TW201311886A - Temperature setting method of polymerase chain reaction - Google Patents

Abstract

A temperature setting method of polymerase chain reaction comprises a preparation step, a temperature-increasing step, and a chain reaction step. In the preparation step, a reaction test tube loaded with a reaction sample is disposed onto a heating base, a main heating member in the heating base is disposed corresponding to the bottom of the reaction test tube, and an auxiliary heating member is corresponded to the middle section of the reaction test tube. The temperature-heating step is to heat the main heating member to the denature temperature and heat the auxiliary heating member to an auxiliary heating temperature to carry out afterwards the chain reaction, wherein the auxiliary heating temperature is smaller than the denature temperature. The present invention utilizes the main heating member and the auxiliary heating member to separately achieve the heating and stabilization of the temperature of each part of the reaction test tube, thereby capable of increasing the chain reaction speed.

Description

Temperature setting method for polymerase chain reaction

The present invention relates to a polymerase chain reaction, and more particularly to a temperature setting method for a polymerase chain reaction.

The amplification of specific nucleic acid sequences by Polymerase Chain Reaction (PCR) is a well-established technology. It is an important technology in medicine and biotechnology. The reaction process has three main stages: "Denaturation". "Primer Annealing" and "Extension", in which the reaction temperatures required for these three stages are different. In today's commercial PCR equipment, the desired reaction sample contains the template DNA to be amplified, an oligonucleotide primer pair complementary to a specific sequence on each strand of the template DNA, a thermostable DNA polymerase, and a deoxynucleoside triphosphate ( dNTP). The PCR device circulates the reaction sample at three different temperatures by repeatedly heating and cooling the reaction sample, thereby amplifying a specific portion of the template DNA nucleic acid sequence.

The first step of the PCR is a denaturation reaction, which is to heat the reaction sample to a high temperature to separate the double-stranded template DNA into a single strand of DNA, typically at a temperature ranging from 90 ° C to 95 ° C.

The second step of PCR is the primer binding reaction, which first cools the reaction sample separated into single strand DNA to a lower temperature, so that the primer binds to the single strand DNA formed in the first step to form a complex of DNA and primer. The temperature of the primer, typically the primer binding reaction, is selected depending on the melting temperature (Tm) of the primer used, and is usually in the range of 35 ° C to 65 ° C.

The third step of PCR is the extension reaction, which is to maintain the reaction sample of the complex forming the DNA and the primer at an appropriate temperature, and the primer is extended by the action of the DNA polymerase to form a new single sheet complementary to the template DNA. The temperature of the strand DNA, typical polymerization, was 72 °C.

Therefore, each cycle consisting of the above three stages can replicate twice the template DNA, and the PCR cycle including the denaturation reaction, the primer binding reaction, and the extension reaction can be repeated for about 20 to 40 times, and can be produced. A replica of millions of target nucleic acid sequences.

Please refer to "Figure 1" and "Figure 2". Another cost-effective technology is thermal convection PCR (CPCR). The reaction sample of conventional thermal convection PCR is filled into a circular capillary 1, a plurality of The capillary tube 1 is neatly arranged on a test tube holder 2 equipped with a heater (not shown) and a cooling device (not shown), wherein the reaction sample driven by the heat convection is acted upon by the heater and the cooling device. A circulating flow is formed in the capillary 1. Therefore, each region of the capillary 1 has a different temperature, and when the reaction sample circulates between the regions, the three steps of the PCR can occur sequentially and repeatedly. However, the heater is only placed at one end of the capillary 1 to be heated and heated, and other parts of the capillary 1 cannot be effectively controlled for the temperature of the primer binding reaction and the extension reaction due to the difference in air convection conditions and the external temperature change at that time. The temperature that causes the chain reaction of the polymerase cannot be effectively raised.

The main object of the present invention is to solve the problem that the PCR reaction in the thermal convection mode is degraded due to insufficient temperature stability of various portions of the capillary.

To achieve the above object, the present invention provides a temperature setting method for a polymerase chain reaction comprising the following steps:

a preparation step of placing a reaction sample into a reaction tube, and fixing one end of the reaction tube to a heating seat, so that a position of a main heating element in the heating seat corresponds to a bottom portion of the reaction tube, and An auxiliary heating member is spaced apart from the main heating member to correspond to an intermediate portion of the reaction tube;

a heating step of raising the temperature of the main heating member and heating to a denaturation temperature, and heating the auxiliary heating member to an auxiliary heating temperature, the auxiliary heating temperature being less than the denaturation temperature;

A chain reaction step of controlling the primary heating element to maintain the denaturation temperature for a chain reaction.

It can be seen from the above description that the heating step is used to heat the main heating element and the auxiliary heating element to the denaturation temperature and the auxiliary heating temperature, respectively, and the temperature of the bottom portion and the middle portion of the reaction tube can be respectively controlled, thereby making The reaction sample in the reaction tube is subjected to a denaturation reaction, a primer adhesion reaction, and an extension reaction in a stable temperature control to increase the rate of the polymerase chain reaction.

The detailed description and technical contents of the present invention will now be described as follows:

Please refer to FIG. 3, FIG. 4 and FIG. 5: the present invention is a temperature setting method for a polymerase chain reaction, but before describing the setting method of the present invention, the method using the present invention will be described first. The mechanism comprises a base 10, a heating base 20 connected to the base 10, an auxiliary heating element 30 connected to the heating base 20, at least one reaction tube 40, and a reaction tube 40 fixed to the auxiliary heating element. A fixed connector 50 on the 30, and an observation module 60. The base 10 includes a light source 11 (in this embodiment, a light-emitting diode) for illuminating a reaction sample containing a fluorescent dye contained in the reaction tube 40, and reacting the fluorescent dye. Light is sent to the observation module 60 for subsequent analysis.

The temperature setting method of the present invention comprises the following steps:

S1: a preparation step, the reaction sample is placed in the reaction tube 40, and one end of the reaction tube 40 is fixed on the heating block 20, and the position of the main heating member 21 corresponds to a bottom portion 41 of the reaction tube 40. And the auxiliary heating member 30 is spaced apart from the main heating member 21 to correspond to an intermediate portion 42 of the reaction tube 40. The auxiliary heating member 30 is made of aluminum in the embodiment, and is permeable to aluminum. The heating temperature of the auxiliary heating member 30 is controlled by a temperature control device 70 connected to the auxiliary heating member 30, wherein the auxiliary heating member 30 is not connected to the reaction tube 40, only by controlling the reaction. The temperature of the intermediate section 42 of the reaction tube 40 is stabilized by the ambient temperature of the tube 40. In order to enable the heat of the main heating member 21 to be more accurately transmitted to the reaction tube 40, a high heat conducting member 80 can be wound around the bottom portion 41 of the reaction tube 40, and the reaction tube 40 can be passed through the high heat conducting member. 80 is in contact with the main heating member 21.

In order to avoid the interference of the external temperature, please refer to the "FIG. 6", and the preparation step further includes a step S1A: the reaction tube 40, the heating base 20 and the auxiliary heating element 30 are disposed in a closed casing. Within 90.

S2: a reverse transcription step, please refer to "Figure 7", which shows a main heating element temperature curve 101 and an auxiliary heating element temperature curve 102, respectively, when the reaction sample is a ribonucleic acid ( This step is required only for the Ribonucleic Acid, RNA) sample. If the reaction sample is already a Deoxyribonucleic Acid (DNA) sample, this step can be skipped for the next stage. This step is to maintain the primary heating element 21 at a reverse transcription stage t1 at an initial temperature and convert the ribonucleic acid sample to a DNA sample having a starting temperature between 45 ° C and 55 ° C.

S3: a heating step, in a warming phase t2, raising the temperature of the main heating member 21 and heating to a denaturation temperature, such as the main heating member temperature curve 101 in "FIG. 7", and making the auxiliary heating member 30 is heated to an auxiliary heating temperature, such as the auxiliary heating element temperature curve 102 in "FIG. 7", the auxiliary heating temperature is less than the denaturation temperature, and more precisely, the denaturation temperature is between 90 ° C and 98 ° C. The auxiliary heating temperature is between 40 ° C and 50 ° C.

S4: a chain reaction step, in the chain reaction stage t3, controlling the main heating element 21 to maintain the denaturation temperature to facilitate the chain reaction, and the auxiliary heating element 30 is maintained at the auxiliary heating temperature, and A good reaction effect, that is, the auxiliary heating member 30 has a temperature rising action in the step S3, and when the chain reaction is performed, the temperature is maintained to be constant to facilitate the chain reaction. On the other hand, please refer to the main heating element temperature curve 101 and the auxiliary heating element temperature curve 102 shown in FIG. 8 because the auxiliary heating element 30 is higher than the preferred temperature range in the heating step of step S3. (40 ° C ~ 50 ° C), so the fan can be used to cool down, the temperature of the auxiliary heating element 30 is controlled between 40 ° C ~ 50 ° C, to avoid a reduction in the reaction rate. Auxiliary heating temperature

In summary, since the heating step 21 is used to heat the main heating member 21 and the auxiliary heating member 30 to the denaturation temperature and the auxiliary heating temperature, the bottom portion 41 and the intermediate portion 42 of the reaction tube 40 can be separately controlled. The temperature, whereby the reaction sample in the reaction tube 40 is subjected to a denaturation reaction, a primer adhesion reaction, and an extension reaction in a stable temperature control to increase the rate of the polymerase chain reaction. Therefore, the present invention is highly progressive and conforms to the requirements of the invention patent application, and the application is filed according to law, and the praying office grants the patent as soon as possible.

The present invention has been described in detail above, but the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the scope of the invention. That is, the equivalent changes and modifications made by the scope of the present application should remain within the scope of the patent of the present invention.

Conventional technology:

1. . . Capillary

2. . . Test tube holder

this invention

10. . . Base

11. . . light source

20. . . Heating seat

twenty one. . . Main heating element

30. . . Auxiliary heating element

40. . . Reaction tube

41. . . bottom

42. . . Middle section

50. . . Fixed connector

60. . . Observation module

70. . . Temperature control device

80. . . High thermal conductivity

90. . . Closed housing

101. . . Main heating element temperature curve

102. . . Auxiliary heating element temperature curve

T1. . . Reverse transcription stage

T2. . . Heating stage

T3. . . Chain reaction phase

Figure 1 is a schematic view of a capillary of the prior art.

Figure 2 is a schematic view of a capillary heating arrangement of the prior art.

Figure 3 is a perspective view of a preferred embodiment of the present invention.

Figure 4 is a partially exploded perspective view of a preferred embodiment of the present invention.

Figure 5 is a partial cross-sectional view showing a preferred embodiment of the present invention.

Figure 6 is a schematic view showing the housing assembly of a preferred embodiment of the present invention.

Fig. 7 is a schematic view showing the temperature control of the first embodiment of the present invention.

Figure 8 is a schematic diagram of temperature control in accordance with a second embodiment of the present invention.

10. . . Base

20. . . Heating seat

30. . . Auxiliary heating element

40. . . Reaction tube

50. . . Fixed connector

60. . . Observation module

70. . . Temperature control device

Claims (7)

A method for setting a temperature of a polymerase chain reaction includes the following steps:
a preparation step of placing a reaction sample into a reaction tube, and fixing one end of the reaction tube to a heating seat, so that a position of a main heating element in the heating seat corresponds to a bottom portion of the reaction tube, and An auxiliary heating member is spaced apart from the main heating member to correspond to an intermediate portion of the reaction tube;
a heating step of raising the temperature of the main heating member and heating to a denaturation temperature, and heating the auxiliary heating member to an auxiliary heating temperature, the auxiliary heating temperature being less than the denaturation temperature; and a chain reaction step controlling the main The heating element is maintained at the denaturation temperature for a chain reaction. The method for setting a temperature of a polymerase chain reaction according to claim 1, wherein the reaction sample is a ribonucleic acid sample, and there is a reverse transcription step between the preparation step and the warming step: The primary heating element maintains the initial temperature and converts the ribonucleic acid sample to a DNA sample. A method for setting a temperature of a polymerase chain reaction as described in claim 2, wherein the starting temperature is between 45 ° C and 55 ° C. The method for setting a temperature of a polymerase chain reaction according to claim 1, wherein in the preparing step, a high thermal conductive member is wound around the bottom of the reaction tube, and the reaction tube is passed through the high thermal conductive member. The main heating element is in contact. The method for setting a temperature of a polymerase chain reaction according to claim 1, wherein the preparing step further comprises a step of disposing the reaction tube, the heating seat and the auxiliary heating member in a closed casing. The method for setting a temperature of a polymerase chain reaction according to claim 1, wherein the auxiliary heating temperature is between 40 ° C and 50 ° C. The method for setting a temperature of a polymerase chain reaction according to claim 1, wherein the denaturation temperature is between 90 ° C and 98 ° C.