Nucleic Acids Research, 1991, Vol. 19, No. 19 5227-5232
© 1991
MOLECULAR BIOLOGY |
Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5' ends of mRNAs and for constructing cDNA libraries by in vitro amplification
Laboratoire de Neurobiologie Cellulaire et Moléculaire CNRS. F-91198, Gif sur Yvette Cedex, France
*To whom correspondence should be addressed
Received July 22, 1991. Accepted September 13, 1991.
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 homopolymerlc tall 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 homopolymerlc 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 104 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.
+Present address: Howard Hughes MedicaJ Institute, BLDG 533, University of Utah, Salt Lake City, UT 84112, USA
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