6533b824fe1ef96bd1281506
RESEARCH PRODUCT
Assay for O6-alkylguanine-DNA-alkyltransferase using oligonucleotides containing O6-methylguanine in a BamHI recognition site as substrate
Franz OeschS Kleinsubject
Molecular Sequence DataOligonucleotidesBiophysicsBiologyCleavage (embryo)Sensitivity and SpecificityBiochemistryHigh-performance liquid chromatographyO(6)-Methylguanine-DNA Methyltransferasechemistry.chemical_compoundHumansLymphocytesMolecular BiologyChromatography High Pressure LiquidBase SequenceOligonucleotideSubstrate (chemistry)MethyltransferasesCell BiologyMolecular biologyPeptide FragmentsRestriction siteRestriction enzymeBiochemistrychemistryBamHIPhosphorus RadioisotopesDNAdescription
Abstract Double-stranded oligonucleotides, 40 bases in length containing an O 6 -methylguanine in a Bam HI restriction site, were developed as substrates for the determination of human O 6 -alkylguanine-DNA-alkyltransferase (AGT). The assay proved highly sensitive and quantitative. After incubation of the 5′-end-labeled oligonucleotides with cell homogenates of peripheral blood lymphocytes, the DNA was digested with Bam HI. Cleavage with this restriction enzyme did not occur in the O 6 -methylguanine-containing oligonucleotide unless the fragment was repaired. The cleaved oligonucleotide was separated from the intact parent oligonucleotide by reverse-phase high-performance liquid chromatography. Calculation of the AGT content was achieved by integrating the radioactivity of the peak corresponding to the digested fragment, which is equal to the molar amount of repaired oligonucleotide and corresponds directly to the molar AGT content in the lymphocyte homogenate.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 1992-09-01 | Analytical Biochemistry |