CN108456748A - The methods, devices and systems controlled are reacted to sequencing - Google Patents
The methods, devices and systems controlled are reacted to sequencing Download PDFInfo
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- CN108456748A CN108456748A CN201710098314.3A CN201710098314A CN108456748A CN 108456748 A CN108456748 A CN 108456748A CN 201710098314 A CN201710098314 A CN 201710098314A CN 108456748 A CN108456748 A CN 108456748A
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- 238000012163 sequencing technique Methods 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 41
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- 238000001514 detection method Methods 0.000 claims abstract description 21
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- 238000012864 cross contamination Methods 0.000 abstract description 22
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- 239000007788 liquid Substances 0.000 description 32
- 238000012360 testing method Methods 0.000 description 18
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- 239000000243 solution Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001917 fluorescence detection Methods 0.000 description 3
- 238000002493 microarray Methods 0.000 description 3
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
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Abstract
The invention discloses the method controlled is reacted to sequencing, sequencing reaction is controlled using Sequence Detection System.Sequence Detection System includes fluid means, and in fluid means, valve component includes the first valve and the second valve, and the first valve includes stator and rotor, and the first valve has common port, has multiple ports on stator, has connectivity slot on rotor, method includes step：Make first port pass through connectivity slot to be connected to common port；The second valve is set to be connected to the first reagent and first port；The first reagent is set to enter reaction unit through the second valve and the first valve successively using drive component, to carry out the first biochemical reaction；Before rotor, the second valve is made to be connected to the first buffer solution and first port；Make the first buffer solution followed by the second valve and the first valve using drive component.In the above method, before rotor, using first the first valve of buffer solution for cleaning, that is, the first reagent in the first valve is replaced, the risk of cross contamination when avoidable rotor switches different reagents.
Description
Technical field
The present invention relates to sequencing technology field more particularly to it is a kind of to the sequencing method that is controlled of reaction and
Sequence Detection System and device.
Background technology
Sequencing is sequenced, include the measurement of nucleic acid sequence.Microarray dataset on the market includes that generation sequencing is flat at present
Platform, two generation microarray datasets and three generations's microarray dataset.
Using the platform for carrying out sequence based on biochemical reaction during sequencing, need on reaction unit
Biochemical reaction is carried out, such as needs different reagents using liquid channel system together or be successively introduced on chip to react.
Currently, to make the liquid channel system compact efficient in platform, liquid channel system be all made of the first valve to switch input/output reagent.
The first presently commercially available valve inevitably occurs different due to its design feature when different reagents switch
The reagent of degree intersects or is mixed into, i.e. reagent cross contamination.Reagent cross contamination can influence the progress of reaction, especially for that
The reaction of amount of reagent very little needed for a little reactions itself, such as single-molecule sequencing, the cross contamination of reagent is fatal.
Therefore, the reagent cross contamination in fluid path how is reduced or avoided, problem to be solved is become.
Invention content
Embodiment of the present invention aims to solve at least one of technical problem present in the relevant technologies or at least provides one
The useful business selection of kind.It finds and imagines based on studying below the fractionation of the structure of the first valve, inventor makes this hair
It is bright.
The first valve on the market, also referred to as sampling valve, multiposition valve, rotary valve or revolving valve at present, as sample collection, liquid
Body sample introduction or flow path conversion etc. component.Its composition generally comprises stator and rotor, passes through combining closely for stator and rotor, energy
It is enough to form effectively sealing.
It is the port that different flow path liquid into or outs can all pass through that first valve, which has common port, common port, and common port is located at
On stator and/or rotor, there is one or more of the other port on stator and/or rotor.By the rotation of rotor, can realize
The connection of rotor and stator access, to be connected to common port and other ports, to reach the function of selection sample introduction or shunting.The
General configuration/standard configurations of one valve are mostly logical selection type, i.e., in the process of running, only a port is connected to common port.
Common port is connected to other ports, generally require one or several public structures by being arranged on rotor come
Connection.When having liquid in the public structure, the seal interface that is connected due to the rotation of rotor, rotor and stator it is relatively living
Dynamic, inevitably at least part liquid in public structure can be brought to the place except public structure, i.e. flow path is converted
When, it can inevitably make the liquid that a upper flow path is carried in next flow path liquid, and if follow-up negative direction flow path conversion, and meeting
The next flow path liquid for being mixed with a flow path liquid is set to be brought in the liquid of lower flow path；In this way, even if micro be mixed into, generate
Cross contamination be difficult to control, influence be difficult to estimate.
Inventor in the result that the manual result of comparative analysis and upper machine measure, repetition test and checking device system
All parts are associated with and based on studying above the fractionation of the structure of the first valve, determine it is above, due to public affairs when flow path convert
At least part liquid in co-structured is incorporated into the i.e. next reaction process of next flow path, the i.e. cross contamination of reagent, to sequence
Measurement result, which generates, unfavorable is difficult to expect influence.For this purpose, embodiment of the present invention provide it is a kind of to sequencing react into
The method and Sequence Detection System and control device of row control.
Embodiment of the present invention provides a kind of method controlled sequencing reaction, the sequencing reaction packet
The first biochemical reaction is included, first biochemical reaction is carried out using the first reagent on reaction unit, and Sequence Detection System is utilized
Sequencing reaction is controlled.The Sequence Detection System includes fluid means, and the fluid means includes valve body
Component and drive component.The valve component includes the first valve and the second valve, and first valve is connected with the reaction unit, institute
It includes the stator and rotor that can be connected to state the first valve, and first valve has common port, has multiple ports, institute on the stator
Stating has connectivity slot on rotor, the common port and at least one port can be made to pass through by the rotation rotor described
Connectivity slot is connected to, and the multiple port includes first port, and second valve can connect the first port, first reagent
And/or first buffer solution, the method includes the steps：
The first port is set to be connected to the common port by the connectivity slot；
Second valve is set to be connected to first reagent and the first port；
First reagent is set to enter successively through second valve and first valve using the driving component described anti-
Device is answered, to carry out first biochemical reaction；
Before rotating said rotor, second valve is made to be connected to first buffer solution and the first port；
Make first buffer solution followed by second valve and first valve using the driving component.
In the above method, before rotor, the first buffer solution is set to flow into the first valve so that the liquid in connectivity slot is revolving
It walks around and is substituted by the first buffer solution before son, in other words, before the rotor rotation of the first valve, reacted using being measured to target sequence
The first buffer solution without influence avoids original in connectivity slot in rotor rotation process instead of the first reagent in connectivity slot
Reagent be brought to stator and rotor linkage interface other positions, and then cross contamination when having avoided switching different reagents
Risk.
A kind of Sequence Detection System of embodiment of the present invention controls sequencing reaction, the sequencing
Reaction includes the first biochemical reaction, and first biochemical reaction is carried out using the first reagent on reaction unit.The sequence is surveyed
It includes control device and fluid means to determine system, and the control device connects the fluid means, and the fluid means includes valve
Body component and drive component.The valve component includes the first valve and the second valve, and first valve is connected with the reaction unit,
First valve includes the stator and rotor that can be connected to, and first valve has common port, has multiple ports on the stator,
There is connectivity slot on the rotor, the common port and at least one port can be made to pass through institute by rotating the rotor
Connectivity slot connection is stated, the multiple port includes first port, and second valve can connect the first port, first examination
Agent and/or the first buffer solution, the control device are used for：
The first port is set to be connected to the common port by the connectivity slot；
Second valve is set to be connected to first reagent and the first port；
First reagent is set to enter successively through second valve and first valve using the driving component described anti-
Device is answered, to carry out first biochemical reaction；
Before rotating said rotor, second valve is made to be connected to first buffer solution and the first port；
Make first buffer solution followed by second valve and first valve using the driving component.
In above-mentioned Sequence Detection System, before rotor, the first buffer solution is set to flow into the first valve so that in connectivity slot
Liquid is substituted before rotor by the first buffer solution, in other words, before the rotor rotation of the first valve, using to target sequence
First buffer solution of the reaction without influence is measured instead of the first reagent in connectivity slot, avoids and is connected in rotor rotation process
Original reagent is brought to the other positions of the linkage interface of stator and rotor in slot, and then while having avoided switching different reagents hands over
Pitch the risk of pollution.
A kind of computer readable storage medium of embodiment of the present invention is held for storing the program executed for computer
Row described program includes the method for completing any of the above-described embodiment.Computer readable storage medium may include：Read-only storage
Device, random access memory, disk or CD etc..
The additional aspect and advantage of embodiment of the present invention will be set forth in part in the description, partly will be from following
Become apparent in description, or the practice of embodiment is recognized through the invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of embodiment of the present invention retouch embodiment from conjunction with following accompanying drawings
It will be apparent and be readily appreciated that in stating, wherein：
Fig. 1 is the flow diagram for the method for embodiment of the present invention controlled sequencing reaction；
Fig. 2 is the structural schematic diagram of the Sequence Detection System of embodiment of the present invention；
Fig. 3 be the port of the first valve of embodiment of the present invention, connectivity slot and common port relation schematic diagram；
Fig. 4 is the structural schematic diagram of the valve component of embodiment of the present invention；
Fig. 5 is the structural schematic diagram of the test platform of embodiment of the present invention；
Fig. 6 is the schematic diagram of the obtained one group of test data of the test platform of embodiment of the present invention；
Fig. 7 is the schematic diagram of the obtained another group of test data of the test platform of embodiment of the present invention；
Fig. 8 is the contrast schematic diagram of the obtained different test data of the test platform of embodiment of the present invention；
Fig. 9 is another flow diagram for the method for embodiment of the present invention controlled sequencing reaction；
Figure 10 is the high-level schematic functional block diagram of the Sequence Detection System of embodiment of the present invention.
Specific implementation mode
Embodiments of the present invention are described below in detail, the example of the embodiment is shown in the accompanying drawings, wherein from beginning
Same or similar element or element with the same or similar functions are indicated to same or similar label eventually.Below by ginseng
The embodiment for examining attached drawing description is exemplary, and is only used for explaining the present invention, and is not considered as limiting the invention.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indicating or implies relative importance or implicitly indicate the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more feature.In description of the invention
In, the meaning of " plurality " is two or more, unless otherwise specifically defined.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, " connection " should do broad sense
Understand, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected；It can be mechanical connection, also may be used
To be electrical connection or can be in communication with each other；It can be directly connected, can also can be indirectly connected through an intermediary two
The interaction relationship of connection or two elements inside element.It for the ordinary skill in the art, can basis
Concrete condition understands the concrete meaning of above-mentioned term in the present invention.
Following disclosure provides many different embodiments or example is used for realizing the different structure of the present invention.In order to
Simplify disclosure of the invention, hereinafter to the component of specific examples and being set for describing.In addition, the present invention can be in different examples
Repeat reference numerals and/or reference letter in son, this repetition are for purposes of simplicity and clarity, itself not indicate to be begged for
By the relationship between various embodiments and/or setting.
So-called " sequencing " the same determining nucleic acid sequence of embodiment of the present invention, including DNA sequencing and/or RNA sequencings,
Including long segment sequencing and/or short-movie section sequencing.So-called " sequencing reaction " same to sequencing reaction.Usually, in nucleic acid sequence
In the measurement of row, a base or a certain types of base, alleged base choosing can be measured by a wheel sequencing reaction
From at least one of A, T, C, G and U.In the sequencing reaction for being sequenced in synthesis and/or being sequenced in connection, so-called one
Wheel sequencing reaction includes extension (base extension), information collects (/ Image Acquisition of taking pictures) and group cuts off (cleave).Institute
" nucleotide analog " the i.e. substrate claimed, also referred to as terminator (terminator) are the analog of A, T, C, G and/or U, energy
It enough follows base complementrity principle and certain types of base pairing, next nucleotide/Binding Capacity can be terminated simultaneously to mould
On plate chain.
It please join Fig. 1 and Fig. 2, embodiment of the present invention provides a kind of method controlled sequencing reaction, sequence
It includes the first biochemical reaction to measure reaction, and the first biochemical reaction is carried out using the first reagent 11 on reaction unit 40, and sequence is utilized
Row measurement system controls sequencing reaction.
Sequence Detection System includes fluid means 100, and fluid means 100 includes valve component 10 and drive component 50.
Valve component 10 includes the first valve 20 and the second valve 30, and the first valve 20 and reaction unit 40 connect, the first valve 20 packet
The stator and rotor that can be connected to are included, the first valve 20 has common port, has multiple ports on stator, has connectivity slot on rotor
21, common port can be made to pass through connectivity slot 21 at least one port by rotor and be connected to, multiple ports include first end
Mouth 22, it includes step that the second valve 30, which can connect first port 22, the first reagent 11 and/or the first buffer solution 60, method,：
S11 makes first port 22 be connected to common port by connectivity slot 21；
S12 makes the second valve 30 be connected to the first reagent 11 and first port 22；
S13 makes the first reagent 11 enter reaction unit 40 through the second valve 30 and the first valve 20 successively using drive component 50,
To carry out the first biochemical reaction；
S14 makes the second valve 30 be connected to the first buffer solution 60 and first port 22 before rotor；
S15 makes the first buffer solution followed by the second valve 30 and the first valve 20 using drive component 50.
In the above method, before rotor, the first buffer solution 60 is set to flow into the first valve 20 so that the liquid in connectivity slot 21
Body is substituted before rotor by the first buffer solution 60, in other words, before the rotor rotation of the first valve 20, using to target sequence
Row measure first buffer solution 60 of the reaction without influence instead of the first reagent 11 in connectivity slot 21, avoid and are rotated through in rotor
Original reagent is brought to the other positions of the linkage interface of stator and rotor in connectivity slot 21 in journey, and then has avoided switching not
With the risk of cross contamination when reagent.
Specifically, in some embodiments, reaction unit 40 can be chip, and reaction unit 40 carries sample.It please tie
Fig. 2 is closed, reaction unit 40 includes first unit 41 and second unit 42, and each unit includes plurality of passages (channel), can
Respectively different types of sequencing reaction, first unit 41 are carried out in the channel of first unit 41 and the channel of second unit 42
Channel in sequencing reaction reacted with the sequencing in 42 channel of second unit be staggered, it is nonsynchronous, mutual
It does not influence.For example, when needing to carry out biochemical reaction to the sample in first unit 41, fluid means 100 can be single to first
Member 41 conveys the reagent of reaction, at this point, identical reagent will not be made to enter second unit 42, vice versa.
In embodiments of the present invention, Fig. 2 please be join, each unit is correspondingly connected with that there are one the first valves 20.Specifically, first
The common port of valve 20 is connected to corresponding unit so that the reagent exported from the common port of the first valve 20 can enter corresponding unit
Carry out biochemical reaction.In this way, the process of sequencing can be accelerated.
In some embodiments, before carrying out sequencing reaction, the first unit 41 of reaction unit 40 and the second list
The sample for needing sequencing has been fixed on the surface in the channel of member 42, has waited for that the sample of sequencing is, for example, to have double-strand or list
The DNA chain of chain structure.
Between first reagent 11 and the second valve 30, between the first buffer solution 60 and the second valve 30, port and the second valve 30 it
Between, can connect and be connected to by hose between the second valve 30 and the first valve 20 and/or between the first valve 20 and reaction unit 40, such as
This, hose can make the configuration of fluid path more flexible.
In some embodiments, multiple-way valve can be used in the first valve 20.Triple valve can be used in second valve 30, such as three are powered
Magnet valve, the normally closed port and normally open of three-way magnetic valve respectively connected the reagent and buffer solution that need to be added.In certain embodiment party
In formula, the first valve 20 can be rotary valve, in this way, reacting having a wide range of application for the method controlled to sequencing.
In some embodiments, common port is opened on stator, multiple ports around common port be arranged, and common port with
One end of connectivity slot 21 corresponds to connection.In other embodiments, common port is opened on rotor, and positioned at connectivity slot 21
One end.
In embodiments of the present invention, step S11 is implemented before step S12, in other embodiments, step S12
It can implement before step S11 or step S11 and step S12 can be implemented simultaneously.
So-called buffer solution is that can maintain liquid pH in the solution of particular range to a certain degree, be weak acid, weak base and/or in
Property solution.In some embodiments, the first buffer solution be do not influence the first biochemical reaction and/or sequencing reaction it is other
The solution of biochemical reaction.
Usually, the sealing of the first valve 20 is substantially by the end face seal between stator and rotor, in rotor, even
Agent liquid in straight slot 21 can be between the stator and the rotor sealing surface have residual.As shown in figure 3, carrying out the first biochemical reaction
When, the first reagent 11 as the port 1 of first port 22, connectivity slot 21 and common port 0 through entering reaction unit 40.And it is needing
When carrying out other biochemical reactions, when the first reagent 11 in connectivity slot 21 does not clean, when rotor goes to port from port 1
When 2, the first reagent 11 in connectivity slot 21 can remain on the region between port 1 and port 2 (the delta in such as Fig. 3
Domain), these remaining first reagents 11 can pollute the other reagents entered through other ports with the rotation of rotor.So
It please join Fig. 2 and Fig. 4, triple valve is externally connected in the port of stator, before rotor, triple valve be made to be connected to the first buffer solution 11
With first port 22, and make the first buffer solution 60 followed by the second valve and the first valve 20 using drive component 50, and then to even
The case where remaining first reagent 11 is cleaned, significantly improves cross contamination in straight slot 21.It is appreciated that the present invention shows
In example, the second valve 30 may include the one or more of triple valve V1-V8.First port 22 may include one in the 1-8 of port or
It is multiple.
