Open AccessThe use of nuclease P1in sequence analysis of end group labeled RNA
Author(s) -
Melvin Silberklang,
Amanda M. Gillum,
Uttam L. RajBhandary
Publication year - 1977
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/4.12.4091
Subject(s) - biology , rna , nuclease protection assay , rnase p , oligonucleotide , ribonuclease t1 , nuclease , microbiology and biotechnology , rnase h , nucleotide , sequence analysis , biochemistry , sequence (biology) , non coding rna , messenger rna , digestion (alchemy) , transfer rna , dna , gene , chemistry , chromatography
A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.