CN107446812A - A kind of reaction instrument for PCR four-temperature region tubular type temperature regulating device and comprising the device - Google Patents
A kind of reaction instrument for PCR four-temperature region tubular type temperature regulating device and comprising the device Download PDFInfo
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- CN107446812A CN107446812A CN201710609865.1A CN201710609865A CN107446812A CN 107446812 A CN107446812 A CN 107446812A CN 201710609865 A CN201710609865 A CN 201710609865A CN 107446812 A CN107446812 A CN 107446812A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1811—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using electromagnetic induction heating
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Abstract
The present invention relates to a kind of four-temperature region tubular type temperature regulating device for PCR, including temperature control post and reaction tube；Temperature control post is divided into three sections, it is followed successively by including denaturation section, repetend and end extends section eventually, not heat conduction between three sections of temperature control post, denaturation section cross-sectional circumference is arranged to denaturation heated at constant temperature area, the cross-sectional circumference of end extension section is arranged to end extension heated at constant temperature area eventually eventually, the cross-sectional circumference of repetend is divided into four heated at constant temperature areas, is followed successively by degeneration area, cooling zone, annealed zone and extension area；Reaction tube includes entrance, outlet and pipe shaft, the pipe shaft of reaction tube is divided into three domains in the longitudinal direction, it is followed successively by denaturation domain, the range of a loop in order from entrance to export direction and end extends domain eventually, respectively coil on three sections of temperature control post in three domains of pipe shaft, and denaturation domain is connected with the range of a loop by being denatured domain connecting tube, the range of a loop is connected with end extension domain eventually by extending domain connecting tube.
Description
Technical field
The present invention relates to molecular biology equipment, more specifically it relates to a kind of tubular type temperature regulating device of three-stage four-temperature region,
And include the PCR instrument of the tubular type temperature regulating device.
Background technology
DNA semi-conservative replication is the important channel of biological evolution and passage.Double-stranded DNA can be with the presence of a variety of enzymes
Denaturation is untwisted into single-stranded, in the presence of archaeal dna polymerase, is copied into two same molecules according to base pair complementarity principle and is carried on the arm
Shellfish.Denaturation can also occur in high temperature and unwind by DNA, can turn into double-strand with renaturation again after temperature reduces.Therefore, temperature is passed through
Change control DNA denaturation and renaturation, add design primer, and archaeal dna polymerase, dNTP cans complete the external multiple of specific gene
System.
PCR (PCR) is to become single-stranded in 95 DEG C of high temperature time variations using DNA, low temperature (often 60
DEG C or so) when primer combined with single-stranded by the principle of base pair complementarity, then temperature regulating is to archaeal dna polymerase optimal reactive temperature
(72 DEG C or so), direction composition complementary strand of the archaeal dna polymerase along phosphoric acid to pentose (5 ' -3 ').Based on polymerase manufacture
Actual PCR instrument is exactly a temperature control device, can be in denaturation temperature, renaturation temperature, is controlled well between elongating temperature.
Since perkin-elmercetus companies First PCR instrument comes out, tens different producers are there is now in state
Inside and outside production and selling PCR instrument.Between short several years, PCR instrument passes through the development of several generations, constantly employs new technology, and enter
One step develops towards the direction of convenient, practical, high-intelligentization and automation.The general principle of round pcr is similar to the natural multiple of DNA
Process processed, its specificity depend on the Oligonucleolide primers complementary with target sequence both ends.PCR is by being denatured, annealing and extending three
Fundamental reaction step is formed：1. the denaturation of template DNA：After template DNA is heated to 95 DEG C or so certain times, make template DNA
Double-strand expands the double-stranded DNA to be formed through PCR and unwind, and is formed single-stranded, so as to the combination of primer, is prepared for lower whorl reaction；②
The annealing (renaturation) of template DNA and primer：For the heated denaturation of template DNA into after single-stranded, temperature is down to 55 DEG C or so, primer and mould
Complementary series pairing single-stranded plate DNA combines；3. the extension of primer：DNA profiling/primer complex is thermophilic poly- in TaqDNase etc.
In the presence of synthase, using dNTP as reaction raw materials, aim sequence is template, by base pair complementarity and semi-conservative replication principle,
The new semi-conservative replication chain complementary with template DNA chain of synthesis one, repeat the above steps and be achieved with more " partly retain multiple
Chain processed ", and this new chain can turn into the template of circulation next time.Often completing a circulation needs 2-4 minutes, and 2-3 hours are with regard to energy
Will millions of times of target gene amplification amplification be expanded.
