Ko M S
Furusawa MorphoGene Project, Research Development Corporation of Japan (JRDC), Tsukuba.
Nucleic Acids Res. 1990 Oct 11;18(19):5705-11. doi: 10.1093/nar/18.19.5705.
The total number of genes in higher organisms is estimated to be under one hundred thousand. However, constructing a cDNA library containing a full set of genes expressed throughout the life time of an organism, without redundancy, is a major challenge for modern biology. Towards this goal, I have tried to make a library of mouse fibroblastoid Ltk- cells with nearly equal representations of cDNA clones. Double-stranded cDNAs (ds-cDNAs) are synthesized from mRNA using an oligo(dT)-Notl primer. After shearing to 200-400 bp, a synthetic linker-primer, which has one blunt and one sticky end and an internal EcoRl site, is ligated to the cDNAs. The cDNAs are amplified by the polymerase chain reaction (PCR) using the ligated linker-primer sequence. After denaturation and reassociation of the ds-cDNAs, and isolation of single-stranded cDNAs (ss-cDNAs) by hydroxylapatite chromatography, the ss-cDNAs are again amplified by PCR. The cDNAs are digested with EcoRl and Notl, and inserted into a plasmid vector. Colony hybridization with eight probes of different abundance showed a reduction in 'abundance variation' from at least 20,000-fold in the original library to 40-fold in the library constructed after three cycles of equalization. This indicates the usefulness of the current procedure for making equalized cDNA libraries.
高等生物中的基因总数估计在十万个以下。然而，构建一个包含生物体整个生命周期中表达的全套基因且无冗余的cDNA文库，是现代生物学面临的一项重大挑战。为了实现这一目标，我尝试构建一个小鼠成纤维样Ltk-细胞文库，其中cDNA克隆的代表性几乎相等。使用oligo(dT)-Notl引物从mRNA合成双链cDNA（ds-cDNA）。将其剪切成200 - 400 bp后，连接一个具有一个平端和一个粘性末端以及一个内部EcoRl位点的合成接头引物。使用连接的接头引物序列通过聚合酶链反应（PCR）扩增cDNA。ds-cDNA变性和重新退火后，通过羟基磷灰石柱层析分离单链cDNA（ss-cDNA），然后再次通过PCR扩增ss-cDNA。用EcoRl和Notl消化cDNA，并将其插入质粒载体。用八个丰度不同的探针进行菌落杂交表明，“丰度变异”从原始文库中的至少20000倍降低到均衡三个循环后构建的文库中的40倍。这表明当前制备均衡化cDNA文库的方法是有用的。
