CN100374579C - Improved system for multicolor real time PCR - Google Patents
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- CN100374579C CN100374579C CNB2004800090267A CN200480009026A CN100374579C CN 100374579 C CN100374579 C CN 100374579C CN B2004800090267 A CNB2004800090267 A CN B2004800090267A CN 200480009026 A CN200480009026 A CN 200480009026A CN 100374579 C CN100374579 C CN 100374579C
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Abstract
The invention is directed to a system for performing multi-color real time PCT, comprising a flexible real time PCT instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixtuer which comprises at least 3, preferably 4-5 and most preferably exactly 4 pairs of FRET hybridization probes. Each pair of said hybridization probes consists of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.
Description
Invention field
The present invention relates to the field of PCR in real time.Particularly, the present invention relates to carry out the system of multiple PCR in real time.
The prior art background
By polymerase chain reaction (PCR) DNA amplification is molecular biological basic fundamental.
Foranalysis of nucleic acids by PCR needs specimen preparation, amplification and product analysis.Although these steps are normally sequentially carried out, amplification and analysis can take place simultaneously.DNA dyestuff or fluorescent probe can be added in the PCR mixture before the amplification, and during increasing, be used to analyze the PCR product.Sample analysis occurs in the interior same test tube of same instrument simultaneously with amplification.This bonded method is not owing to will sample shift out from their airtight containers and be used for further analysis, thus reduced sample preparation, saved the time, and greatly reduced and be used for the contaminated risk of the product of afterreaction.To increase and product analysis bonded notion be known as " in real time " PCR.See, for example, U.S. Patent No. 6,174,670.
Monitor fluorescence in each PCR cycle period and relate to the use ethidium bromide at first.(HiguchiR, G Dollinger, PS Walsh and R.Griffith, Simultaneous amplificationand detection of specific DNA sequences, Bio/Technology 10 (1992) 413-417; Higuchi R, C Fockler G Dollinger and R Watson, Kinetic PCRanalysis:real time monitoring of DNA amplification reactions, Bio/Technology 11 (1993) 1026-1030).In this system, the relative measurement value of first order fluorescence as production concentration measured in each circulation.Ethidium bromide detects double-stranded DNA; If there is template, fluorescence intensity increases along with temperature cycle so.In addition, at first detecting the cycle number of fluorescence increase and the logarithm of initial template concentrations increases inversely.Other fluorescing system that can provide about the other data of nucleic acid concentration and sequence has been provided.
In dynamic real-time PCR, the formation of monitoring PCR product in each PCR circulation.
During amplified reaction, usually measure amplification at thermal cycler with the device that is used for measuring fluorescent signal in addition.
The PCR in real time instrument
The real-time detection thermal cycler of several types known in the state of the art.
For example, EP 0 640 828 discloses a kind of instrument that is used for monitoring simultaneously a plurality of nucleic acid amplifications.It is characterized in that it comprises a metal block thermal cycler, comprising a heat conduction component that has in order to hold porous flat plate such as microtiter plate a plurality of recesses that form within it.Method by the CCD photographic camera obtains to detect, and described photographic camera is arranged for detecting the light that (simultaneously) launched from all described recesses.Selectively, fibre optics is used in suggestion.Depend on the fluorescence dye of use, the more important thing is and depend on the filter wheel that exists near the CCD photographic camera, this system has the ability of analyzing multiplex amplification reaction, and wherein in a reaction chamber, the hybridization probe by 2 or more a plurality of not isolabelings detects one or more different amplicons.Yet EP 0 640 828 is not also not suggestion of expection both, which mark or test format can be used for this multiple/the polychrome method.
US 6,015,674 discloses a kind of PCR in real time that is used for detects and quantitative instrument and system, and feature is that it can detect first and second fluorescent indicators, in order to detect different amplicons in same reaction vessel, described indicator can be used as the mark of different hybridization probes.Yet US 6,015, and 674 openly are not used to have the system of the multiple experiment of higher complexity.
Another general example is Roche Diagnostics LightCycler (Cat.No.20110468).It is an a kind of fast PCR system, can carry out dynamically online (on-line) PCR quantitatively and the analysis of PCR product melting curve subsequently.The optical system that current commercially available LightCycler is 1.2 editions comprises a light source, a blue light-emitting diode (470nm LED) and three sense channels.By only when they methods of the fluorescently-labeled hybridization probe of emitting fluorescence signal or also detect amplified production in some cases just during in conjunction with target nucleic acid by method in conjunction with the fluorescence dye of double-stranded DNA.To the defined signal threshold value of all reaction assay that will analyze, and for target nucleic acid and for reference nucleic acid such as standard or house-keeping gene measure and to reach the needed cycle number Cp of this threshold value.Can on the basis of the Cp value that target nucleic acid and reference nucleic acid are obtained, measure the absolute or relative copy number of target molecule.
To be divided into the different wave length that can in one of three sense channels (530/640/710nm), be recorded by sample institute emitted fluorescence by a series of dichroic mirrors and colour filter.This allows the detection of double-stranded DNA bonded dyestuff SybrGreenI, the monochrome detection of TaqMan probe form and the double-colored detection of hybridization probe (HybProbe) form.The details of Lightcycler system is disclosed in WO 97/46707, WO 97/46712 and WO 98/46714.Here the full content with these application is incorporated herein by reference.
A very important feature of LightCycler instrument is a color compensating software.This in principle software allows to carry out accurately quantitative and curve analysis by the method for the spectra overlapping of the fluorescent radiation of proofreading and correct the temperature dependency mode of being monitored.At US 6,197, ins and outs are disclosed in 520.
With the LightCycler system class seemingly, Corbett Rotor-Gene PCR in real time thermal cycler (www.corbettresearch.com) is a kind of 4 channel and multiple systems, it comprise 4 different LEDs as excitaton source and corresponding photorectifier as fluorescence detection unit.Therefore, although this instrument hardware has in a reaction vessel and maximum 4 hybridization probe of isolabeling abilities of carrying out multiple experiment not at least in theory, yet there is not the open scheme of successful Application separately up to now.
Another PCR in real time instrument is a Biorad iQ multicolor real time PCR detection system (Cat.No:170-8740), and it allows fluorophore exciting and launch from 400nm to 700nm.This system is based on the porous heat block of the routine that is used for thermal cycling, and as the tengsten lamp of excitaton source, the filter wheel of the excitation wavelength that is used to provide suitable is used to select second filter wheel of suitable emission wavelength, and as the CCD photographic camera of detecting unit.This instrument has been successfully used to detect the multiple assay of 4 different amplicons that produce from the target of volumetric molar concentration such as similar, wherein in same reaction vessel, use 4 not TaqMan probe (Pedersen of isolabeling, S., Bioradiations 107 (2001) 10-11).
In another method that improves multiple ability, US 6,369,893 disclose a kind of PCR in real time thermal cycler, and it comprises first Optical devices with at least two light sources and comprises at least two second Optical devices that are used to detect and distinguish the detector of different emission light.Particularly, disclose with 4 different LEDs as light source and the specific embodiments of 4 different photorectifiers as detector.Therefore, 6,369, disclosed instrument can be used for having the PCR in real time of extensive selection to detect to different fluorescence dyes well known in the prior art in principle in 893.Yet US 6,369, and 893 had not both had expection to advise how designing comprising each any method with the multiple experiment of a plurality of different probes of different fluorescence entity marks yet.
The PCR in real time test format
Generally speaking, exist to be used for detecting in real time the multi-form of institute's DNA amplification, wherein followingly be commonly known in the art and be normally used:
A) dna binding dye form
Because the amount of double-stranded amplified production surpasses the amount of the nucleic acid that exists at first usually in sample to be analyzed, so can use double-stranded DNA specificity dyestuff, described dyestuff excites with suitable wavelength to show the fluorescence enhancing only after they are attached on the double-stranded DNA.Preferably, only use those dyestuffs that does not influence the efficient of PCR reaction, for example, SybrGreenII.
All other forms well known in the prior art need design fluorescently-labeled hybridization probe, and it is emitting fluorescence after being attached on its target nucleic acid only.
B) TaqMan probe
With two kinds of composition mark strand hybridization probes.According to the principle of FRET (fluorescence resonance energy transfer), when using first kind of composition of optical excitation of suitable wavelength, the energy of absorption is transferred in second kind of composition, promptly so-called quencher.During the annealing steps of PCR reaction, hybridization probe is attached on the target DNA, and is degraded by 5 '-3 ' exonuclease activity of Taq polysaccharase in the extension stage subsequently.As a result, excited fluorescent composition and quencher spatially are separated from each other, thereby can measure the fluorescent emission (US 5,538,848) of first kind of composition.
C) molecular beacon
Also with first kind of composition with these hybridization probes of quencher mark, described mark is preferably located in the two ends of probe.As the result of probe secondary structure, two kinds of compositions are spatially adjacent in solution.With after the target nucleic acid hybridization two kinds of compositions are separated from each other, thereby with the fluorescent emission that can measure first kind of composition after the optical excitation of suitable wavelength (US 5,118,801).
D) single label probe (SLP) form
This test format by 5 '-or 3 '-end form (WO 02/14555) with single oligonucleotide of single fluorochrome label.Can be with two different oligonucleotide marks that are designed for: G-quenching probe and nitroindoline-go quenching probe (Nitroindole-Dequenching probe).
In G-quenching probe embodiment, fluorescence dye is attached on the C of 5 ' of oligonucleotide-or 3 '-end.When the hybridization of probe and target, if two G are positioned on the target chain relative with C and on the other position 1 of complementary oligonucleotide probe, fluorescence just significantly reduces.
Go in the quencher embodiment at nitroindoline, fluorescence dye is attached on the nitroindoline of 5 ' of oligonucleotide-or 3 '-end.Nitroindoline reduces the fluorescent signal of free probe in some way.When the hybridization of probe and target DNA owing to go quenching effect to make the fluorescence increase.
E) FRET hybridization probe
FRET hybridization probe test form is especially to all types of homology hybridization assays useful (Matthews, J.A. and Kricka, L.J., Analytical Biochemistry 169 (1988) 1-25).That it is characterized in that using simultaneously and right with two strand hybridization probes of adjacent site complementary of the same chain of institute amplifying target nucleic acid.Two probes are with different fluorescent component marks.When using the optical excitation of suitable wavelength, principle according to FRET (fluorescence resonance energy transfer), first kind of composition transferred to the energy that is absorbed on second kind of composition, and the result can measure the fluorescent emission of second kind of composition when two hybridization probes are attached to the consecutive position of detected target molecule.
In the time of on being annealed to target sequence, hybridization probe each other must be very close, with series arrangement from the beginning to the end.Usually, the gap between the 5 ' end of the 3 ' end of the mark of first probe and the mark of second probe is as far as possible little, that is, and and 1-5 base.This permission FRET compound donator and FRET acceptor compound are close, and it generally is the 10-100 dust.
As substituting that the fluorescence of monitoring the FRET receptor component increases, also may monitor the quantitative measurment of the fluorescence minimizing of FRET donor composition as hybridisation events.
Especially, the target DNA that increases in order to detect can use FRET hybridization probe form in PCR in real time.In all known test format in PCR in real time field, proved that FRET hybridization probe form is highly sensitive, accurate and (WO 97/46707 reliably; WO 97/46712; WO 97/46714).Yet, limited by the special characteristics of the target nucleic acid sequence that will detect.
As to using substituting of two FRET hybridization probes, also may use fluorescently-labeled primer and the oligonucleotide probe of a mark (Bernard, P.S. is etc., AnalyticalBiochemistry 255 (1998) 101-107) only.In this, it can at random be selected, and no matter primer is with the FRET donor or with FRET acceptor compound mark.
Except PCR and PCR in real time, the FRET hybridization probe is used for curve analysis.In this mensuration, amplifying target nucleic acid in general PCR reaction at first with suitable amplimer.Hybridization probe may exist during amplified reaction or add afterwards.After the PCR reaction was finished, composing type ground increased the temperature of sample, and as long as hybridization probe is attached on the target DNA and just can detects fluorescence.When melting temperature(Tm), hybridization probe is discharged by the target from them, and fluorescent signal drops to background level immediately.With suitable fluorescence the figure of temperature-time is monitored this reduction, can measure first devived value like this, observe the maximum value that fluorescence reduces therein.
Exist many known different fluorescence dyes right in the prior art, according to the present invention its mainly as FRET donor/FRET acceptor to and can work together.Yet, before the present invention, there is not the example of maniflest function, described example aspects is successfully to have used 4 different FRET right in multiple detection assay.Except other reason, this may be owing to lack suitable plant and instrument, in addition, is functional other fluorescent chemicals interferential fact that is present in the same reaction mixture owing to the right FRET method of special FRET.
As discussed above, there is the different real-time detection thermal cycler device that is used for multiple/polychrome detection with maximum 4 detector channels.Yet, since with several (at least more than 2) not the probe of isolabeling set up have enough sensitivity and specific real-time polychrome multiple assay trial up to now not successfully, so it is very limited to be used for the effectiveness of all these instruments of multiple detection up to now.
Therefore, the purpose of this invention is to provide a kind of improved system, its allow optimization and simultaneously flexible design multiple/the polychrome test experience.In one aspect, the problem that will solve relates to the design that improves suitable hybridization probe.In yet another aspect, the problem that will solve relates to the improvement of apparatus.
Summary of the invention
Therefore, new invention relates to a kind of real-time PCR system with applicable test format and the expansion of polychrome feature.This system comprises and is used for special reagent mixture that multiple/polychrome detects and the improved PCR in real time instrument with the optical detection system that comprises a plurality of detecting units.
Design excitaton source and the sense channel of this optical system, for example: SybrGreenI, multiple FRET hybridization probe and multiple TaqMan probe to be provided for multiple test format.
More properly, the present invention relates to carry out the system of multicolor real time PCR, specific group compound or reaction mixture that it comprises the PCR in real time instrument and is used to carry out multiplex PCR:
Composition of the present invention or reaction mixture comprise at least 3 pairs, preferably 4-5 to and 4 pairs of FRET hybridization probes definitely most preferably.Described every pair of hybridization probe by the FRET donor probe that carries FRET donor part and carry have 550 and 710nm between the FRET acceptor probe of FRET acceptor portion of emission maximum form.
In one embodiment, at least 3, preferably at least 4 and most preferably definitely 4 FRET donors partly be identical.Most preferably, all FRET donor parts are identical.In other words, if all right FRET donor parts of different FRET all are identical, their available same excitaton sources excite so.
In another embodiment of the invention, at least 3, preferably at least 4 and most preferably definitely 4 FRET donors partly be fluorescein.Most preferably, all FRET donor parts all are fluoresceins.
In a specific embodiment, at least one other FRET donor partly is selected from Atto425 and WI343.
In another specific embodiment, it does not repel not mutually for above-mentioned disclosed those embodiments, a kind of FRET acceptor portion be selected from LC-Red 705, Cy5.5, JA286 and as at US 6,027,709, disclosed sulfonated cyanine dye in the formula 1.
Can select Cy5 as the 2nd FRET acceptor portion.
Can use LC-Red 640 as the 3rd FRET acceptor portion.
Select LCRed derivative L C-Red 610 as the 4th FRET acceptor portion.
In a special embodiment, the 5th FRET acceptor portion is selected from Rh6G and TAMRA.
The feature of the instrument part of system is that described PCR in real time instrument comprises
At least one light source, preferably LED
At least 4,5-6 fluorimetric detector entity preferably, described each entity have each other and differ at least 25, and preferably wavelength is detected at the center of 30nm at least
Feature is that described detector entity can
Detect at least 3 simultaneously, preferably 4 and 5 right maximum fluorescence emission of FRET hybridization probe of isolabeling not most preferably,
Detect simultaneously at least 2 not the TaqMan hybridization probe of isolabeling maximum fluorescence emission and
Detect the maximum fluorescence emission of SybrGreenI
The device of heating and cooling
The a plurality of reaction vessels that hold reaction mixture.
In a preferred embodiment of the invention, the feature of system instrument part is that described PCR in real time instrument comprises a light source definitely.
In this context, can detect the fluorescent emission of FRET form, TaqMan form and SybrGreen form although should be understood that this instrument, but term " simultaneously " meaning be in same thermal cycling scheme (promptly, in the once operation of instrument), detect at least 3 not isolabeling the FRET hybridization probe or to few 2 different TaqMan probe or the SybrGreenI of person.
Preferably, this instrument comprises at least 24, more preferably 32 and 48 described reaction vessels most preferably.
Equally preferably, the wavelength region that wavelength is selected from 520-540nm, 545-565nm, 570-590nm, 600-620nm, 630-650nm, 660-680nm and 700-720nm is detected at described center.
Equally preferably, the feature of each reaction vessel is that same axle along described reaction vessel obtains fluorescence excitation that is caused by described fluorescence light source and the fluorescence monitoring of being undertaken by the fluorimetric detector entity.
If verified described light source and described detector entity are positioned at independent shell then particularly advantageous.
If also verified described heating and cooling device is force fluid or the device that forces gas then particularly advantageous preferably forces device of air.
If also further proof reaction vessel is fixed in the carousel of rotation then particularly advantageous.
Aspect in the end the present invention also relates to be used to increase and detects the correlation method of a plurality of target DNA sequences, comprises
A) provide as above-mentioned disclosed reaction mixture,
B) described reaction mixture is carried out the thermal cycling scheme, so that the amplification of described a plurality of target sequences can take place
C) after many amplification cycles, at least once monitor the hybridization of described every pair of FRET hybridization probe.
In order to carry out curve analysis, hybridization that also can at least monitoring temperature dependency mode.
Detailed Description Of The Invention
A) instrument
Aspect first, system according to the present invention comprises the instrument that is suitable for PCR in real time.Randomly, this instrument also is applicable to curve analysis, that is, and and the temperature dependency combination of monitoring DNA binding entity such as hybridization probe or dsDNA combination dye.
Instrument is made up of opticator and the device that is used for thermal cycling basically, its can make they separately a plurality of amplification solution in the reaction vessel carry out multiple thermal cycling process so that polymerase chain reaction (PCR) can take place.
Preferably, the feature of this system instrument part is that described PCR in real time instrument comprises
At least one light source, preferably photodiode
At least 4 and 5-6 fluorimetric detector entity preferably, described each entity have each other and differ at least 25, and preferably wavelength is detected at the center of 30nm at least
Feature is that described detector entity can
Detect at least 3 simultaneously, preferably 4 and 5 right maximum fluorescence emission of FRET hybridization probe of isolabeling not most preferably,
Detect simultaneously at least 2 not the TaqMan hybridization probe of isolabeling maximum fluorescence emission and
Detect the maximum fluorescence emission of SybrGreenI
The device of heating and cooling and
The a plurality of reaction vessels that hold reaction mixture.
Most preferably, the feature of this system instrument part is that described PCR in real time instrument comprises a kind of light source definitely.
Can at random select the device of thermal cycling.For example, can use metal block circulation instrument commonly known in the art.Yet, be favourable if the device of thermal cycling provides the device of initiatively heating and active refrigerative independent device.If also the device of verified described heating and cooling is force fluid or the device that forces gas then particularly advantageous in addition.In a specific embodiment, described device uses and forces gas.
If described thermocirculator can carry out rapid thermal cycling in LightCycler technology (WO97/46712) as it, it also is preferred so.As can from WO97/46712, inferring, this technology based on be heated with ambient air coil heats be feature force the gas heating and with provide ambient air be feature forced air cooling but.
Further improvement about rapid thermal cycling is owing to having than higher surface/volume, so use kapillary as reaction vessel.The inventor has proved that external diameter is less than 5mm, preferably less than 3.2mm and most preferably less than the kapillary particularly advantageous of 1.6mm, this is because their allow cumulative volume still can carry out each round-robin reaction times less than 2 minutes thermal cycling scheme rapidly at the reaction mixture of 10 to 100 μ l.In addition, each reaction vessel needs at least in part or fully by clearly material such as glass or plastics manufacturing on the optics.
The location of each reaction vessel (kapillary) in monitoring position preferably carries out fluorescence excitation with the same axle along described reaction vessel and monitoring is a feature.Because the phenomenon of total internal reflection ratio, so such principle improves the signal conduction.
For whole a plurality of reaction vessels all produce unified thermal cycling feature, reaction vessel is fixed in the carousel of rotation then particularly advantageous if further proved.
The opticator of instrument is made up of the detecting unit that excites the unit and be used for detecting from described reaction vessel fluorescent emission that has for one or more suitable light sources of the fluorescent chemicals that exists in the provocative reaction solution basically.
According to WO 97/46712, under the prerequisite of the dichroic mirror that uses proper number, may make up a kind of optical system, wherein will excite unit and detecting unit to place the common shell.Yet in the context of the present invention, if comprise exciting the unit and comprising that the detecting unit of detector entity is arranged in independent shell of light source, the PCR in real time instrument of result's improvement comprises two independent optical units, proves favourable so.
For exciting the unit, can use monochromatic excitation or polychrome to excite according to the present invention.For monochromatic excitation, can use monochromatic LED (photodiode) or one-wavelength laser.Also may use to have two or more LEDs or one-wavelength laser, but in single measurement, once only use one instrument.Excite for polychrome, can use White LED, halogen lamp or xenon lamp.In this case, need filter wheel be installed in instrument or be used for suitable colour filter shift-in and shift out excitation beam other the device so that the light with different choice excitation wavelength can be provided.
To excite unitary optical characteristics be to select according to the different parameters that must consider according to of the present invention:
At first, the shortest excitation wavelength should be longer than 360nm, preferably is longer than 400nm, and this is because short wavelength radiation has the low relatively conventional optical material that is used for the real-time PCR reactions container that uses that penetrates, as the transmissivity of glass or plastics.In addition, with the higher wavelength fluorescence interference that excites the reaction vessel material internal of having avoided to hinder reaction monitoring of wavelength in the scope of 400-500nm for example.
In a preferred embodiment of the invention, excite the unit only to be made up of a light source, because according to the present invention, it also may carry out multiple detection in PCR in real time even have single light source.In addition, multiple detection even may be with single, monochromatic source in PCR in real time.
In one embodiment, excite the unit to be included in the blue led of 470nm place emission.Fig. 1 shows the excitation spectrum of the spendable different fluorescence dyes according to the present invention.In these dyestuffs, the single photodiode of the available 470nm of having excitation wavelength excites as the fluorescein of the FRET compound donator that often uses or TaqMan well known by persons skilled in the art report dyestuff, and only excites the FRET acceptor dye of frequent use reluctantly with 470nm LED.
In addition, can randomly use 400 and 430nm between, preferably excite and compare other FRET compound donator with fluorescein with littler excitation maximum at the 2nd UV-LED of 415nm emission.
More properly, blue 470nm LED can the fluorescence excitation element, its with the HybProbe form as with 4 kinds of isoacceptor (report) dyestuff bonded donor dye not.On the other hand, general FRET acceptor dye such as LC-Red-640 or Cy5 are only excited reluctantly by 470nm LED.Measure Smalt LED at double-colored TaqMan and also excite report dyestuff FAM and HEX or VIC.In addition, fluorescein with single label probe (SLP) form with the dyestuff of making reports.In addition, for not based on the detection of the amplified production of probe, use blue led to excite SybrGreenI.
Can use optional UV LED to excite and suitable report dyestuff bonded short wavelength donor dye with the HybProbe form at relatively shorter wavelength emission fluorescence.In addition, can be included in the third LED of 580nm emission.
For detecting unit, can use different detection mode well known in the prior art according to the present invention.For example, use several photorectifiers to be well known in the prior art, it is characterized in that each photorectifier can detect the fluorescence of special wavelength.In one aspect of the invention, provide to have 5 or 6 passages definitely, use the detection system of photorectifier as detector.As being shown, this embodiment can have been carried out 4 kinds of color FRET hybridization probes simultaneously and detected.
Optical signature according to detecting unit of the present invention is selected according to following different parameters:
At first, the longest emission wavelength that should be detected is approximately less than 800nm, and preferably less than 730nm, and this is because the detection of higher wavelength signals is subjected to the influence of the possible background of the infrared radiation of inferring that produced by instrument heating part branch.In addition, long wavelength infrared dyestuff well known in the prior art has inferior the suitableeest quantum yield, instability, therefore more difficult being coupled on the desired hybridization probe.
As a result, different sense channels should 500 and 800nm between, preferably 520 and 730nm between have the center and detect maximum value.In this context, Fig. 2 shows the emmission spectrum of the spendable different fluorescence dyes according to the present invention.
500 and the scope of 800nm in, can define sense channel definitely by selecting suitable colour filter, light beam must be earlier by described colour filter and then enter separately photorectifier.For appropriate selection, the inventor has identified the following parameters that should consider:
At first, should select the centre wavelength of each passage to detect maximum value according to the maximum value of the emmission spectrum of those fluorescence dyes that will mainly detect.According to rule of thumb, for suitable detection sensitivity, with center in the maximum value of the fluorescent emission of detected dyestuff and each passage detect wavelength do not differ should surpass+/-10nm.
In addition, all sense channels should detect discontinuous emission, that is, spectra overlapping should be minimum as far as possible.In this, if the half-band width of each passage is 40nm or littler, then be favourable.More advantageously be that the half-band width of each passage is 20nm or littler.
Preferably, in close passage with the phase mutual interference (crosstalk) of detected fluorophore less than 70%, more preferably less than 50%, most preferably less than 30%.In this, another important criterion is trap seat (blocking value), its indication detected maximum strength outside the appointment transmission range of passage (colour filter).Preferably, this value is less than 10
-3
Based on these standards, if verified according in the 5-6 of the present invention passage each the center detect wavelength and be separated by each other above 25nm, preferably even to surpass 30nm then be favourable.
In one embodiment, the wavelength region that wavelength is selected from 520-540nm, 545-565nm, 570-590nm, 600-620nm, 630-650nm, 660-680nm and 700-720nm is detected at described center.
In addition, in a specific embodiments of forming by 6 detector channels, the center of described passage detects wavelength and is about 530,580,610,640,670 and 710nm+/-5nm or 530,555,610,640,670 and 710nm+/-5nm or preferably+/-2nm.
In another specific embodiments of forming by 5 detector channels, the center of described passage detects wavelength and is about 530,610,640,670 and 710nm+/-5nm or preferably+/-2nm.
In the 3rd specific embodiments of forming by 4 detector channels, the center of described passage detects wavelength and is about 610,640,670 and 710nm+/-5nm or preferably+/-2nm.
As an example, Fig. 3 shows 6 sense channels of preferred embodiment and the emmission spectrum of spendable different fluorescence dyes according to the present invention.In this embodiment, make 6 passages be separated by at least the 25nm spectrum intervals so that each report dyestuff enters the phase mutual interference minimum that the proximity detection passage carries out.Detecting unit can detect SybrGreenI and TaqMan report dye fluorescence element/FAM in having the passage that centre wavelength detection maximum value is about 530nm.In addition, this optimum system choosing can detect HEX/VIC in the second passage of about 560nm.Long wavelength's the sense channel SLP report dyestuff that can detect the HybProbe acceptor dye and randomly detect the long wavelength more.
Excite unit and detecting unit by the connection of many forks (multiple-leg) fibrous bundle.Use photoconductive tube with the light uniformly distributing of reaction vessel (for example, glass capillary) emission and to be sent to 6 glass fibre intrafascicular.The single glass fibrous bundle of these 50 μ m is sent in 6 sense channels each with light.
Compare with disclosed optical unit in WO 97/46712, this layout that excites unit and detecting unit to be positioned at independent shell provides two advantages: the light that will launch is evenly distributed to all 6 sense channels and excites the unit and the mechanical decoupling of detecting unit.This can make and excite the unit accurately to locate (for example, the kapillary top) towards the reaction vessel height that will monitor, and need not mobile detecting unit.In addition, make the number minimum of essential dichroic mirror.An example that has shown this layout in Fig. 4, it discloses a possible embodiment of the present invention.As can be seen, excite unit and detecting unit to be positioned at different shells.
In addition, system according to the present invention comprises as at US 6,197, and detailed disclosed color compensating instrument in 520 is incorporated it among the application as a reference in full.Corresponding software makes it possible to the temperature dependency spectra overlapping measuring the fluorescence of whole temperature range and proofread and correct above-mentioned disclosed 6 detector channels.
This realizes through the following steps substantially
A) be provided at the calibration solution of each the fluorescence entity that mixes in the multiple experiment that will carry out
B) in order to measure temperature dependent mutual interference volume between the different passages, in each detector channel the temperature dependency fluorescence of each described solution of monitoring and
C) produce the color compensating data set that comprises correction value.
B) selection of check form and dyestuff
In the context of the present invention, term " multiplex PCR " is appreciated that to react as the PCR of feature by using 2 pairs or more methods to amplimer to produce 2 or more a plurality of different amplified productions in same PCR reaction.
Similarly in the context of the present invention, term " multicolor real time PCR " is defined as to detect at multiplex PCR or one or more different amplified productions of being produced in substance PCR (only using 1 pair of amplimer) with the hybridization probe by isolabeling not and measures as the PCR in real time of feature.
Main aspect of the present invention is based on the hybridizing reagent that uses isolabeling not, and every kind of reagent comprises 1 pair and comprises according to the right FRET hybridization probe of the interactional two kinds of fluorescence dyes of FRET (fluorescence resonance energy transfer) (FRET) principle.According to the present invention, can select suitable dyestuff to be used for mark can be at least 3 or 4, the hybridizing reagent that is used to detect in the sense channel preferably as much as possible.
More properly, this hybridizing reagent is made up of two adjacent hybridization oligonucleotides, suitably carries out mark so that they can be according to working together as disclosed FRET-HybProbe test format in WO 97/46707, WO 97/46712 and WO97/46714.In many cases, if hybridizing reagent is made up of single oligonucleotide or in the situation of FRET HybProbe form, to forming, then be enough by the oligonucleotide that works together as donor probe and acceptor probe.Yet, in other situation, in the target sequence that needs detect, may have many other sequence variants.Therefore can not be by only using a pair of all members' of FRET oligonucleotide hybridization probe in detecting sequence.
For those situations, hybridizing reagent can by 1,2 or more a plurality of hybridization probe form, its similar in appearance to and in conjunction with homologous sequence, but each other because 1,2,3 or more a plurality of mononucleotide, dinucleotides or trinucleotide exchange, disappearance or add and different.If hybridizing reagent is a pair of FRET hybridization probe, so described hybridizing reagent can by 1,2,3 or more a plurality of FRET donor oligonucleotide probe and/or 1,2,3 or more a plurality of acceptor oligonucleotide probe form.In this case, all donor probes may be similar, but different because mononucleotide, dinucleotides or trinucleotide exchange, lack or add each other.Equally, all acceptor probes may be similar, but different because mononucleotide, dinucleotides or trinucleotide exchange, lack or add each other.
In addition, if carrying out polychrome FRET detects, wherein use as at Bernard, P.S., Deng, the primer of disclosed suitable mark is replaced a member of the hybridization probe centering of described mark among Analytical Biochemistry 255 (1998) 101-107, and it is also within the scope of the invention so.In addition, the fluorescent emission of FRET acceptor compound increases if not monitoring separately, reduce as indication and have the target nucleic acid that will detect but monitor one, fluorescent emission several or all FRET donors part, it is also within the scope of the invention so.