Below to test the cross contamination performance to illustrate to improve front and back.In this test, the first valve 20 is with rotary valve
For illustrate.
First, existing two rotary valve in the market is chosen, and builds test platform as shown in figure 5, using test platform
Assess the cross contamination performance of two rotary valve (calling rotary valve A1 and rotary valve B1 in the following text).Incorporated by reference to Fig. 4, adjacent port is selected
1,2 and 8 as testing, and port 1 is connected to fluorescent reagent 1, and port 2 and port 8 are buffer solution, reaction unit flow cell
There are two parallel channel A and B, test operation details as follows：
(1) the channel A of selection reaction unit flow cell, makes fluorescent reagent 1 flow through channel A, so using drive component 50
After rotate clockwise rotor, with port switching, make 21 communications ports 2 of connectivity slot and common port 0, make excess using drive component 50
Buffer solution through port 2 enter rotary valve, ensure rotary valve in the fluid path including common port 0 and connectivity slot 21 fluorescence examination
Agent 1 all cleans up；
(2) fluid path is switched to the channel B of reaction unit flow cell, is first taken using single molecule fluorescence detection system
The background of channel B, statistics fluorescence points n1；Then rotor counterclockwise makes the switching of connectivity slot 21 be communicated to port 8, that is, holds
Mouth 8 is connected to common port 0, so that a certain amount of buffer solution is entered rotary valve through port 8 using drive component 50 and is flowed to reaction dress
The channel B for setting flow cell uses the same regions single molecule fluorescence detection system photographs channel B, statistics fluorescence points n2；
(3) since enter rotary valve through port 2 and port 8 is that buffer solution, unstressed configuration point, therefore n2-n1 can be considered
Rotary valve is switched to port 2 (clockwise) from port 1 and is switched to caused cross contamination during port 8 (counterclockwise) again,
The increase of n2-n1, that is, fluorescence points must be that fluorescent reagent 1 is mixed into the buffer solution entered through port 8 and is detected glimmering
Spot number, therefore the numerical value can assess cross contamination severity when rotating Vavle switching.
It is the initial data of 8 groups of tests shown in Fig. 6, it can be seen that either rotary valve A1 or rotary valve B1 can not
The cross contamination for avoiding reagent, when being happened at rotor due to the pollution, after being passed through fluorescent reagent 1, even if connectivity slot
21 be switched to port 2 carry out rotary valve cleaning after be switched to port 8 again, can not also avoid always fluorescent reagent 1 be mixed into through end
Mouthfuls 8 buffer solutions entered and generate pollution, therefore, common cleaning process also can not be solved the problems, such as thoroughly.It should be noted that
In Fig. 6, in the block diagram shown in the same test group, the data of n1, the column on the right before the histogram graph representation rotation on the left side
Shape figure indicates the data of n2 after rotation.
In embodiment of the present invention, before rotor, the second valve 30 is made to be connected to the first buffer solution 60 and first port 22,
And make the first buffer solution 60 followed by the second valve 30 and the first valve 20 using drive component 50, above-mentioned cross contamination feelings can be improved
Condition.Specifically, Fig. 4 please be join, illustrated so that the second valve 30 is three-way magnetic valve as an example.The normally closed port of three-way magnetic valve and normally opened
Mouth respectively connected the reagent and buffer solution for needing to be added, for example, solenoid valve V1 powers on (port 1 is connected to reagent 1 at this time),
After reagent 1 introduces rotary valve by drive component 50, solenoid valve V1 (port 1 is connected to buffer solution at this time) is immediately closed, is utilized
What drive component 50 made to remain in the connectivity slot 21 that a small amount of buffer solution (determining specific amount according to pipeline situation) washes
Reagent 1 there will be no to remain in the end of rotary valve when reagent 1, in this way cleaning and then rotor switching different port
On face, although remaining is buffer solution, on biochemical reaction without influence, the method can be substantially reduced and be avoided in other words buffer solution
Reagent cross contamination caused by being rotated due to rotary valve.
Similarly, using single molecule fluorescence detection system, the cross contamination situation after assessment improvement, initial data such as Fig. 7
It is shown.The comparison for improving front and back n2-n1 is as shown in Figure 8.Known to Fig. 8, it can be seen that compared to before rotary valve A1 and
The method of B1, embodiment of the present invention so that the cross contamination of rotary valve is obviously improved, embodiment of the present invention
Method prevent reagent cross contamination from source, be very suitable for applying in the occasion very sensitive to micro cross contamination, than
Such as unimolecule gene sequencer system.It should be noted that in Fig. 7, in the block diagram shown in the same test No., the left side
Histogram graph representation rotation before n1 data, the right histogram graph representation rotation after n2 data.In Fig. 8, in the same test
In block diagram shown in group, the data of the n2-n1 after the histogram graph representation improvement on the left side, before intermediate histogram graph representation improves
The data of the n2-n1 of rotary valve A1, the histogram graph representation on the right improve the data of the n2-n1 of preceding rotary valve B1.
In some embodiments, Fig. 9 please be join, sequencing reaction includes the second biochemical reaction, and the second biochemical reaction is adopted
It being carried out on reaction unit 40 with the second reagent 12, valve component 10 includes third valve 31, and multiple ports include second port 23,
It includes step that third valve 31, which can connect second port 23, the second reagent 12 and/or the second buffer solution, method,：
S16, rotor make connectivity slot 21 be connected to second port 23 and common port；
S17 makes third valve 31 be connected to the second reagent 12 and second port 23；
S18 makes the second reagent 12 enter reaction unit 40 through third valve 31 and the first valve 20 successively using drive component 50,
To carry out the second biochemical reaction；
S19 makes third valve 31 be connected to the second buffer solution and second port 23 before rotor；
S20 makes the second buffer solution followed by third valve 31 and the first valve 20 using drive component 50.
