Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis.
The ability to detect DNA sequence heterogeneity quickly and reliably is becoming increasingly important as more genes involved in disease processes are discovered. We have assessed the ability of a high pressure liquid chromatography technique (HPLC) termed temperature-modulated heteroduplex analysis (TMHA) to detect a collection of 20 point mutations distributed throughout a 279 base pair fragment spanning the exon 8 region of the human HPRT gene. All mutant/wild type heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved from their parental homoduplexes. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA and DGGE are measuring similar melting characteristics in heteroduplex molecules. TMHA appears to be a robust approach for detecting and/or purifying a wide variety of mutations in a defined region of DNA, provided that the melting characteristics of the fragment under study are carefully considered.
Skopek
T R
TR
Genetic and Cellular Toxicology Department, Merck Research Laboratories, West Point, PA 19486, USA. thomas_skopek@merck.com
Glaab
W E
WE
Monroe
J J
JJ
Kort
K L
KL
Schaefer
W
W
D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry*; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors
D004273:DNA, Neoplasm; D009692:Nucleic Acid Heteroduplexes; D007041:Hypoxanthine Phosphoribosyltransferase