Except the various possibilities of carrying out detecting based on the polychrome of FRET principle, system of the present invention also allows to carry out other test format, detects target nucleic acid as the method that detects by SybrGreenI, the multiple detection of using the molecular beacon of isolabeling not, single label probe or double-colored TaqMan.As be well known in the art, double-colored TaqMan measures may be based on the Cy5 (Amersham) that uses as the quencher compound, and it is with the FAM that reports dyestuff as first standard with as the HEX of the second report compound or selectively VIC combination.
In principle, have the multiple possibility of fluorochrome combinations, it may be as FRET to work together (summary: Resonance Energy Transfer Editors:Meer, B Wiebvan der etc., VCH Publishers INC., 1994).Yet based on the prerequisite of instrument, to those skilled in the art, exploitation is not nonsensical with the polychrome test that the sensitivity that influences the experiment of PCR in real time separately and/or specific mode make up a plurality of non-interfering FRET hybridizing reagents.
About the right selection of suitable FRET hybridization probe, the dyestuff that for example is used for FRET donor and FRET acceptor can not be optional, but needs to satisfy following at least requirement:
Enough solubleness and can being coupled on the oligonucleotide
The stability of dyestuff under the PCR thermal cycle conditions
Fully and repeatably emissive porwer and quantum yield
The low temperature dependency of emissive porwer
Under different electrochemical conditions, there is not spectrum to move basically
Basically there is not spectrum to move in the time of on being coupled to different oligonucleotide sequences
Discontinuous spectral emissions maximum value
Can utilize suitable mating partner dyestuff to carry out the FRET process
As a result, system according to the invention important aspect focuses on evaluation can be used for a plurality of FRET (fluorescence resonance energy transfer) (FRET) dyestuff of many as far as possible sense channels with the HybProbe form right.In Fig. 1-3, shown and to have set up right excitation spectrum and the emmission spectrum of dyestuff FRET dyestuff that 4 or 5 heavy polychromes detect.Will be described below alternative method.
In this, following table summed up be successfully used to up to now shown in carry out various FRET acceptor dyes and the TaqMan report dyestuff that 4 look FRET hybridization probe assays or double-colored TaqMan measure in the sense channel:
Table 1
| Passage | The FRET acceptor dye | TaqMan reports dyestuff | |
| 530 (also detecting SybrGreenI) | - | Fluorescein (Fam) |
|
| 555 | Rh6GTamra | HexVicTamra | |
| 580 | |
||
| 610 | | Texas Red | |
| 640 | |
||
| 670 | |
||
| 710 | Ja286Cy5.5LCRed705 |
Yet, for several different report entity shown in top, different FRET donor parts (donor) and TaqMan quencher compound (Q) must depend on the excitation wavelength a kind of owing to existing, that two or three different LED s obtains and use respectively.Therefore, more fully summed up in the table below suitable FRET to or the different possibilities of TaqMan combination:
Table 2
| |
530 | 555 | 580 | 610 | 640 | 670 | 710 | |
| FRET： | ||||||||
| Donor: Atto425WI343 | 410 | - | Rh6GTAMRA | - | LCRed610 | LCRed640 | Cy5Bodipy655Alexa647 | JA286LCRed705Cy5.5 |
| Donor: fluorescein | 470 | - | - | - | LCRed610 | LCRed640 | Cy5Bodipy655Alexa647 | JA286LCRed705Cy5.5 |
| Donor: LCRed640 | 590 | - | - | - | - | - | Cy5Bodipy655Alexa647 | JA286LC705Cy5.5 |
| TaqMan： | ||||||||
| Q: optional | 410 | Tonka bean camphor | - | - | - | - | - | - |
| Q：Tamra | 470 | Fam | HexVic | - | - | - | - | - |
| Q．Cy5 | 470 | Fam | HexVicTAMRA | TAMRARox | - | - | - | - |
| Q： BHQ | 470 | Fam | HexVicTAMRA | TAMRARox | - | - | - | - |
| Q： Cy5 | 590 | - | - | - | Texas Red | - | - | - |
| Q：BHQ | 590 | - | - | - | Texas Red | - | - | - |
The approach that obtains the dyestuff mentioned in the above-mentioned table is as follows:
LC-Red-705 and LC Red-640 can buy from Roche Applied Science (Cat.No.2 015 161 and 2 157 594)
According to the standard scheme use as at US 5,750,409, disclosed fluorescence dye among the Compound I I, synthetic LC-Red 610
JA 286 is disclosed in the embodiment 1 of EP 0 747 447.For this dyestuff,, then be favourable if this dyestuff is linked to each other with hybridization oligonucleotide by the non-hybridization transcribed spacer part of the length of 5 nucleotide residues.
Cy-5 NHS-ester can have been bought from Amersham (Cat.No.PA 15100), and the Cyc5.5 phosphoramidite also can have been bought from Amersham (Cat.No 27179901).
Atto 425-NHS ester can have been bought from Attotec (Cat.No.AD 425-3).
WI 343 is derivatives (Aldrich, Cat.No.55 804 654) of tonka bean camphor 343, according to standard method with itself and joint (β alanine (beta alanin)) coupling and be converted into succinimide acyl ester:
In addition, can all TaqMan dyestuffs have for example been bought among the Molecularprobes from different suppliers well known by persons skilled in the art.
BlackHole quencher (BHQ) can have been bought from Biosearch Technologies (Cat.No.BHQ 1-3).
Can carry out suitable mark to oligonucleotide by ordinary method well known by persons skilled in the art.Particularly, can use the fluorescent chemicals that comprises activatory NHS ester or the fluorescent chemicals that is connected on the phosphoramidite carries out mark, thereby can during the de novo synthesis of oligonucleotide own, carry out mark.The glass particle in hole that in addition, can be by using fluorescein control prepares fluorescein-labeled probe as oligonucleotide synthetic solid support (Roche Applied Science Cat.No.3 138 178).
C) method and test kit
In yet another aspect, the present invention relates to be undertaken the method for multiplex PCR by all different embodiments of using above-mentioned disclosed system, instrument and composition.
Particularly, the present invention relates to increase and detect the method for a plurality of target DNA sequences, comprise
Provide according to composition of the present invention or reaction mixture,
Make described reaction mixture carry out the thermal cycling scheme, so as can to take place described a plurality of target sequences amplification and
After many amplification cycles, at least once monitor the hybridization of described every pair of FRET hybridization probe.