Capillary Electrophoresis (capillary electrophoresis, CE) is called HPCE (HPCE), is near
One of analysis method with fastest developing speed over year.Jorgenson and Lukacs in 1981 proposes in 75 μm of internal diameter capillary tube posts first
It is interior to be separated with high voltage, found modern Capillary Electrophoresis.Terabe in 1984 etc. establishes micella capillary electric power
Learn chromatogram.Hjerten in 1987 establishes capillary isoelectric focusing, and Cohen and Karger propose capillary gel electrophoresis.
There is first Capillary Electrophoresis commercial apparatus in 1988-1989.In a few years, due to CE met using bioengineering as
To polypeptide, protein (including enzyme, antibody), nucleotides or even DNA (DNA) in each field of life science of representative
Separation analysis require, obtained rapid development.
At present, although round pcr has obtained very big development, but all PCR instruments are all to control by the following method
PCR reactions processed：PCR pipe equipped with PCR reaction systems is placed in the temperature-control hole of fixed temperature control base, and is close to temperature-control hole
Inwall, control the temperature of PCR pipe and its internal reaction system filled by controlling the temperature of temperature control hole wall.This kind of device
Although having generalized, however it there is some it is unconquerable the shortcomings that.
First, time-consuming.Normal PCR reactions generally comprise 30 circulations, wherein denaturation 30s, annealing 30s, extension
30s/kb.Plus denaturation 5min, end extension 10min eventually, expand 1kb fragment only need one and a half hours or so when
Between.However, when being reacted in PCR instrument, it has been found that expand 1kb fragment generally require at least over 2 hours when
Between.The reason is that the position of sample is fixed in PCR instrument, therefore it is necessary to which the temperature for manipulating temperature control base changes to reaction
Temperature required for the stage proceeded to.30 circulations, every time circulation have 3 alternating temperatures, that is to say, that need to carry out 90 times or so
Alternating temperature.This needs to consume the substantial amounts of time close to the heating and cooling process of 100 times.
Secondly, heating and cooling seem inverse process, and it is higher than the degree of difficulty of heating in fact to cool, for the ease of cooling, PCR
Fan also have to be set in instrument, the volume of PCR instrument is increased, improve cost.
Again, according to the principle of current PCR instrument and setting, in order to ensure the heat conduction efficiency in PCR system, PCR bodies
System can not be too big, and the system of single PCR reactions is no more than 200 microlitres, and which limits a large amount of preparations of specific gene.
The flux that PCR instrument device expands amplification relies primarily on the number for increasing the hole reacted, and current high-throughout PCR instrument device has been accomplished
384 holes, 384 samples can be expanded or quantified simultaneously, but face the amplification and detection of thousands of or up to ten thousand samples, just
It is relatively difficult.
In addition, capillary detection technology (CE) realizes high flux detection, and the operation of automation is realized, still,
Current PCR instrument is unfavorable for forming integrated operation with capillary detection due to the hard defects in structure and setting.
Therefore, it is necessary to a kind of new PCR instrument and new temperature regulating device.
The content of the invention
To solve problem above, the invention provides a kind of tubular type temperature regulating device for PCR, it include temperature control post with instead
Ying Guan；
The temperature control post is divided into three sections, is followed successively by including denaturation section, repetend and end extends section, the temperature control eventually
Not heat conduction between three sections of post, the denaturation section cross-sectional circumference are arranged to denaturation heated at constant temperature area, added
Hot temperature is 90-98 DEG C, and the cross-sectional circumference of the extension of the end eventually section is arranged to end extension heated at constant temperature area, heating-up temperature eventually and is
70-75 DEG C, the cross-sectional circumference of the repetend is divided into four heated at constant temperature areas, is followed successively by degeneration area, cooling zone, annealed zone
And extension area；
The reaction tube includes entrance, outlet and pipe shaft, and the pipe shaft of the reaction tube is divided into three domains in the longitudinal direction,
It is followed successively by denaturation domain, the range of a loop in order from entrance to export direction and end extension domain, three domains of the pipe shaft is divided eventually
Do not coil correspondingly on three of temperature control post sections, and the denaturation domain is connected with the range of a loop by being denatured domain
Adapter connection, the range of a loop are connected with the extension domain of end eventually by extending domain connecting tube.
Three sections of temperature control post can be coaxially disposed or non-coaxial setting.
Temperature control post is divided into three sections by the tubular type temperature regulating device of the present invention, and pipe shaft correspondingly will be divided into three domains, so as to
PCR denaturation stage, cycle stage and whole last extension stage are carried out in three relatively independent sections respectively, reduced
The step of needing programme-control, controlling pump or valve are needed when avoiding using only one section, so that PCR reaction systems are being followed
Stop the time needed for the denaturation stage in degeneration area before ring reaction, and stopped eventually in extension area after circular response terminates
Time needed for the last extension stage.
The division in four heated at constant temperature areas of repetend, adds cooling zone between degeneration area and annealed zone.This is set
The reason for be that denaturation temperature is 95 DEG C or so, annealing temperature is 60 DEG C or so, to annealing needs to cool from denaturation, and the thing that cools
In reality it is one and compares the process that heating is more difficult to.If 60 DEG C of the environment that reaction system directly enters from 95 DEG C of environment, then
Its temperature less easily drops to 60 DEG C, and can be higher than the temperature.In order to overcome this shortcoming, we are in degeneration area and annealed zone
Between add shorter cooling zone, the temperature control of cooling zone is at 45-50 DEG C or so.When reaction system is by cooling zone
When, temperature is reduced to annealing temperature nearby and slightly below annealing temperature, and enter annealed zone at once, and temperature of reaction system is at once
Raise and return to annealing temperature, annealed, extension area is entered after the scheduled time.So that a PCR is formed in reaction tube
Temperature needed for the three phases of circulation, and in repetend, these three warm areas are all constant temperature, it is not necessary to it is warm-natured becoming
Cyclic switching is carried out between degree-annealing temperature-elongating temperature.Reaction system flows successively through cooling zone, annealing since degeneration area
Area, extension area, PCR temperature cycles are completed, it is anti-so as to often once complete the PCR of a circulation around repetend operation
Should.Therefore, the problem of what the division emphasis of four-temperature region to be solved is the cooling difficulty from denaturation to annealing.
In one embodiment, three sections of the temperature control post are that cylinder or cylindrical shape, diameter are equal or different.
Cylinder is easy to being bonded for reaction tube pipe shaft and temperature control post, does not produce space, so as to more effectively conduct heat.It can also adjust as needed
The respective diameter of the section of section three, to obtain suitable length, so that pump keeps uniform rotating speed, nothing during the course of the reaction
Controlling pump is needed to change rotating speed.
In a preferred embodiment, the junction of the denaturation domain connecting tube and the range of a loop is located at the extension
Area and the boundary of the degeneration area, or the boundary positioned at the degeneration area Yu the cooling zone.The setting to react
System enters from denaturation domain enters the cycle stage after the range of a loop, or, because the first step of cycle stage is denaturation,
Just it is identical with the temperature needed for the denaturation stage, so the denaturation position of first circulation can be integrated into denaturation domain
In, during to the range of a loop, it is directly entered annealed zone.
In a preferred embodiment, the junction of the extension domain connecting tube and the range of a loop is located at the extension
Area and the boundary of the degeneration area.
In one embodiment, the extension domain connecting tube has a branch of more than three, one of branch with it is described
End extension domain connection eventually, other branches are connected with the range of a loop at away from the denaturation domain connecting tube difference bung flange number respectively,
And the junction of the extension domain connecting tube and the range of a loop is provided with valve.Valve can have two states, the first state
To make the bung flange on the valve both sides connect (reaction system moves to next bung flange)；Second of state is to make the range of a loop in valve
(reaction system leaves the range of a loop and enters end eventually by extending domain connecting tube with corresponding extension domain connection vessel branches connection for position
Extend domain).Set valve state make reaction system by the specific number of turns and from corresponding extension domain connecting tube branch into
Eventually in end extension domain, so as to carry out specific PCR reaction cycle numbers in the cycle stage.For example, the range of a loop can in repetend disk
Enclosed around 30-60, and be arranged to whole end prolong by extending the branch of domain connecting tube at away from the different number of turns of denaturation domain connecting tube
Domain connection is stretched, setting the state of valve makes reaction system branch into end extension domain eventually by the specific number of turns and from corresponding, from
And carry out specific PCR reaction cycle numbers.
In one embodiment, four heated at constant temperature areas of the repetend cross-sectional circumference are the constant temperature of regular length
Heating zone.The temperature of the degeneration area is 90-98 DEG C, and the temperature of the cooling zone is 40-50 DEG C, and the temperature of the annealed zone is
55-70 DEG C, the temperature of the extension area is 70-75 DEG C, and what the entrance of the reaction tube was located at the degeneration area leans on the cooling
The one end in area.The division in four heated at constant temperature areas to be formed in reaction tube needed for the three phases of a PCR cycle
Solves the problems, such as the cooling difficulty from denaturation to annealing while temperature.And in the temperature regulating device, this four warm areas are all
Constant temperature, it is not necessary to carry out cyclic switching between denaturation temperature-annealing temperature-elongating temperature.Reaction system is since degeneration area
Cooling zone, annealed zone, extension area are flowed successively through, completes PCR temperature cycles, it is once i.e. complete so as to often be run around temperature control post
Into the PCR reactions of a circulation.
In a preferred embodiment, the range of a loop of pipe shaft heated at constant temperature residing for distinguishes section, often encloses spiral
Correspondingly it is divided into denaturation section, cooling section, annealing section and extension of section successively, and by hot exhausted between adjacent section
Short tube made of edge material connects.By taking the short tube of heat insulation to connect between adjacent section, pipe is preferably avoided
Heat transfer between the different constant temperature sections of body.So-called heat insulator refers to the relatively low material of pyroconductivity in the present invention,
Rather than absolute athermanous material.
In a preferred embodiment, the temperature control post surface has the bung flange to match with the reaction tube pipe shaft recessed
Groove, the pipe shaft are embedded in the bung flange groove.The setting makes it possible to more preferably precisely control the temperature of reaction tube pipe shaft.
In a further preferred embodiment, separated between heated at constant temperature area corresponding to the bung flange groove by heat insulator.More preferably
The heat transfer that ground isolates between adjacent heated at constant temperature area.
In another embodiment, the repetend cross-sectional circumference is made up of multiple independent heated at constant temperature units,
The temperature of each heated at constant temperature unit is independently controlled, and the multiple independent heated at constant temperature unit is divided
For four heated at constant temperature areas.In different PCR reaction cycles, required denaturation time, cool time, annealing time and extension
Time may be different, can adjust heated at constant temperature unit and reaction system that each heated at constant temperature area includes as needed in pipe shaft
Translational speed so that reaction system stops the required reaction time at corresponding temperature.
The invention further relates to a kind of PCR instrument, and it includes above-mentioned tubular type temperature regulating device, and the discharge of sample feeding pipe, reaction product
Pipe and pump, the sample feeding pipe are connected with the entrance of the reaction tube, the outlet of the reaction product discharge pipe and the reaction tube
Connection, the reactant that the pump is driven into the sample feeding pipe move to the reaction product discharge pipe.The PCR instrument construct with
The entirely different new reaction mechanism of the original fixed position PCR reaction mechanisms in this area so that be more beneficial for PCR reactions, remove
Heating and cooling time needed for the temperature handoff procedure of alternating temperature fixed position PCR cycle reaction.According to PCR reaction principles and institute
The three phases needed, are divided into three sections by temperature regulating device, and often section realizes respective function, reduce the control of course of reaction Program
Intervention, instrument manufacturing cost is reduced, and also improve durability simultaneously.
In a preferred embodiment, the PCR instrument includes multiple tubular devices and corresponding sample feeding pipe
With reaction product discharge pipe, and in each two heater extension area length it is identical or different.Set specific degeneration area-
Cooling zone-annealed zone-extension section length so that pump can be kept at the uniform velocity in cyclic process, according to the fragment length of required amplification,
Different temperature regulating devices is selected in instrument is reacted obtains different extension of time.
In a preferred embodiment, the PCR instrument also includes controller, pump and the temperature control post and controller electricity
Connection, when the extension domain connecting tube is provided with valve with the range of a loop junction, the valve electrically connects with the controller.
The parameters of the controllable reaction instrument of controller, are carried out so that reaction is accurate.
In the tubular type temperature regulating device of the present invention, the temperature of reaction tube is controlled by controlling the temperature of temperature control post.Make anti-
System is answered to enter from reaction tube inlet, by pipe shaft, the temperature of reaction system is by heat transfer and the pipe shaft temperature with present position
Spend identical, and reacted at such a temperature.Such tubular type temperature regulating device can build isothermal reaction and temperature cycles change is anti-
The reaction environment answered.After the repetend cross-sectional circumference of temperature control post is divided into multiple different heated at constant temperature areas, the present invention
Tubular type temperature regulating device particularly suitable for building the reaction environment of temperature cycles change, such as PCR, the temperature regulating device need not
The process of the temperature of cyclic switching temperature control base is needed in normal PCR instrument, the plenty of time saved for PCR reactions.Simultaneously as
Use tube designs so that temperature regulating device of the invention after being combined with pump, be advantageous to the sampling device of front end and after
The separator at end is combined, and is thus reacted to realize that amplification of DNA fragments is formulated into PCR from system again to PCR reaction products
Separation full-automation.
Brief description of the drawings
Fig. 1 is the schematic diagram of an example of temperature regulating device；
Fig. 2 is denaturation section and the viewgraph of cross-section for being coiled in denaturation domain thereon；
Fig. 3 is that end extends the viewgraph of cross-section for saving and extending domain with the end eventually being coiled in thereon eventually；
Fig. 4 is the viewgraph of cross-section of an example of the repetend with being coiled in the range of a loop thereon；
Fig. 5 is the viewgraph of cross-section of another example of the repetend with being coiled in the range of a loop thereon；
Fig. 6 is the viewgraph of cross-section of another example of the repetend with being coiled in the range of a loop thereon；Fig. 7 is temperature regulating device
An example schematic diagram；
Fig. 8 is the schematic diagram of an example of reaction instrument.
In accompanying drawing, the list of parts representated by each label is as follows：
101st, denaturation section, 102, repetend, 1020, heated at constant temperature unit, 1021, degeneration area, 1022, cooling zone,
1023rd, annealed zone, 1024, extension area, 103, end extension section eventually, 201, denaturation domain, 202, the range of a loop, 2021, degeneration area
Section, 2022, cooling section, 2023 annealing sections, 2024, extension of section, 2025, insulation short tube, 203, end extension domain eventually, 204,
It is denatured domain connecting tube, 205, extension domain connecting tube, 3, entrance, 4, outlet, 5, sample feeding pipe, 6, reaction product discharge pipe, 7, valve.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
Embodiment 1
As shown in figures 1 to 6, the tubular type temperature regulating device for PCR of the present embodiment, it includes temperature control post and reaction tube；
The temperature control post is divided into three sections, is followed successively by including denaturation section 101, repetend 102 and end extends section eventually
103, not heat conduction between three sections of the temperature control post, it is coaxially disposed or non-coaxial setting, the denaturation section 101 is horizontal
Cross sectional periphery is arranged to denaturation heated at constant temperature area, and heating-up temperature is 90-98 DEG C, the cross section of the extension of the end eventually section 103
Periphery is arranged to end extension heated at constant temperature area eventually, and heating-up temperature is 70-75 DEG C, and the cross-sectional circumference of the repetend 102 divides
For three heated at constant temperature areas, degeneration area 1021, cooling zone 1022, annealed zone 1023 and extension area 1024 are followed successively by.Degeneration area
1021 heating-up temperature is 90-98 DEG C, the heating-up temperature of cooling zone 1022 is 40-50 DEG C, and the heating-up temperature of annealed zone 1023 is
50-70 DEG C, the heating-up temperature of extension area 1024 is 70-75 DEG C.
The reaction tube includes entrance 3, outlet 4 and pipe shaft, the pipe shaft of the reaction tube and is divided into three in the longitudinal direction
Domain, it is followed successively by denaturation domain 201, the range of a loop 202 in order from entrance to export direction and end extends domain 203 eventually.The pipe
Three domains of body are respectively coiled on three sections of the temperature control post, and the coiled coil of denaturation domain 201 becomes in initial
Property section 101 on, the coiled coil of the range of a loop 202 is in repetend 102, and the end extension coiled coil of domain 203 is in end extension section 103 eventually eventually
On, and the denaturation domain 201 is connected with the range of a loop 202 by being denatured domain connecting tube 204, the range of a loop 202
Connected with the extension domain 203 of end eventually by extending domain connecting tube 205.
In running, reaction system enters from entrance 3, successively by denaturation domain 201, the range of a loop 202 and whole end
Extend domain 203, and from the outflow of outlet 4, the temperature of reaction system by heat transfer and identical with the pipe shaft temperature of present position, and
Reacted at such a temperature.Such tubular type temperature regulating device can be used for building the anti-of isothermal reaction and temperature cycles reacting condition
Answer environment.
Reaction system completes the PCR denaturation stage in denaturation domain 201, is completed in end extension domain 203 eventually
PCR extension stage end last.In the range of a loop, the division in three heated at constant temperature areas make it that a PCR is formed in reaction tube to follow
The warm area of three kinds of temperature needed for ring, and in the temperature regulating device, these three warm areas are all constant temperature, it is not necessary to it is warm-natured becoming
Cyclic switching is carried out between degree-annealing temperature-elongating temperature.Reaction system flows successively through cooling zone, annealing since degeneration area
Area, extension area, PCR temperature cycles are completed, it is anti-so as to often once complete the PCR of a circulation around range of a loop operation
Should.
Embodiment 2
As shown in figure 5, the range of a loop of the temperature regulating device of the present embodiment heated at constant temperature residing for distinguishes section, spiral phase is often enclosed
Answer ground to form four constant temperature sections, be followed successively by denaturation section 2021, cooling section 2022, annealing section 2023 and extension of section
2024, and connected between adjacent section by short tube made of heat insulator 2025.By between adjacent section
Take the short tube 2025 of heat insulation to connect, preferably avoid the heat transfer between the different constant temperature sections of the range of a loop 202.This reality
Apply so-called heat insulator in example and refer to the relatively low material of pyroconductivity, rather than definitely athermanous material.
Embodiment 3
In a preferred scheme, the temperature control post surface has the bung flange to match with the reaction tube pipe shaft recessed
Groove, the reaction tube pipe shaft are embedded in the bung flange groove.The setting makes it possible to more preferably precisely control reaction tube pipe shaft
Temperature.It is highly preferred that being separated between heated at constant temperature area corresponding to the bung flange groove by heat insulator, preferably isolate phase
Heat transfer between adjacent heated at constant temperature area.So-called heat insulator refers to the relatively low material of pyroconductivity in the present embodiment, and
Non- definitely athermanous material.
Embodiment 4
As shown in fig. 6, the periphery of the range of a loop cross section of the temperature regulating device of the present embodiment is by multiple heated at constant temperature units
1020 compositions, the multiple heated at constant temperature unit 1020 can be divided into four heated at constant temperature by control on demand in use
Area, is followed successively by degeneration area 1021, cooling zone 1022, annealed zone 1023 and extension area 1024, and each heated at constant temperature area includes more
Individual heated at constant temperature unit 1020, the temperature of the heated at constant temperature unit in the degeneration area 1021 is 90-98 DEG C, the cooling zone
The temperature of heated at constant temperature unit in 1022 is 40-50 DEG C, and the temperature of the heated at constant temperature unit in the annealed zone 1023 is 55-
70 DEG C, the temperature of the heated at constant temperature unit in the extension area 1024 is 70-75 DEG C, the denaturation domain connecting tube 204 and circulation
The junction in domain is located at the boundary of the degeneration area 1021 and the annealed zone 1024, and the extension domain connecting tube 205
It is located at the boundary of the extension area 1024 and the degeneration area 1021 with the junction of the range of a loop 202.The device can use
In PCR instrument, the temperature regulating device as PCR reactions.
In different PCR reaction cycles, required denaturation time, annealing time and extension of time are different, can be according to need
The translational speed of heated at constant temperature unit and reaction system that each heated at constant temperature area includes in pipe shaft is adjusted, so that instead
System is answered to stop the required reaction time at corresponding temperature.
Embodiment 5
As shown in fig. 7, the extension domain connecting tube 205 has the branch of more than three, one of branch and the end eventually
Extension domain 203 connects, and other branches are respectively from the range of a loop 202 at away from the different bung flange numbers of the denaturation domain connecting tube 204
Connection, and the junction of the extension domain connecting tube 205 and the range of a loop 202 is provided with valve 7.Valve 7 can have two kinds of shapes
State, the first state are that the bung flange for making the both sides of valve 7 connects (reaction system moves to next bung flange)；Second of state be
Making the range of a loop 202, (reaction system leaves the range of a loop 202 simultaneously with corresponding extension domain connecting tube 205 branch connection in the position of valve 7
Enter end extension domain 203 eventually by extending domain connecting tube 205).Setting the state of valve 7 makes reaction system pass through the specific number of turns simultaneously
Branched into from corresponding extension domain connecting tube 205 into end extension domain 203 eventually, so as to carry out specific PCR in the cycle stage
Reaction cycle number.For example, the range of a loop 202 can be coiled in repetend 1023 30-60 circle, and away from denaturation domain connecting tube 204 not
It is arranged to be connected with end extension domain eventually by extending the branch of domain connecting tube 205 at the same number of turns, setting the state of valve makes reaction
System branches into end extension domain eventually by the specific number of turns and from corresponding, so as to carry out specific PCR reaction cycle numbers.
Embodiment 6
As shown in figure 8, the present embodiment is the invention further relates to a kind of PCR instrument, it includes above-mentioned tubular type temperature regulating device, and
Sample feeding pipe 5, reaction product discharge pipe 6 and pump, the sample feeding pipe 5 are connected with the entrance 3 of the reaction tube, the reaction product row
Outlet pipe 6 is connected with the outlet 4 of the reaction tube, and the reaction system that the pump is driven into the sample feeding pipe 5 is produced to the reaction
Thing discharge pipe 6 moves.
In running, reaction system is added into sample feeding pipe 5, opens pump, pump driving reaction system passes through from sample feeding pipe 5
Cross reaction tube pipe shaft to run to reaction product discharge pipe 6, reaction system completes reaction during by reaction tube pipe shaft.
The PCR instrument constructs the new reaction mechanism entirely different with the original fixed position PCR reaction mechanisms in this area, makes
PCR reactions must be more beneficial for, when eliminating the heating and cooling needed for the temperature handoff procedure of alternating temperature fixed position PCR cycle reaction
Between.According to PCR reaction principles and required three phases, temperature regulating device is divided into three sections, often section realizes respective function, reduces
The intervention of course of reaction Program control, instrument manufacturing cost is reduced, and also improve durability simultaneously.Also,
Pressue device also can be set in the reaction instrument to control the pressure inside reaction tube, the raising of pressure can avoid the steaming of reaction system
Hair.
Embodiment 7
PCR instrument in the present embodiment includes multiple tubular type temperature regulating devices and corresponding sample feeding pipe 3 and reaction product row
Outlet pipe 4, and in each two tubular type temperature regulating device extension area 1024 length it is identical or different.Specific degeneration area-cold is set
But area-annealed zone-extension section length so that pump can be kept at the uniform velocity in cyclic process, according to the fragment length of required amplification,
Different temperature regulating devices is selected in reaction instrument obtains different extension of time.
Preferably, the above-mentioned PCR instrument also includes controller, and respectively section is electrically connected pump and the temperature control post with the controller
Connect, when the junction of the range of a loop 202 and the extension domain connecting tube 205 is provided with valve 7, the valve 7 and the control
Device electrically connects.The parameters of the controllable reaction instrument of controller, are carried out so that reaction is accurate.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (10)
1. a kind of tubular type temperature regulating device for PCR, it is characterised in that including temperature control post and reaction tube；
The temperature control post is divided into three sections, is followed successively by including denaturation section, repetend and end extends section eventually, the temperature control post
Not heat conduction between three sections, the denaturation section cross-sectional circumference are arranged to denaturation heated at constant temperature area, the end
The cross-sectional circumference of end extension section is arranged to end extension heated at constant temperature area, the cross-sectional circumference of the repetend eventually and is divided into four
Heated at constant temperature area, it is followed successively by degeneration area, cooling zone, annealed zone and extension area；
The reaction tube includes entrance, outlet and pipe shaft, and the pipe shaft of the reaction tube is divided into three domains in the longitudinal direction, from entering
Mouth is followed successively by denaturation domain, the range of a loop in order to export direction and phase is distinguished in end extension domain, three domains of the pipe shaft eventually
Domain is answered to be around on three sections of the temperature control post, and the denaturation domain and the range of a loop are by being denatured domain connecting tube
Connection, the range of a loop are connected with the extension domain of end eventually by extending domain connecting tube.
2. tubular type temperature regulating device according to claim 1, it is characterised in that three sections of the temperature control post are cylinder
Or cylindrical shape, diameter are equal or different.
3. tubular type temperature regulating device according to claim 1, it is characterised in that the denaturation domain connecting tube and the range of a loop
Junction be located at the boundary of the extension area and the degeneration area, or point positioned at the degeneration area and the cooling zone
At boundary.
4. tubular type temperature regulating device according to claim 1, it is characterised in that the extension domain connecting tube and the range of a loop
Junction be located at the boundary of the extension area and the degeneration area.
5. tubular type temperature regulating device according to claim 4, it is characterised in that the extension domain connecting tube has more than three
Branch, one of branch are connected with the extension domain of end eventually, and other branches are respectively with the range of a loop away from the denaturation domain
Connected at connecting tube difference bung flange number, and the junction of the extension domain connecting tube and the range of a loop is provided with valve.
6. the tubular type temperature regulating device according to any one of claim 1-5, it is characterised in that outside the repetend cross section
Four heated at constant temperature areas in week are the heated at constant temperature area of regular length.
7. the tubular type temperature regulating device according to any one of claim 1-5, it is characterised in that outside the repetend cross section
Week is made up of multiple independent heated at constant temperature units, and the temperature of each heated at constant temperature unit is independently controlled, and
The multiple independent heated at constant temperature unit is divided into four heated at constant temperature areas.
8. a kind of PCR instrument, it is characterised in that including the tubular type temperature regulating device any one of claim 1-7, and sample introduction
Pipe, reaction product discharge pipe and pump, the sample feeding pipe are connected with the entrance of the reaction tube, the reaction product discharge pipe and institute
The outlet connection of reaction tube is stated, the reactant that the pump is driven into the sample feeding pipe moves to the reaction product discharge pipe.
9. reaction instrument according to claim 8, it is characterised in that the PCR instrument include multiple tubular devices with
And corresponding sample feeding pipe and reaction product discharge pipe, and in each two heater extension area length it is identical or different.
10. PCR instrument according to claim 8 or claim 9, it is characterised in that also including controller, pump and the temperature control post and institute
Controller electrical connection is stated, when the extension domain connecting tube is provided with valve with the range of a loop junction, the valve and the control
Device electrical connection processed.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710609865.1A CN107446812A (en) | 2017-07-25 | 2017-07-25 | A kind of reaction instrument for PCR four-temperature region tubular type temperature regulating device and comprising the device |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710609865.1A CN107446812A (en) | 2017-07-25 | 2017-07-25 | A kind of reaction instrument for PCR four-temperature region tubular type temperature regulating device and comprising the device |
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| CN107446812A true CN107446812A (en) | 2017-12-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710609865.1A Pending CN107446812A (en) | 2017-07-25 | 2017-07-25 | A kind of reaction instrument for PCR four-temperature region tubular type temperature regulating device and comprising the device |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446811A (en) * | 2017-07-25 | 2017-12-08 | 新疆昆泰锐生物技术有限公司 | A kind of reaction instrument for PCR tubular type temperature regulating device and comprising the device |
| CN110394143A (en) * | 2019-08-21 | 2019-11-01 | 兖州煤业股份有限公司 | A kind of coal resolution tubular type reaction unit |
| CN110394143B (en) * | 2019-08-21 | 2024-06-07 | 兖矿能源集团股份有限公司 | Tubular reaction device is cleared up to coal |
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| CN107446811A (en) * | 2017-07-25 | 2017-12-08 | 新疆昆泰锐生物技术有限公司 | A kind of reaction instrument for PCR tubular type temperature regulating device and comprising the device |
| CN110394143A (en) * | 2019-08-21 | 2019-11-01 | 兖州煤业股份有限公司 | A kind of coal resolution tubular type reaction unit |
| CN110394143B (en) * | 2019-08-21 | 2024-06-07 | 兖矿能源集团股份有限公司 | Tubular reaction device is cleared up to coal |
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Effective date of registration: 20211210 Address after: 237012 room 323, engineering innovation center, Wanxi University, Gulou Street, Yu'an District, Lu'an City, Anhui Province Applicant after: Lu'an kuntairui Technology Development Co.,Ltd. Address before: 830000 room 425, incubation building, Xinjiang biomedical innovation and entrepreneurship Park, No. 181, Xicai Road, high tech Industrial Development Zone (new urban area), Urumqi, Xinjiang Uygur Autonomous Region Applicant before: XINJIANG QUINTARABIO BIOTECHNOLOGY CO.,LTD. |
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Application publication date: 20171208 |