Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5' ends of mRNAs and for constructing cDNA libraries by in vitro amplification.
Cloning full length cDNAs is a difficult task especially if mRNAs are not abundant or if tissue is only available in limited amounts. Current strategies are based on in vitro amplification of cDNAs after adding a homopolymeric tail at the 3' end of the ss-cDNA. Since subsequent amplification steps yield unspecific amplified DNA mostly due to non-specific annealing of the reverse primer containing a homopolymeric tail, we have devised a new strategy based on the ligation of single-stranded oligodeoxyribonucleotide to the 3' end of single-stranded cDNAs. The efficiency of the strategy was assessed by analyzing the 5' ends of the rat pineal gland tryptophan hydroxylase messenger. The 5' end of the least abundant messenger (0.005% of total mRNAs) could be cloned without selection. Sixty percent of the analyzed clones correspond to TPH. This technique revealed a 5-nt stretch not apparent using dG tailing strategy. The potentiality of the method for generating cDNAs libraries was tested with 10(4) PC12 cells. In this library, the abundance of tyrosine hydroxylase clones (0.03%) correlated well with the abundance of the corresponding messenger, showing that no major distortion was introduced into the construction of the library.
Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS. Gif sur Yvette Cedex, France.
J B
JB
Edwards
J
J
Delort
J
J
Mallet
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism*; D015723:Gene Library; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010870:Pineal Gland / Q000201:enzymology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D014365:Tryptophan Hydroxylase / Q000235:genetics; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014365:Tryptophan Hydroxylase