In this way, the method for embodiment of the present invention can be applied in sequencing reacts need to carry out multiple and different types
Biochemical reaction expands the application range of the method for embodiment of the present invention.
Specifically, in the present example, incorporated by reference to Fig. 4, second port 23 may include one or more in the 1-8 of port
A, third valve 31 may include one or more of triple valve V1-V8.It should be pointed out that the second valve 30 and third valve 31 are answered
Select the different valves in triple valve V1-V8.First port 22 and second port 23 answer the different port in selection port 1-8.
It should be noted that the second buffer solution is the solution for not influencing the first biochemical reaction, the first buffer solution 60 is not shadow
Ring the solution of the second biochemical reaction.
In the example of Fig. 2 of the present invention, the second buffer solution and the first buffer solution 60 are same buffer solution.Certainly, second is slow
Fliud flushing and the first buffer solution are alternatively chosn to different buffer solutions.In one example, the first buffer solution and the second buffer solution are
Same buffer solution is the buffer solution of " 150mM HEPES, 150mM NaCl, pH=7.0 ", is not influenced on sequencing reaction.
In some embodiments, one of stator of the first valve 20 port 70 can be connected to outside air, with side
Just air is introduced pipeline is carried out to remove agent liquid.
In embodiments of the present invention, step S16 is implemented before step S17, in other embodiments, step S17
It can implement before step S16 or step S16 and step S17 can be implemented simultaneously.
In some embodiments, the first biochemical reaction includes extension.
Specifically, extension be connected on the sample for waiting for sequencing based on base complementrity, by specific substrates, and
The type in conjunction with upper substrate is measured using the detectable group carried on substrate, to measure sequence.In one example, may be used
Detection moiety includes fluorophor, can send out fluorescence under the laser of specific wavelength.
In embodiments of the present invention, so-called first reagent is terminator reagent, i.e. reaction substrate, including A, T, C and G
Base analogue, specifically, the structure of so-called base analogue, that is, terminator is that A/T/C/G- terminates group-connection unit-
Luminophore, that is, so-called first reagent is that the reagent comprising A- termination group-connection unit-luminophores (calls A examinations in the following text
Agent), comprising T- terminate group-connection unit-luminophore reagent (calling T reagents in the following text), comprising C- terminate group-connection list
The reagent (calling C reagents in the following text) of member-luminophore and/or the reagent that group-connection unit-luminophore is terminated comprising G- (call G in the following text
Reagent).Termination group therein be light and/or chemistry can cleavable groups, make substrate with hair by connection unit (linker)
Light group.
In a specific example, the luminophores of four kinds of terminator institute bands is the same structure or is sent out when being excited
The detectable light of the same color, four kinds of base analogues are contained in respectively in different reagent bottles.When sequencing, sequentially add
A, one kind in T, C and G terminator, every four kinds of terminators reaction are known as a cycle.The reagent bottle for holding different terminators is logical
Cross triple valve, the first valve is connect with reaction unit.
In an example below in conjunction with Fig. 4 to illustrate the present invention, the adding procedure of mentioned reagent.
In Fig. 4, reagent 1 is A reagents, and reagent 2 is T reagents, and reagent 3 is C reagents, and reagent 4 is G reagents.Prolonged
When stretching reaction, triple valve V1 is powered on, and triple valve V2-V8 is closed, and port 1 is connected to A reagents, 21 communications ports 1 of connectivity slot and public
Mouth 0, drive component 50 makes A reagents be reacted in triple valve V1 and the first valve 20 entrance reaction unit 40, in rotor
Before, triple valve V1 is closed, and port 1 is connected to buffer solution, and drive component 50 makes buffer solution flow through triple valve V1 and the first valve 20.Rear
Continuous when needing to change T reagents, C reagents, G reagents and/or the other reagents of addition, rotor makes connectivity slot 21 be connected to common port 0
With corresponding port, and carried out according to above-mentioned process.
In some embodiments, the second biochemical reaction includes group excision.
It specifically, need to will be on the terminator of a upper structure when adding in the terminator to reaction unit 40 of different structure
The terminator of another structure is added after luminophore excision again.For example, incorporated by reference to above-mentioned example, A reagents are being added to reaction
When in device 40, exciting light is sent out to reaction unit 40, to excite luminophore to send out using light-emitting device (such as laser)
Fluorescence, and taken pictures using imaging device to acquire fluorescence, and image is formed to carry out sequencing.After the completion of taking pictures, A need to be tried
Other reagents are added again after the luminophore excision of agent.Further, in this example embodiment, reagent 5 is that the reagent of excision (is called in the following text
Cut off reagent).
After the completion of taking pictures, when addition cuts off reagent, rotor makes 21 communications ports 5 of connectivity slot and common port 0, and three
Port valve V5 is powered on, and triple valve V1-V4 and V6-V8 are closed, and the connection of port 5 excision reagent, drive component 50 makes excision reagent through three
Port valve V5 and the first valve 20, which enter in reaction unit 40, carries out excision reaction, and before rotor, triple valve V5 is closed, port 5
It is connected to buffer solution, drive component 50 makes buffer solution flow through triple valve V5 and the first valve 20.
In some embodiments, extension is carried out using ligase and/or polymerase.
In some embodiments, the second biochemical reaction includes capping.
Specifically, so-called to cap the group/key being exposed after predominantly blocking group excision.In one example,
First biochemical reaction includes extension, and the second biochemical reaction includes group excision, and base can be broken by light and/or chemical ablation
After group, the group being exposed is sulfydryl, by capping such as by the way that alkylating reagent is added, sulfydryl can be protected not oxidized.
Incorporated by reference to above-mentioned example, further, in this example embodiment, reagent 6 (calls in the following text to cap added reagent and caps examination
Agent).When addition caps reagent, rotor makes 21 communications ports 6 of connectivity slot and common port 0, triple valve V6 power on, threeway
Valve V1-V5 and V7-V8 are closed, and the connection of port 6 caps reagent, and drive component 50 makes to cap reagent through triple valve V6 and the first valve 20
Reaction is capped in into reaction unit 40, before rotor, triple valve V6 is closed, and port 6 is connected to buffer solution, driving group
Part 50 makes buffer solution flow through triple valve V6 and the first valve 20.
