ABSTRACT
Cloning of cDNAs plays an important role in current molecular biology.1-7 Construction of a cDNA library is a highly sophisticated technology that involves a series of enzymatic reactions. The quality and integrity of the cDNA library greatly impact the success or failure in isolation of the cDNAs of interest.2 The general principles begin with mRNA that is transcribed into the „rst-strand DNA, called a complementary DNA or cDNA, which is based on nucleotide bases complementary to the mRNA template. This step is catalyzed by AMV reverse transcriptase using oligo(dT) primers. The second strand DNA is copied from the „rst strand cDNA using DNA polymerase I, thus producing a double-stranded cDNA molecule. Subsequently, the double-stranded cDNA is ligated to an adapter and then to an appropriate vector via T4 DNA ligase. The recombinant vector-cDNA molecules are then packaged in vitro and cloned in a speci„c host, generating a cDNA library. Speci„c cDNA clones can be “„shed” out by screening the library with a speci„c probe.1,4-10
cDNA cloning takes advantage of the 3′ hairpins produced by AMV reverse transcriptase during the synthesis of the „rst strand cDNAs. The hairpins are used to prime the second strand cDNA that is catalyzed by Klenow DNA polymerase and reverse transcriptase. Hence, S1 nuclease is needed to cleave the hairpin loops. However, the digestion is dif„cult and causes a low cloning ef„ciency and the loss of signi„cant sequence information corresponding to the 5′ end of the mRNA.2 To solve this problem, 4 mM sodium pyrophosphate is utilized to suppress the formation of hairpins during the synthesis of the „rst-stranded cDNA. The second strand cDNA is then produced by RNase H to create nicks and gaps in the hybridized mRNA template, generating 3′-OH priming sites for DNA synthesis using DNA polymerase I. After treatment with T4 DNA polymerase to remove any remaining 3′ protruding ends, the blunt-ended, double-stranded cDNAs are ready for ligation with adaptors or linkers. Therefore, SI digestion is eliminated and the cloning ef„ciency is much higher than that obtained with the classic method. Besides, the sequence information is optimally reserved.
