Anchoring a Defined Sequence to the 5′ Ends of mRNAs

Abstract

Among numerous applications, the PCR (1,2) provides a convenient means to clone 5′ ends of rare messengers and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods (e.g., screening of cDNA libraries). Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5′ end of the first strand cDNAs (corresponding to the 3′ end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5′ end of rare messenger). Several strategies have been devised to tag the 3′ end of the ss-cDNAs (corresponding to the 5′ end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) (“anchor PCR” [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency. More importantly, however, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplification, a phenomenon that prevents the isolation of low abundance mRNA species (3), and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks two approaches have been devised. First, a strategy based on “a cRNA enrichment” procedure has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification caused by the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 3′ end of ss-cDNAs. This strategy is referred to as “SLIC” for single-strand ligation to ss-cDNA (8).