It should be pointed out that in some embodiments, the first reagent may include not having to the biochemical reaction in sequencing
The reagent of influence, at this point, in the reagent after the second valve and the first valve 20 enter reaction unit 40, and before rotor, and
The second valve and the first valve 20 need not be flowed through with fliud flushing or buffer solution, in this way, the time of sequencing reaction can be saved.
In some embodiments, drive component 50 includes pump, and pump is connected to common port by reaction unit 40.
In this way, the driving to reagent and buffer solution can be achieved using pump, control method is simple and practicable.
Specifically, in the present example, pump includes the first pump 51 and the second pump 52, and the first pump 51 passes through first unit 41
It is connected to the common port of one of them the first valve 20, the second pump 52 is connected to the public of another the first valve 20 by second unit 42
Mouthful, so that the first reagent and the first buffer solution is entered first unit 41 through the second valve 30 and the first valve 20 successively using the first pump 51,
The first reagent and the first buffer solution is set to enter second unit 42 through the second valve 30 and the first valve 20 successively using the second pump 52.
In this way, it is single to realize that the agent liquid for exporting the first valve 20 is input to first respectively using the first pump 51 and the second pump 52
Member 41 and/or second unit 42, are conveniently operated.
Specifically, the first pump 51 connects first unit 41 and second unit 42 with 52 difference pipeline of the second pump, for example, passing through
Hose connects.
First pump 51 is connected to the common port of one of them the first valve 20 by first unit 41, and the second pump 52 is single by second
Member 42 is connected to the common port of another the first valve 20, and when work, the first pump 51 provides negative pressure to first unit 41, so that first
Other doses (including buffer solution and/or other reagents) that unit 41 obtains the first reagent and/or connect with the port of the first valve 20
Biochemical reaction and/or cleaning are carried out, after first unit 41 has obtained dose liquid, the first pump 51 stops providing negative pressure.
First pump 51 makes any agent liquid enter first unit 41, depends on：1) which port connectivity slot 21 is connected to；With it is 2) right
In that port (calling communications ports in the following text) being connected to connectivity slot 21, communications ports and which triple valve being connect with communications ports make
Kind agent liquid connection.For example, incorporated by reference to Fig. 4,21 communications ports 1 of connectivity slot, the triple valve V1 being connect with port 1 makes port 1 and examination
Agent 1 is connected to, then when the first pump 51 provides negative pressure, reagent 1 enters first unit 41 through triple valve V1 and the first valve 20.
Similarly, the operation of the second pump 52 can join the operation of the first pump 51.
Further, in some embodiments, drive component 50 further includes the 4th valve 53, the 5th valve 54 and waste liquid bottle
55.4th valve, 53 pipeline is connected between the first pump 51 and first unit 41, while going back pipeline connection waste liquid bottle 55.5th valve 54
Pipeline is connected between the second pump 52 and second unit 42, while going back pipeline connection waste liquid bottle 55.
First pump 51 is connected to first unit 41 or waste liquid bottle 55 through the 4th valve 53, to which the first pump 51 extracts first unit 41
After the waste liquid for inside having completed sequencing reaction, waste liquid can be injected to waste liquid bottle 55, so that the first pump 51 carries out down
Negative pressure once is provided to first unit 41, to carry out sequencing reaction.The setting identical as the 4th 53 structure of 5th valve 54, herein
It repeats no more.In some instances, the 4th valve 53 and the 5th valve 54 can be triple valve.
In some embodiments, fluid means 100 includes control unit, and control unit is electrically connected valve component 10 and drives
Dynamic component 50 is run with application valve body component 10 and drive component 50.
So, it can be achieved that valve component 10 and drive component 50 automation control, and then improve efficiency.
Specifically, in the present example, control unit is electrically connected the first valve 20, the second valve 30, third valve 31 and driving
Component 50 is run with controlling the first valve 20, the second valve 30, third valve 31 and drive component 50.Control unit can be include single
The devices such as piece machine, computer processor or central control processor control the first valve 20, triple valve V1-V8 using control unit
It is run with drive component, realizes 100 automatic running of fluid means, improve efficiency.
In some embodiments, incorporated by reference to Fig. 2 and Fig. 4, multiple port distributions are rounded, and common port is set with circular shape concentric
It sets.
In this way, connectivity slot when multiple ports of rounded distribution and common port ensure that rotor with circular shape concentric setting
21 accuracys being connected to corresponding port and common port.
In some embodiments, incorporated by reference to Fig. 2 and Fig. 4, connectivity slot 21 is in linear.It is being connected in this way, agent liquid can be reduced
Flow path in slot 21, and then realize and ensure quickly sequencing.
Specifically, be in linear connectivity slot 21, can with shorter path be connected to positioned at 21 both ends of connectivity slot port and
Common port.In the present example, linear is linear.
It please join Figure 10, a kind of Sequence Detection System 300 of embodiment of the present invention controls sequencing reaction,
Sequencing reaction includes the first biochemical reaction, and the first biochemical reaction is carried out using the first reagent 11 on reaction unit 40.
Sequence Detection System 300 includes control device 302 and fluid means 100,302 connecting fluid device of control device
100, fluid means 100 includes valve component 10 and drive component 50.
Valve component 10 includes the first valve 20 and the second valve 30, and the first valve 20 and reaction unit 40 connect, the first valve 20 packet
The stator and rotor that can be connected to are included, the first valve 20 has common port, has multiple ports on stator, has connectivity slot on rotor
21, common port can be made to pass through connectivity slot 21 at least one port by rotation rotor and be connected to, multiple ports include first end
Mouth 22, the second valve 30 can connect first port 22, the first reagent 11 and/or the first buffer solution 60, control device 302 and be used for：
First port 22 is set to be connected to common port by connectivity slot 21；
The second valve 30 is set to be connected to the first reagent 11 and first port 22；
So that the first reagent 11 is entered reaction unit 40 through the second valve 30 and the first valve 20 successively using drive component 50, with into
The first biochemical reaction of row；
Before rotor, the second valve 30 is made to be connected to the first buffer solution 60 and first port 22；
Make the first buffer solution 60 followed by the second valve 30 and the first valve 20 using drive component 50.
In above-mentioned Sequence Detection System 300, before rotor, the first buffer solution 60 is made to flow into the first valve 20 so that even
Liquid in straight slot 21 is substituted before rotor by the first buffer solution 60, in other words, before the rotor rotation of the first valve 20,
First buffer solution 60 of the reaction without influence is measured instead of the first reagent 11 in connectivity slot 21 using on target sequence, is avoided
Original reagent is brought to the other positions of the linkage interface of stator and rotor in connectivity slot 21 in rotor rotation process, in turn
The risk of cross contamination when having avoided switching different reagents.
It should be noted that reacting the method controlled to sequencing in any of the above-described embodiments and examples
Technical characteristic and the explanation and illustration of advantageous effect be also applied for the Sequence Detection System 300 of present embodiment, it is superfluous to avoid
It is remaining, it is no longer developed in details herein.
In some embodiments, sequencing reaction includes the second biochemical reaction, and the second biochemical reaction is using the second examination
Agent 12 carries out on reaction unit 40, and valve component 10 includes third valve 31, and multiple ports include second port 23, third valve 31
Second port 23, the second reagent 12 and/or the second buffer solution, control device 302 can be connected to be used for：
Rotor makes connectivity slot 21 be connected to second port 23 and common port；
Third valve 31 is set to be connected to the second reagent 12 and second port 23；
So that the second reagent 12 is entered reaction unit 40 through third valve 31 and the first valve 20 successively using drive component 50, with into
The second biochemical reaction of row；
Before rotor, third valve 31 is made to be connected to the second buffer solution and second port 23；
Make the second buffer solution followed by third valve 31 and the first valve 20 using drive component 50.
In some embodiments, the first biochemical reaction includes extension.
In some embodiments, the second biochemical reaction includes group excision.
In some embodiments, extension is carried out using ligase and/or polymerase.
In some embodiments, the second biochemical reaction includes capping.
In some embodiments, drive component 50 includes pump, and pump is connected to common port by reaction unit 40.
In some embodiments, fluid means 100 includes control unit, and control device 302 connects control unit, control
Unit is electrically connected valve component 10 and drive component 50 and is run with application valve body component 10 and drive component 50.
Specifically, control unit can receive the control signal of control device 302, and according to control signal to valve component
10, other components of drive component 50 and fluid means 100 are controlled.In this way, in this way can be by the part of control device 302
Function executes realization by control unit, reduces the load of control device 302.In some embodiments, control unit and
Control device 302 can be integrated in component, module or a device, to improve the integrated level of Sequence Detection System 300, reduce at
This.
In some embodiments, multiple port distributions are rounded, and common port is arranged with circular shape concentric.
In some embodiments, connectivity slot 21 is in linear.
It please join Figure 10, embodiment of the present invention provides a kind of device 302 controlled sequencing reaction, device
302 include：
Storage device 304, for storing data, data include computer executable program；
Processor 306, for executing computer executable program, it includes completing above-mentioned to execute computer executable program
The method of one embodiment.
A kind of computer readable storage medium of embodiment of the present invention is held for storing the program executed for computer
Line program includes the method for completing any of the above-described embodiment.Computer readable storage medium may include：Read-only memory, with
Machine memory, disk or CD etc..
In the description of this specification, reference term " embodiment ", " certain embodiments ", " schematically implementation
What the description of mode ", " example ", " specific example " or " some examples " etc. meant to describe in conjunction with the embodiment or example
Particular features, structures, materials, or characteristics are contained at least one embodiment or example of the present invention.In this specification
In, schematic expression of the above terms are not necessarily referring to identical embodiment or example.Moreover, the specific spy of description
Sign, structure, material or feature can be combined in any suitable manner in any one or more embodiments or example.
Expression or logic and/or step described otherwise above herein in flow charts, for example, being considered use
In the order list for the executable instruction for realizing logic function, may be embodied in any computer readable storage medium,
For instruction execution system, device or equipment (system of such as computer based system including processor or other can be from finger
Enable the system for executing system, device or equipment instruction fetch and executing instruction) it uses, or combine these instruction execution systems, device
Or equipment and use.For the purpose of this specification, " computer readable storage medium " can be it is any can include, store, communicating,
Propagate or transmission program for instruction execution system, device or equipment or in conjunction with these instruction execution systems, device or equipment and
The device used.
In addition, each functional unit in each embodiment of the present invention can be integrated in a processing module, also may be used
To be that each unit physically exists alone, can also two or more units be integrated in a module.It is above-mentioned integrated
The form that hardware had both may be used in module is realized, can also be realized in the form of software function module.The integrated module
If being realized in the form of software function module and when sold or used as an independent product, a calculating can also be stored in
In machine read/write memory medium.
Although embodiments of the present invention have been shown and described above, it is to be understood that the above embodiment is
Illustratively, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be right
The above embodiment is changed, changes, replacing and modification.
