CZ2016837A3 - A device for amplification of DNA by polymerase chain reaction in a miniature reactor - Google Patents

Abstract

An apparatus that allows thermal cycling of the PCR reaction mixture by means of a thermostatic element immersed in the mixture, and wherein at the same time evaporation is reduced by placing the reaction vessel upside down. The device is suitable for performing PCR amplification in miniature containers, for example in microfluid analyzer input ports, where the amplified product can be immediately analyzed without the need for a pipetting step.

Description

Technical field

The present invention relates to the field of molecular genetics and diagnostics using a polymerase chain reaction to amplify DNA fragments.

BACKGROUND OF THE INVENTION

The polymerase chain reaction (PCR) method is one of the basic techniques used in molecular biology, genetics and related fields. PCR is used to artificially amplify a specific region based on the artificial in vitro DNA replication process for further processing or detection. The basic principle is based on repeated synthesis of complementary strands of single-stranded DNA fragments resulting from denaturation of the double-stranded product from the previous reaction step. In the PCR reaction, the steps of (i) denaturation, (ii) initiation (annealing of short single-stranded DNAs, so-called primers), and (iii) self-synthesis (extension of the complementary strand) are cyclically repeated. Buffered mixture of reaction components containing, in addition to template for copying (template DNA) mainly mixture of building units of synthesized fragments (G-, Τ-, A-, C- deoxyribonucleotides) and enzyme PCR polymerase is heated to 3 different temperatures during PCR reaction

for 60 seconds (denaturation), 54rC for 30 seconds (primer annealing)

CM (1) (chain extension) and these temperatures are alternated sequentially over a typically 20 to 25 cycle.

In the most widespread configuration, the PCR reaction takes place in a reaction vessel, typically in a volume of 5 gL to 20 μι, inside a PCR cycler in which the vessel is placed in a thermostatic block that is heated and cooled from the outside. The rate of heat transfer between the block and the reaction mixture and especially the heat transfer through the wall of the reaction vessel is often a limiting factor for increasing the rate of PCR reaction. Upon completion of amplification, the product (amplified DNA fragment) is removed from the vial to verify proper reaction or further processing or detection. For example, a frequent procedure is to visualize the PCR amplification by applying a portion of the volume of the reaction product to an agarose gel followed by electrophoretic separation. Recently, instead of classical gel electrophoresis, dedicated devices have been used, where electrophoretic separation takes place ρώ in the microfluidic chip format, such as the Bioanalyzer 1200 system from Agilent Technologies or the Experion system from Bio-Rad Laboratories ² .

SUMMARY OF THE INVENTION

In the solution proposed here, instead of the reaction vessel heated and cooled by external contact with the thermostatic block, a thermostatic element is used which is immersed in the reaction vessel and converts the heating or cooling inside the reaction mixture from the external temperature cycler as shown in Figure 1. is that it allows thermal cycling inside any geometric shapes of the reaction vessel, which can be used for example to perform PCR reactions in miniature vessels or inlet ports of microfluidic analyzers, etc. In the case of small dimensions of the reaction vessel (up to approx. 20 pL) at the same time place the reaction vessel upside down (due to surface tension the liquid remains in the vessel) and thereby reduce the rate of evaporation of the heated mixture.

DETAILED DESCRIPTION OF THE INVENTION

One example of the use of the proposed device is to perform PCR amplification directly in the input port of the Bioanalyzer 1200 Microchip Analyzer from Agilent Biotechnologies. The principle of the device of Figure 1 has been adapted so that a microfluidic chip used in the Bioanalyzer system can be inserted from one side of the device (Figure 2). At the same time, the bottom of the device was fitted with conical columns of heat conducting material (copper), which fit exactly into a 96-well block of a conventional PCR thermocycler into which the device was inserted as shown in Figure 2. The PCR cycler was then set to temperature program and running cycling is carried out by the proposed device for amplification directly in the input port of the microchip. After the program has finished, the chip containing the amplified product can be removed from the device and inserted directly into the microchip analyzer without the need for another pipetting intermediate step. Thanks to the block dimensions for standard 96-well microtiter plates, the proposed device can be used for amplification in 2 microfluidic chips at a time.

¹ TC Lorenz, Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies, J. Vis.

Exp. JoVE. (2012) e3998. doi: 10.3791 / 3998.

² Dolnik, V., Liu, S., Applications of capillary electrophoresis on microchip. J. Sep. Sci. 2005, 28,1994-2009.

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Claims (5)

A system for amplifying nucleotide sequences based on a polymerase chain reaction, characterized in that a thermostatic element is immersed in the reaction vessel, which heats and cools the reaction mixture in terms of a thermocyclic amplification reaction. 2. The system described in claim 1, wherein the reaction vessel is a dosing compartment of a microfluidic separation device. 3. The system described in Jaoda I and II. characterized in that the thermostatic element is separated from the direct contact with the reaction mixture by an impermeable heat-conducting barrier. 4. The system described in points I to III. characterized in that the reaction vessel is turned upside down and the thermostatic element is immersed in the reaction mixture from below so as to suppress evaporation. 5. The system described in points I to III. characterized in that the thermostatic element is used as a reaction stirrer,