Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library-猫眼课题宝

DOI：

10.1016/s0378-1119(97)00411-3

发表时间：

1997-10-24

期刊：

GENE

影响因子：

3.5

通讯作者：

中科院分区：

生物学3区

文献类型：

--

作者：

Suzuki, Y;YoshitomoNakagawa, K;Sugano, S

使用“寡加帽的”mRNA [Maruyama，K.，Sugano，S.，1994.寡核苷酸加帽：一种用寡核苷酸取代真核生物mRNA帽结构的简单方法。Gene 138，171-174]，其帽结构被合成的寡核苷酸取代，我们构建了两种类型的cDNA文库。一个是“全长富集cDNA文库”，其具有高含量的全长cDNA克隆，另一个是“5 '末端富集cDNA文库”，其具有高含量的cDNA克隆及其mRNA起始位点。构建了5 ′-末端富集文库，特别用于分离长mRNA的mRNA起始位点。为了表征这些文库，我们对来自两个文库的随机选择的cDNA克隆进行了一次测序（全长富集的cDNA文库的84个克隆和5 '末端富集的cDNA文库的159个克隆）。多肽链延伸因子1 α的cDNA克隆是最常见的（9个克隆）分离，其中80%以上（8个克隆）含有该基因的mRNA起始位点。此外，两个文库中约80%的cDNA克隆（其序列与已知基因匹配）具有已知的5 ′末端或已知5 ′末端上游的序列（全长富集文库35个中的28个，5 ′末端富集文库62个中的51个）。在28个克隆中，最长的全长cDNA克隆约为3300 bp。相反，来自5 ′-末端富集的cDNA文库的7个克隆（在具有mRNA起始位点的51个克隆中）来自长度大于3500 bp的mRNA。这些cDNA文库可能有助于利用EST数据库中缺乏的mRNA起始位点信息来产生5'EST。(C)1997年Elsevier Science B.V.

Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database. (C) 1997 Elsevier Science B.V.