Claims (9)
1. a kind of reacting the method controlled to sequencing, which is characterized in that the sequencing reaction includes the first life
Change reaction, first biochemical reaction is carried out using the first reagent on reaction unit, using Sequence Detection System to the sequence
Row measure reaction and are controlled,
The Sequence Detection System includes fluid means, and the fluid means includes valve component and drive component,
The valve component includes the first valve and the second valve, and first valve is connected with the reaction unit, the first valve packet
The stator and rotor that can be connected to are included, first valve has common port, has multiple ports on the stator, have on the rotor
There is connectivity slot, the common port and at least one port can be made to pass through the connectivity slot by the rotation rotor and connected
Logical, the multiple port includes first port, and second valve can connect the first port, first reagent and/or the
One buffer solution, the method includes the steps：
The first port is set to be connected to the common port by the connectivity slot；
Second valve is set to be connected to first reagent and the first port；
First reagent is set to be filled successively into the reaction through second valve and first valve using the driving component
It sets, to carry out first biochemical reaction；
Before rotating said rotor, second valve is made to be connected to first buffer solution and the first port；
Make first buffer solution followed by second valve and first valve using the driving component.
2. the method as described in claim 1, which is characterized in that the sequencing reaction includes the second biochemical reaction, described
Second biochemical reaction is carried out using the second reagent on the reaction unit, and the valve component includes third valve, the multiple
Port includes second port, and the third valve can connect the second port, second reagent and/or the second buffer solution, institute
The method of stating includes step：
Rotating said rotor makes the connectivity slot be connected to the second port and the common port；
The third valve is set to be connected to second reagent and the second port；
Second reagent is set to be filled successively into the reaction through the third valve and first valve using the driving component
It sets, to carry out second biochemical reaction；
Before rotating said rotor, the third valve is made to be connected to second buffer solution and the second port；
Make second buffer solution followed by the third valve and first valve using the driving component.
3. the method as described in claim 1, which is characterized in that first biochemical reaction includes extension；
Optional, second biochemical reaction includes group excision；
Optional, the extension is carried out using ligase and/or polymerase；
Optional, second biochemical reaction includes capping.
4. the method as described in claim 1, which is characterized in that the driving component includes pump, and the pump passes through the reaction
Device is connected to the common port；
Optional, the fluid means includes control unit, and described control unit is electrically connected the valve component and the driving
Component is run with controlling the valve component and the driving component；
Optional, the multiple port distribution is rounded, and the common port is arranged with the circular shape concentric；
Optional, the connectivity slot is in linear.
5. a kind of Sequence Detection System controls sequencing reaction, which is characterized in that the sequencing, which reacts, includes
First biochemical reaction, first biochemical reaction are carried out using the first reagent on reaction unit,
The Sequence Detection System includes control device and fluid means, and the control device connects the fluid means, described
Fluid means includes valve component and drive component,
The valve component includes the first valve and the second valve, and first valve is connected with the reaction unit, the first valve packet
The stator and rotor that can be connected to are included, first valve has common port, has multiple ports on the stator, have on the rotor
There is connectivity slot, the common port and at least one port can be made to pass through the connectivity slot by the rotation rotor and connected
Logical, the multiple port includes first port, and second valve can connect the first port, first reagent and/or the
One buffer solution, the control device are used for：
The first port is set to be connected to the common port by the connectivity slot；
Second valve is set to be connected to first reagent and the first port；
First reagent is set to be filled successively into the reaction through second valve and first valve using the driving component
It sets, to carry out first biochemical reaction；
Before rotating said rotor, second valve is made to be connected to first buffer solution and the first port；
Make first buffer solution followed by second valve and first valve using the driving component.
6. system as claimed in claim 5, which is characterized in that the sequencing reaction includes the second biochemical reaction, described
Second biochemical reaction is carried out using the second reagent on the reaction unit, and the valve component includes third valve, the multiple
Port includes second port, and the third valve can connect the second port, second reagent and/or the second buffer solution, institute
Control device is stated to be used for：
Rotating said rotor makes the connectivity slot be connected to the second port and the common port；
The third valve is set to be connected to second reagent and the second port；
Second reagent is set to be filled successively into the reaction through the third valve and first valve using the driving component
It sets, to carry out second biochemical reaction；
Before rotating said rotor, the third valve is made to be connected to second buffer solution and the second port；
Make second buffer solution followed by the third valve and first valve using the driving component.
7. system as claimed in claim 5, which is characterized in that first biochemical reaction includes extension；
Optional, second biochemical reaction includes group excision；
Optional, the extension is carried out using ligase and/or polymerase；
Optional, second biochemical reaction includes capping.
8. system as claimed in claim 5, which is characterized in that the driving component includes pump, and the pump passes through the reaction
Device is connected to the common port；
Optional, the fluid means includes control unit, and the control device connects described control unit, described control unit
It is electrically connected the valve component and the driving component and is run with controlling the valve component and the driving component；
Optional, the multiple port distribution is rounded, and the common port is arranged with the circular shape concentric；
Optional, the connectivity slot is in linear.
9. a kind of reacting the device controlled to sequencing, including：
Storage unit, for storing data, the data include computer executable program；
Processor, for executing the computer executable program, it includes completing as weighed to execute the computer executable program
Profit requires 1-4 any one of them methods.
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| CN201710098314.3A CN108456748B (en) | 2017-02-22 | 2017-02-22 | Method, device and system for controlling sequence determination reaction |
| PCT/CN2018/077387 WO2018153377A1 (en) | 2017-02-22 | 2018-02-27 | Method, device, and system for controlling sequencing reaction |
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| CN110904206A (en) * | 2019-12-18 | 2020-03-24 | 深圳市真迈生物科技有限公司 | Liquid path system, biomolecule analysis system and nucleic acid sequence measuring system |
| CN114682310A (en) * | 2020-12-31 | 2022-07-01 | 深圳市真迈生物科技有限公司 | Liquid path system, sequencing system and method |
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| WO2021120651A1 (en) * | 2019-12-18 | 2021-06-24 | 深圳市真迈生物科技有限公司 | Liquid path system, biomolecule analysis system and nucleotide sequencing system |
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| CN114682310B (en) * | 2020-12-31 | 2023-12-05 | 深圳市真迈生物科技有限公司 | Liquid path system, sequencing system and method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108456748B (en) | 2023-04-25 |
| WO2018153377A1 (en) | 2018-08-30 |
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