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PMID: 7003544 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA.

Nucleic acids research ·Vol. 8 ·No. 20 ·1980-10-24 ·Pages 4763-76

Kuo KC, McCune RA, Gehrke CW, Midgett R, Ehrlich M

Abstract

We have developed a method to accurately determine (< 3% RSD) the complete major and modified base composition of a few micrograms of unlabeled DNA. The DNA samples were quantitatively hydrolyzed with DNase 1, Nuclease P1, and bacterial alkaline phosphatase. The resulting deoxyribonucleosides were directly separated in 70 min by reversed-phase high performance liquid chromatography with detection by ultraviolet absorption at 254 nm and 280 nm (RP-HPLC). The highly sensitive and selective dual wavelength quantitation greatly enhances the precision and accuracy of the chromatographic analysis. Contamination of DNA preparations with RNA does not interfere with the DNA analysis due to the high resolution of the chromatography. We have used this method for the quantitation of m5dCyd in 5 microgram of calf thymus and salmon sperm DNA in which the m5dCyd comprises only 1 to 2% of the total bases. This method should be a useful research tool in studies on various DNAs and DNA subfractions and should help to elucidate the functions of methylation of DNA.

MeSH Terms
Alkaline Phosphatase Animals Cattle Chromatography, High Pressure Liquid/methods DNA Deoxyribonucleases Deoxyribonucleosides/analysis Escherichia coli/analysis Male Microchemistry Salmon Spermatozoa Thymus Gland
Chemicals
Deoxyribonucleosides DNA Deoxyribonucleases Alkaline Phosphatase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Kuo K C
McCune R A
Gehrke C W
Midgett R
Ehrlich M
References (34)
34 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/8.20.4763
Published
1980-10-24
Pages
4763-76
Language
English
Region
England
NLM ID
0411011
PMCID
PMC324386
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/8.20.4763 PMC Full Text View journal details
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