Determination of DNA mutation load in human tissues using denaturing HPLC-based heteroduplex analysis.
Since its introduction more than a decade ago, denaturing HPLC has been widely used to screen nuclear and mitochondrial DNA for mutations. We recently developed a quantitative method based on denaturing HPLC to measure DNA mutation load, using tRNA Leu(UUR) region of the mitochondrial DNA as an example. The mutation load is determined based on the quadratic function that governs DNA reannealing and the formation of heteroduplex and homoduplex DNAs. We have used this assay to measure heteroplasmy level for several mitochondrial mutations in DNAs obtained from human tissue samples. This quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.
Lim
Kok Seong
KS
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Naviaux
Robert K
RK
Haas
Richard H
RH
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000235:genetics*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D008928:Mitochondria / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics
D004272:DNA, Mitochondrial; D012346:RNA, Transfer, Amino Acyl