In specific embodiment, the hybridization of at least monitoring temperature dependency mode.
Composition or reaction mixture mainly comprise at least 3 pairs, preferably 4-5 is right, most preferably be 4 pairs of FRET hybridization probes definitely, the every pair of hybridization probe all by the FRET donor probe that carries FRET donor part and carry have 550 and 710nm between the FRET acceptor probe of FRET acceptor portion of emission maximum form.
In addition, can comprise one or several or preferably all compounds and the reagent that is selected from the following tabulation according to this composition of the present invention or reaction mixture:
Damping fluid can be used for polymerase chain reaction
Deoxynucleoside triphosphate
The template dependent dna-polymerases, preferably heat-stable
At least one pair of or several to amplimer
In yet another aspect, the present invention relates to comprise the test kit of at least the first group amplimer and at least 3,4 or 5 pairs of FRET hybridization probes.
In addition, this test kit can comprise one or several other compound and the reagent that is selected from the following tabulation according to the present invention:
Damping fluid can be used for polymerase chain reaction
Deoxynucleoside triphosphate
The template dependent dna-polymerases, preferably heat-stable
At least one pair of or many to amplimer
This test kit also can comprise internal control dna, and it can use the identical primer that is used to detect any target nucleic acid and probe to increase and detect.Above-mentioned disclosed every kind of composition can be housed in the single storage container.Yet, also may in same container, preserve the composition of arbitrary combination.
D) Fa Ming best mode
Best mode of the present invention known for inventor is as follows when applying date of the present invention:
In this embodiment preferred, system according to the present invention provides the photometer with one or two excitaton source and 6 sense channels.With blue (470nm) and randomly UV-light (410nm) photodiode with have 530,555,610,640,670 and the center of the 710nm sense channel that detects wavelength make up.
The photometer of the PCR in real time instrument of improvement comprises two independently optical units.As disclosed in Fig. 4, excite unit and detecting unit to be arranged in different shells.
(I) have the unit that excites of one or two LED ' s: one at the blue led of 470nm emission and the purple LED that randomly launches at 415nm.Blue led can excite SybrGreenI and fluorescein.Fluorescein is as donor dye in the HybProbe form, with 4 not isoacceptor (report) dye combinations: Red 610, Red 640, Cy 5, Red 705.
In addition, measure Smalt LED at double-colored TaqMan and excite report dyestuff FAM and HEX or VIC.In addition, fluorescein in single label probe (SLP) form with the dyestuff of making reports.Can be to use optional purple LED in the HybProbe form to excite the short wavelength's donor dye (Atto 425) with report dyestuff (Rh6G) combination of 555nm.
(II) have the detecting unit of 6 passages, it uses photorectifier as detector.6 passages are separated by 25nm spectrum intervals at least so that the phase mutual interference that every kind of report dyestuff is entered adjacent sense channel reduces to minimum.This detecting unit can detect SybrGreenI and fluorescein/FAM in the 530nm passage.In addition, this system can detect HEX/VIC and RH6G in the 555nm passage.Long-wavelength detection passage (610,640,670,710nm) can detect the HybProbe acceptor dye and randomly be used to detect long wavelength SLP report dyestuff.
Excite unit and detecting unit by 6 fork fibrous bundles connections.Use photoconductive tube with the emission light uniformly distributing of glass capillary and be sent in 6 glass fiber bundles.These 50 μ m single glass fibrous bundles are sent in 6 sense channels each with light.This device is compared with LC 1.2 optical units two advantages are provided: the light of emission is evenly distributed in all 6 sense channels and the mechanical decoupling that excites unit and detecting unit.This can make and excite the unit accurately to locate towards kapillary top height, and need not mobile detecting unit.Effectively the color compensating function allows to detect simultaneously a plurality of targets in different passages.
Make up these hardware and software characteristics, detect, set up the polychrome ability of system in order to carry out multiple FRET hybridization probe.In addition, system according to the present invention allows other non-FRET-HybProbe test format.
Based on the hybridization probe test format, system can carry out at least 4 looks multiple real-time quantitative and carry out curve analysis.Consider the parsing of the melting curve at each 6 peak that unwind of passage, use and to distinguish 24 different PCR products at most according to photometer of the present invention.In order to realize this point, for the center emission wavelength 610 and the sense channel of 670nm developed new before the FRET acceptor dye of the unknown.In addition, optional 410nm LED provides a kind of selection of short wavelength's donor dye of the acceptor dye combination of using and launching at 555nm, and therefore the 5th HybProbe sense channel be provided.In Fig. 1 and 2, show exciting and emmission spectrum of particularly useful dyestuff.
Use fluorescein as the FRET donor dye, can identify that difference can detected two new FRET acceptor dyes at 610nm passage (Red610) with in 670nm passage (Cy5).When using with HybProbe FRET form, two dyestuffs all show high signal kinetics.In addition, the spectral quality of dyestuff meets top disclosed photometer fully, and the phase mutual interference that therefore will enter in the different sense channels reduces to minimum.Use hyperchannel color compensating algorithm flexibly, shown that the specific detection of all the 4 kinds of FRET acceptor dyes (comprising existing dyestuff LC Red 640 and LC Red 705) from single reaction is feasible in 40 ℃ to 95 ℃ temperature range.
When attempting to identify the 5th kind of FRET acceptor dye that is used for the 555nm passage, the FRET signal kinetics that the result is good is suppressed by two effects:
(I) the fluorescein donor dyestuff shows the high phase mutual interference that enters the 555nm passage, has therefore covered the signal of the increase of potential FRET acceptor dye in the PCR process.
(II) the FRET acceptor dye itself in the 555nm emission is directly excited by 470nm LED, thereby has significantly increased background fluorescence, so reduced detection sensitivity.
The solution of finding these shortcomings is by using the 410nm ultraviolet leds to excite the short wavelength's donor dye (Atto 425) that makes up with suitable 555nm acceptor dye (Rh6G).In this combination, will reduce to minimum from phase mutual interference and directly the exciting of FRET acceptor that donor enters the 555nm sense channel, and behind color compensating, obtain sufficient signal kinetics.
In addition, the result is short wavelength's donor Atto 425 even can be used in combination with all long wavelength's acceptor dyes.Signal kinetics when being used in combination with the FRET signal kinetics of long wavelength's acceptor of Atto 425 combination and fluorescein unexpectedly, is suitable.This observed data provides a kind of selection of only using a kind of donor dye the polychrome feature to be expanded to 5 channel forms.
