Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.
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.
Suzuki
Y
Y
International and Interdisciplinary Studies, The University of Tokyo, Japan.
Yoshitomo-Nakagawa
K
K
Maruyama
K
K
Suyama
A
A
Sugano
S
S
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004398:Dyneins / Q000737:chemistry / Q000235:genetics; D004798:Enzymes / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004798:Enzymes; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011506:Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D004398:Dyneins