WO2001081619A2 - Conductivity pcr - Google Patents

Abstract

The invention is based on the concept of using the change in conductivity to be measured during the PCR as a marker for the progression of the PCR and for the quantification of the starting quantity of DNA. This apparatus determines the changes in conductivity "online" during the course of a PCR by means of small microelectrodes (4) which are immersed in the PCR solution. Using this parameter, the apparatus is able to calculate the quantity of DNA or RNA before the conclusion of the PCR.

Description

Description:

CONDUCTIVITY PCR (conductivity PCR)

An important question in molecular biology is that of the activity of individual genes. To determine individual genes in their activity, a PCR (polymerase chain reaction) or an RT-PCR (reverse transcriptase polymerase chain reaction) is often carried out. One difficulty is that the PCR reaction is difficult to quantify, that is, the amount of DNA / RNA before the PCR reaction is complete can only be estimated.

Previously customary "online" PCR apparatus either stained the DNA formed in the course of the PCR or used fluorescent-labeled oligonucleotide primers to quantify the course of the PCR. A complex optical system for measuring the respective fluorescence was required.

This invention is based on the consideration of using the amount of phosphate which arises when the PCR reaction proceeds and the associated changes in conductivity as markers for the amplification factor of the PCR reaction. During the course of a PCR reaction, this apparatus determines the phosphate increase “online” by means of small microelectrodes which are immersed in the PCR solution. Because of this parameter, the apparatus is able to measure the amount of DNA or RNA before the PCR reaction The amount of phosphate is selected as a marker for detection on the basis of its good specific electrical conductivity and on the basis of its rise in the course of the annealing and extension phase of the PCR, and a microprocessor can then determine the phosphate concentration after the end using a previously determined calibration curve and the determined measured values of the PCR reaction, the amplification factor of the PCR reaction and the DNA or RNA concentration before the start of the PCR reaction, using the time course of the increase in conductivity as a function of the number of cycles and the temperature to determine the original concentration of DNA ,

An advantage of this invention is that an electronically usable measure is used directly with the conductivity. The amount of phosphate is also a particularly sensitive parameter. The conductivity changes within each cycle depending on the temperature in accordance with the three phases (annealing, extension, denaturation). Overall, the conductivity decreases in the course of the PCR. In the annealing phase, the conductivity decreases relatively strongly. In the first approximately ten cycles, the conductivity increases linearly ("linear phase") up to the "trashhold cycle". This cycle is particularly suitable for quantifying the polymerase chain reaction. The exponential phase begins from the Trashhold cycle. This is characterized by a total decrease in conductivity. Nevertheless, a phase-wise increase in conductivity remains during the denaturation phase and the extension phase. The changes in conductivity of the individual phases are also suitable for PCR quantification.

Possibly many mononucleotides and many magnesium ions and other ions bind to the DNA at the annealing temperature. With the beginning of the extension phase, the conductivity increases continuously until the end of the extension phase. This increase is thought to be caused by the release of phosphate. And must be measured. The denaturation follows the elongation phase, here the conductivity increases to a maximum. Presumably, all ions dissociate in this phase. Explanation based on an embodiment: drawing

1) Thermal cycler

2) PCR reaction tube

3) PCR reaction solution

4) Microelectrodes for conductivity measurement

5) Microprocessor for registering the conductivity measurements

6) contacts

Claims

Conductivity PCR Patent Claims Main Independent Claim: 1.) An apparatus for carrying out a PCR reaction (polymerase chain reaction) in a so-called "thermal cycler", characterized in that this apparatus "online" during the course of a PCR reaction by means of small microelectrodes immersed in the PCR solution the change in the electrical The conductivity of the solution is monitored continuously or discontinuously (for example only during the annealing / extension phase or only during the first cycles). Dependent subsidiary claims: 2.) A thermal cycler according to protection claim 1), characterized in that this thermal cycler uses the transition from the linear increase in conductivity to the first decrease in conductivity as a trash-hold cycle for quantification. 3.) reaction vessels for carrying out a polymerase chain reaction, characterized in that microelectrodes are integrated in the reaction vessels used. 4.) A multi well plate for carrying out a polymerase chain reaction, characterized in that microelectrodes are integrated in the individual wells of the multi well plate. 5.) A thermal cycler according to protection claims 1-4, additionally characterized in that in addition to the electrical conductivity, the current temperature in the Reaction solution is measured that the various known heating / cooling options (e.g. air heating / cooling or Peltier elements) are used and the contacts for using the reaction vessels Protection claims 3 and 4 are available. 6.) An electrical apparatus, characterized in that it registers and sensibly combines the parameters measured in the course of the polymerase chain reaction (these are the electrical conductivity, temperature, number of cycles and time). 7.) A thermal cycler according to protection claims 1-4 with the possibility of a final melting curve analysis, additionally characterized in that the electrical conductivity is measured during the melting curve analysis.