A general method for detection and characterization of an mRNA using an oligonucleotide probe.
A general method for the detection and characterization of an mRNA using an oligodeoxynucleotide probe is described. The results presented indicate that a G-dT or a dG-U base pair within a short DNA-RNA hybrid does not significantly reduce the stability of the hybrid. On this basis, we propose that 11 amino acids, including Trp and Met, can be used in selecting a peptide sequence suitable for use in designing an oligodeoxynucleotide probe complementary to a given mRNA. To test this hypothesis, we have synthesized an oligodeoxynucleotide (oligo II) complementary to the region of gastrin mRNA coding for Trp-Met-Asp-Phe and have compared its effectiveness as a hybridization probe and as a primer for the synthesis of gastrin-specific cDNA with another oligonucleotide (oligo I) complementary to the region of gastrin mRNA coding for Trp-Met-Glu-Glu. Unlike oligo I, oligo II functions as a primer in specific cDNA synthesis only when the mRNA is denatured prior to initiation of synthesis. Similarly, oligo II can be used as a specific hybridization probe for gastrin mRNA only when the RNA is denatured and partially cleaved with NaOH before hybridization. A simple procedure for denaturing gastrin mRNA to ensure efficient interaction with oligodeoxynucleotide probes is described. This procedure should be useful in studies with other oligonucleotides and mRNAs as well.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D005753:Gastric Mucosa / Q000378:metabolism; D005755:Gastrins / Q000235:genetics; D008722:Methods; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D013329:Structure-Activity Relationship; D013552:Swine
D005755:Gastrins; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase