Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction.
In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
D
D
Riesner
G
G
Steger
U
U
Wiese
M
M
Wulfert
M
M
Heibey
K
K
Henco
D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D018620:PrPSc Proteins; D017096:Prion Diseases / Q000235:genetics; D011328:Prions / Q000235:genetics; D013696:Temperature
D009419:Nerve Tissue Proteins; D018620:PrPSc Proteins; D011328:Prions; D004247:DNA