Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage

✍ Scribed by Shelby L. Berger

• Book ID: 102988643
• Publisher: Elsevier Science
• Year: 1987
• Tongue: English
• Weight: 906 KB
• Volume: 161
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(87)90451-9

✦ Synopsis

A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H. Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied. Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage. Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment. When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA. The sum of the fragment lengths equaled the length of the mRNA. Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA. With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones.