Rapid in vitro labeling procedures for two-dimensional gel fingerprinting

Journal Article · Fri Jan 15 00:00:00 EST 1982 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(82)90690-X· OSTI ID:5532437

Lee, Y F; Fowlks, E R

Improvements of existing in vitro procedures for labeling RNA radioactively, and modifications of the two-dimensional polyacrylamide gel electrophoresis system for making RNA fingerprints are described. These improvements are (a) inactivation of phosphatase with nitric acid at pH 2.0 eliminated the phenol-cholorform extraction step during 5'-end labeling with polynucleotide kinase and (..gamma..-/sup 32/P)ATP; (b) ZnSO/sub 4/ inactivation of R Nase T/sub 1/ results in a highly efficient procedure for 3'-end labeling with T4 ligase and (5'-/sup 32/P)pCp; and (c) a rapid 4-min procedure for variable quantity range of /sup 125/I and RNA results in a qualitative and quantitative sample for high-molecular weight RNA fingerprinting. Thus, these in vitro procedures become rapid and reproducible when combined with two-dimensional gel electrophoresis which eliminates simultaneously labeled impurities. Each labeling procedure is compared, using tobacco mosaic virus, Brome mosaic virus, and polio RNA. A series of Ap-rich oligonucleotides was discovered in the inner genome of Brome mosaic Virus RNA-3.

• Research Organization: Bishop College, Dallas, TX
• OSTI ID: 5532437
• Journal Information: Anal. Biochem.; (United States), Vol. 119:2
• Country of Publication: United States
• Language: English

Related Subjects

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
PHOSPHATASES
INACTIVATION
RNA
LABELLING
RNA-ASE
AMIDES
ELECTROPHORESIS
GELS
IN VITRO
ORGANIC POLYMERS
PATTERN RECOGNITION
PH VALUE
PHOSPHORUS 32
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
COLLOIDS
DAYS LIVING RADIOISOTOPES
DISPERSIONS
ENZYMES
ESTERASES
HYDROLASES
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS ISOTOPES
POLYMERS
RADIOISOTOPES
400702* - Radiochemistry & Nuclear Chemistry- Properties of Radioactive Materials