Schweinfest C W, Henderson K W, Gu J R, Kottaridis S D, Besbeas S, Panotopoulou E, Papas T S
Laboratory of Molecular Oncology, National Cancer Institute, Frederick, MD 21701-1013.
Genet Anal Tech Appl. 1990 May;7(3):64-70. doi: 10.1016/0735-0651(90)90042-e.
cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.
通过消减cDNA克隆已分离出结肠癌和肝细胞癌中差异表达mRNA的cDNA克隆。消减后的材料至少富集了90倍的差异表达序列,可用于构建消减cDNA文库和聚合酶链反应(PCR)扩增以生成差异探针。由于携带克隆cDNA的单链噬菌体可在体内切除,且λ文库便于后续筛选和操作,因此使用市售的λZAP II构建初级文库。利用该方法可分离出差异表达的稀有mRNA(丰度低于0.01%)。