Identification of modified bases and their locations in DNA
I. The locations of modified and common bases in an oligodeoxyribonucleotide may be determined by a simple two-dimensional separation on a polyethyleneimine-cellulose thin layer sheet. Chromatography in the first dimension fractionates by chain length a nested set of fragments that are generated by subjecting the oligomer to partial spleen phosphodiesterase degradation followed by labelling their non-common ends with /sup 32/P using polynucleotide kinase. A subsequent in situ treatment with nuclease Bal 31 produces labelled mononucleotides, and these are identified by chromatography in the second dimension. Since the method does not identify the 3' terminal nucleotide, a convenient procedure involving 3' end labelling followed by enzymatic digestion to monomers has been developed for this purpose. II. A two-dimensional method which can be used to conjunction with a standard sequencing technique to identify the positions of modified bases in polydeoxyribonucleotides is described. In the first dimension, a set of DNA fragments is separated by length, and in the second dimension, the 5' termini are identified. Autoradiography then shows a pattern of spots corresponding to the nucleotides, including those with modified bases, at each position of the polynucleotide chain. In case the resolution of the spots after the two-dimensional separation does not allow precise location of a particular base, comparison of the autoradiograph with the sequence as determined by a standard technique allows the location to be determined without ambiguity.
- Research Organization:
- Purdue Univ., Lafayette, IN (USA)
- OSTI ID:
- 5402170
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
OLIGONUCLEOTIDES
DNA SEQUENCING
PHOSPHORUS 32
TRACER TECHNIQUES
AUTORADIOGRAPHY
DNA
PHOSPHOTRANSFERASES
THIN-LAYER CHROMATOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
RADIOISOTOPES
SEPARATION PROCESSES
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques