified. These probes, which were derived from 452 independent recombinants, should provide an effective saturation of human chromosome 3.
我们利用含有完整3号染色体或带有中间缺失（去除了75%长臂序列）的3号染色体的体细胞杂种（仓鼠/人），构建了λ噬菌体和黏粒克隆载体中的3号染色体特异性重组文库。这些文库包含2×10⁵至5×10⁶个独立重组体。这些文库中约2%的重组体含有人类DNA插入片段。通过将重组体与放射性标记的总人类DNA杂交来鉴定这些片段。从这些不同文库中分离出了超过2500个含有人类DNA的重组体，并从每个重组体中制备了DNA。这代表了80000kb的克隆3号染色体序列。三分之一的DNA用EcoRI或HindIII消化，使用随机六核苷酸引物对不含重复序列的片段进行放射性标记，并作为独特序列杂交探针进行检测。检测了超过6500个片段，其中758个是具有最小或无背景杂交的独特序列探针。验证了它们仅与3号染色体杂交。这些来自452个独立重组体的探针应能有效地覆盖人类3号染色体。
