A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA.
In this report we present a strategy for generating a representative cDNA library from prohibitively low amounts of mRNA template. A defined DNA adapter, which carries an EcoRI site, is ligated to both ends of the products of a cDNA synthesis reaction. This allows low levels of cDNA to be amplified by a polymerase chain reaction. In studies with pg amounts of rabbit globin mRNA, the amplified cDNA product is shown to be full-length. Globin cDNA recombinants are positively identified in lambda gt10. The protocol should be widely applicable to mRNAs of low abundance, whose sequences have not been determined, and to limited samples from patients or animals. It may also be useful for generating representative libraries of low titer or variant viral sequences.
Akowitz
A
A
Section of Neuropathology, Yale University School of Medicine, New Haven, CT 06510.
Manuelidis
L
L
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D005591:Chemical Fractionation; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D015342:DNA Probes; D004259:DNA-Directed DNA Polymerase; D015723:Gene Library; D005914:Globins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
D015342:DNA Probes; D012333:RNA, Messenger; D005914:Globins; D004247:DNA; D004259:DNA-Directed DNA Polymerase