Determination of 5-methylcytosine in DNA by gas chromatography-electron-capture detection.
The following sequence of analytical steps was used to determine the amount of 5-methylcytosine (mol-%) in calf thymus and human lymphocyte DNA:acid hydrolysis of the DNA, derivatization (pentafluorobenzyl bromide, solid phase extraction, pivalic anhydride), internal standard addition, solid phase extraction, high-performance liquid chromatography, and gas chromatography with electron-capture detection. The steps were carefully optimized, leading to a recovery of 30 +/- 1.0% starting with a nucleobase standard containing 1.25 ng of 5-methylcytosine. A second analysis of this sample gave a 30 +/- 0.3%, demonstrating a high precision for the method. In good agreement with earlier work by others, 1.2 +/- 0.10 mol-% of 5-methylcytosine was then found in a 350 ng sample of calf thymus DNA, and values of 0.9 +/- 0.07 and 0.8 +/- 0.04 mol-% (two runs) were found in hyman lymphocyte DNA.
Fisher
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DH
Department of Medical Laboratory Science, College of Pharmacy and Allied Health Professions, Northeastern University, Boston, MA 02115.
Giese
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RW
D044503:5-Methylcytosine; D000215:Acylation; D000478:Alkylation; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000032:analysis*; D003902:Detergents; D004563:Electrochemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D012822:Silicon Dioxide; D013056:Spectrophotometry, Ultraviolet
D003902:Detergents; D007202:Indicators and Reagents; D044503:5-Methylcytosine; D012822:Silicon Dioxide; D003596:Cytosine; D004247:DNA