Batra S K, Metzgar R S, Hollingsworth M A
Duke University Medical Center, Durham, North Carolina.
Genet Anal Tech Appl. 1991 Jun;8(4):129-33. doi: 10.1016/1050-3862(91)90029-q.
A simple method is described for the construction of subtracted cDNA libraries. The technique was used to create a human pancreatic tumor cDNA library that was screened using either hybridization with cDNA probes or antibodies. cDNA from a well-differentiated tumor cell line (CD-11) was subtracted against RNA from an undifferentiated tumor cell line (Panc-1). The subtracted cDNA was purified from RNA-cDNA hybrids by oligo-dA cellulose affinity chromatography. Single-stranded subtracted cDNA was used as a template for random primed second-strand synthesis using the Klenow's fragment of DNA polymerase. After ligation with Eco R1 adapters, cDNA was inserted into lambda gt11. A library of 140,000 primary pfu was obtained that contained 92% recombinants. A small portion of this library (40,000 pfu) was subjected to probe screening with a mucin cDNA probe known to be differentially expressed by CD-11 cells. The ratio of mucin cDNA clones to actin cDNA clones was increased by greater than 300-fold in the subtracted cDNA library compared to a standard cDNA library from the same cell line. The absolute number of mucin cDNA clones per 40,000 pfu was also increased 32-fold in the subtracted library. Pancreatic tumor mucin cDNAs were also identified in the subtracted library by antibody screening. The subtraction procedure yielded a 50-fold enrichment in differentially expressed cDNA detected by antibodies, compared to a nonsubtracted library from the same cell line.
本文描述了一种构建扣除cDNA文库的简单方法。该技术用于创建一个人类胰腺肿瘤cDNA文库，该文库可使用cDNA探针杂交或抗体进行筛选。来自高分化肿瘤细胞系（CD - 11）的cDNA与未分化肿瘤细胞系（Panc - 1）的RNA进行扣除。通过寡聚dA纤维素亲和层析从RNA - cDNA杂交体中纯化扣除的cDNA。单链扣除cDNA用作模板，使用DNA聚合酶的Klenow片段进行随机引物第二链合成。与Eco R1接头连接后，将cDNA插入λgt11。获得了一个包含92%重组体的140,000个初级噬菌斑形成单位（pfu）的文库。该文库的一小部分（40,000 pfu）用已知在CD - 11细胞中差异表达的粘蛋白cDNA探针进行探针筛选。与来自同一细胞系的标准cDNA文库相比，扣除cDNA文库中粘蛋白cDNA克隆与肌动蛋白cDNA克隆的比例增加了300多倍。扣除文库中每40,000 pfu的粘蛋白cDNA克隆的绝对数量也增加了32倍。通过抗体筛选在扣除文库中也鉴定出了胰腺肿瘤粘蛋白cDNAs。与来自同一细胞系的未扣除文库相比，扣除程序使通过抗体检测到的差异表达cDNA富集了50倍。
