Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. II. End-labeling and sequencing.
T4 polynucleotide kinase has been used to end-label specific RNA purified by multiple hybridizations to nitrocellulose-bound DNA. The pico moles of ends of a specific mRNA transcribed from the chromosome, even from several liters of Escherichia coli, give concentrations perhaps 2000-fold below the Km value of the kinase-RNA substrate. In such a reaction, optimal incorporation was observed with increasing ATP concentration to greater than or equal to 7 microM (greater than or equal to 15 mCi of carrier-free [32P]ATP in a 300-500 microliter reaction). The unreacted ATP (greater than 150-fold excess) could best be eliminated by multiple gel filtrations rather than by precipitation, ion exchange chromatography or dialysis. The [5'-32P]RNA was digested with T1 or pancreatic RNase and the [5'-32P]oligonucleotides separated by size in a 20% polyacrylamide gel. Oligonucleotides of a specific size were separated sufficiently by a second dimension electrophoresis on cellulose acetate. We have used partial alkali digestion in sequencing the purified oligonucleotides. As opposed to other digestions, alkali produces 5',3'-diphospho-oligonucleotides whose mobilities can differ from those of the monophosphates, e.g., much longer running times in conventional homochromatography.
V J
VJ
Cannistraro
B M
BM
Wice
D E
DE
Kennell
D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003101:Collodion; D004926:Escherichia coli / Q000235:genetics*; D005374:Filtration; D008722:Methods; D012329:RNA, Bacterial / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D003101:Collodion; D000469:Alkaline Phosphatase