A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments
- 1 November 1976
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 3 (11) , 2895-2914
- https://doi.org/10.1093/nar/3.11.2895
Abstract
A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3′-terminally (3H)- labeled oligonucleotide-(3′) dialcohols and 5′-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5′-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5′) monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 – 0.3 A260 unit of tRNA is needed to sequence a11 fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1–4 required 4–6 A260 units.Keywords
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