Method for detecting DNA point mutations (single nucleotide polymorphism (SNP) analysis) and associated arrangement
The single nucleotide polymorphism analysis involves the utilization of a DNA hybridization process as well as the use of a DNA chip. A liquid DNA sample to be analyzed is guided over a DNA chip in a defined time course. After successful hybridization, the temperature is modified in a defined manner under low stringency conditions such that scavenger/target DNA hybrids are melted, whereby the melting of the scavenger/target DNA hybrids is detected and evaluated according to the temperature. In addition to the DNA chip, at least one device is provided that controls and regulates the temperature, and another device is provided that controls a lateral flow of liquid against the surface of the DNA chip. Factors for matching hybrids and mismatching/single point mismatching hybrids can be detected and evaluated using appropriate measuring device(s).
1. A method for detecting DNA point mutations utilizing capture/target DNA hybrids at hybridization positions, the capture/target DNA hybrids formed by binding target DNA to be detected to capture DNA immobilized position-specifically on a DNA chip, the method comprising,
performing a washing phase and a measurement phase alternately at different temperatures,
the washing phase including:
a) passing a washing liquid at a controlled flow rate over the DNA chip,
b) changing the temperature at the hybridization positions in a defined manner as a function of time, with a continuous increase in temperature as a function of time being carried out in ramps with subsequent temperature holding times, and
c) melting the capture/target DNA hybrids temperature-dependently, and removing melted target DNA from the hybridization positions using the flowing washing liquid, such that non-melted target DNA remains on the DNA chip,
the measurement phase including:
d) stopping the flow of washing liquid during the temperature holding times,
e) position-specifically detecting the non-melted target DNA at a current temperature of the temperature holding time, and
f) analyzing signals according to a predetermined program, the analyzed signals being used to detect the DNA point mutations.
2. The method as claimed in claim 1 , wherein method steps a) to e) are repeated more than once.
3. The method as claimed in claim 1 , wherein the detection of the non-melted target DNA takes place label-free.
4. The method as claimed in claim 3 , wherein the label-free detection takes place optically.
5. The method as claimed in claim 4 , wherein the optical detection makes use of an intrinsic change in the UV absorption based on melting of DNA double strands.
6. The method as claimed in claim 3 , wherein label-free detection takes place electrically.
7. The method as claimed in claim 6 , wherein the label-free detection takes place by way of an intrinsic guanine oxidation process.
8. The method as claimed in claim 7 , wherein the label-free detection takes place by way of electrochemical impedance methods.
9. The method as claimed in claim 7 , wherein the label-free detection takes place gravimetrically.
10. The method as claimed in claim 1 , wherein the detection of the non-melted target DNA takes place with use of a label.
11. The method as claimed in claim 10 , wherein the detection takes place by use of an optical label.
12. The method as claimed in claim 10 , wherein the detection takes place by use of a magnetic label.
13. The method as claimed claim 10 , wherein the detection takes place by use of an enzymatic label.
14. The method as claimed in claim 13 , wherein the washing liquid includes enzyme substrate.
15. The method as claimed in claim 14 , wherein an active enzyme, which is stable in the temperature range used, is used.
16. The method as claimed in claim 13 , wherein the enzymatic label catalyzes an optically detectable reaction.
17. The method as claimed in claim 13 , wherein the enzymatic label catalyzes an electrochemically detectable reaction.
18. The method as claimed in claim 17 , wherein the electrochemical detectable reaction is detectable using a redox cycling-amplified current measurement.
19. The method as claimed in claim 10 , wherein a change in a concentration of at least one of a substrate and a product is detected and analyzed.
20. The method as claimed in 1 , wherein the analysis and representation of the signal takes place as a function of temperature.
21. The method as claimed in claim 20 , wherein a melting curve is standardized to a defined temperature in the analysis.
22. The method as claimed in claim 1 , wherein the analysis is computer-controlled.
23. The method as claimed in claim 22 , wherein the analysis is carried out by software.