Use the TaqMan test format, can carry out double-colored application based on standard TaqMan report dyestuff FAM (detecting) and HEX or VIC (detecting) at 560nm at 530nm, and need not more modification TaqMan chemical process well known in the prior art.Detection sensitivity that is obtained and COBAS TaqMan instrument (Roche Molecular Systems) are suitable.
Single label probe (SLP ' s) form (WO 02/14555) that can in system according to the present invention, be used in addition, curve analysis.This form is based on the quencher of its target sequence hybridization back probe mark or goes quencher separately.This form only needs a kind of with the end-labelled probe of homogencous dyes.Therefore compare with HybProbe or TaqMan form, SLP ' s makes it possible to be useful on the determinator of the significantly low cost of mononucleotide polymorphic (SNP ' s) analysis-by-synthesis.By the report melting temperature(Tm), can in a reaction, distinguish the not isoallele of SLP clearly.By using different report dyestuffs that multiple choices are provided.
In following table, summed up the possibility of polychrome PCR detection in real time in the disclosed instrument in the above:
Table 3
| The mensuration form | Sense channel (report dyestuff) |
| SYBRGreenI | 530nm(SYBR Green I) |
| HybProbes | 610nm(LC RED 610)640nm(LC RED 640)670nm(LC RED 670)710nm(LC RED 705) |
| Hydrolysis probes (TaqMan probe) | 530nm(FAM)555nm(VIC，HEX) |
| Single probe | 530nm (fluorescein) |
Provide the following example, reference, sequence table and accompanying drawing to be used for helping to understand the present invention, in claims, set forth true scope of the present invention.Should be understood that and not depart from spirit of the present invention and in the method for being set forth, make amendment.
Description of drawings
The excitation spectrum of Fig. 1 FRET donor of the present invention and acceptor dye.
The emmission spectrum of Fig. 2 FRET donor of the present invention and acceptor dye.
The emmission spectrum and the sense channel of Fig. 3 FRET donor of the present invention and acceptor dye.
Fig. 4 photometer device
The 4 look PCR in real time quantitative experiments of Fig. 5 embodiment 1
Primer/probe design of Fig. 6 embodiment 2
The 4 look PCR in real time melting curves experiment of Fig. 7 embodiment 2
The double-colored TaqMan PCR in real time quantitative experiment of Fig. 8 embodiment 3
Use quantitative PCR in real time with the right factor VDNA of the different FRET hybridization probes of different fluorescent chemicals marks
For the amplification of factor V dna fragmentation, set up 4 kinds of following differences, 100 μ l real-time PCR reactions mixtures:
Contain factor V gene plasmid 10
6Individual copy
3mM MgCl
2
The primer of each 500nM
200nM is according to the FRET 3 ' hybridization probe of Seq.A
200nM is respectively according to the FRET 5 ' hybridization probe of Seq.B1, B2, B3 or B4
In addition, use LightCycler DNA Hyb probe reagent box (Roche AppliedScience, PCR component Cat.No.2158825).
The primer and the probe that use are as follows:
The primer of factor V, forward:
5 '-GAG AGA CAT CGC CTC TGG GCT A-3 ' (22 aggressiveness) is (Seq.ID.No:1)
The primer of factor V, reverse:
5 '-TGT TAT CAC ACT GGT GCT AA-3 ' (20 aggressiveness) is (Seq.ID.No:2)
The fluorescein-labeled hybridization probe of A:3 '
5 '-AAT ACC TGT ATT CCT CGC CTG TC-3 ' (23 aggressiveness) is (SEqId.No:3)
B1:5 ' Red610 hybridization probe
5 '-AGG GAT CTG CTC TTA CAG ATT AGA AGT AGT CCT ATT-3 ' (36 aggressiveness)
B2:5 ' Red640 hybridization probe
5 '-AGG GAT CTG CTC TTA CAG ATT AGA AGT AGT CCT ATT-3 ' (36 aggressiveness)
B3:5 ' Cy5 hybridization probe
5 '-AGG GAT CTG CTC TTA CAG ATT AGA AGT AGT CCT ATT-3 ' (36 aggressiveness)
B4:5 ' Red705 hybridization probe
5 '-AGG GAT CTG CTC TTA CAG ATT AGA AGT AGT CCT ATT-3 ' (36 aggressiveness)
(B1-B4：Seq.Id.NO：4)
According to following thermal cycling scheme, in above-mentioned disclosed LightCycler instrument, increase as best way of the present invention:
| Temperature [℃] | Time [second] | Climbing speed [℃/second] | Obtain thing | Circulation | |
| The sex change amplification | 95 95 55 72 | 30 10 20 20 | 20.0 20.0 20.0 20.0 | There is not no |
1 45 |
With with without the color compensating algorithm, and use by measuring second of fluorescent signal in the sense channels threshold method of deriving 45 cycle periods, and the arithmetic background correction that is used for initial fluorescence background strength criterionization is monitored in real time.
The results are shown among Fig. 5 a-5d.As can seeing in the drawings, behind the color compensating can corresponding 610,640,670 and the 710nm sense channel in obtain the special and quantitative amplification signal of the FRET acceptor dye of all uses.In addition, when carrying out the appropriate color compensation, show that the spectra overlapping of the different dyes in the adjacency channel does not influence specificity and quantitative signal.
4 look curve analysis
The 479bp fragment of certain plasmid that contains the people NAT-2 gene fragment of coding N-acetyl-transferring enzyme isozyme with primer amplified, and detect by fluorescence, 4 pairs of not homospecific FRET hybridization probes wherein used.Wherein 3 probes carry out mark at 3 ' end with fluorescein, and 4 detection probes carry out mark and hold 3 ' modifying by phosphorylation at 5 ' end with Light-Cycler-Red 610,640,705 or Cy5.The synoptic diagram that in Fig. 6, has shown this experimental establishment.As can from figure, inferring, 4 not FRET acceptor probes of isolabeling are arranged, but three detection site are only arranged.This causes two detection probes annealed competitions.
Condition determination is with disclosed substantially the same in embodiment 1, change be to use 3x10
6The target DNA of individual copy.
Forward primer:
5 ' TGC CTT GCA TTT TCT GCT T-3 ' (19 aggressiveness) (Seq.Id.No:5)
Reverse primer:
5 ' GAG TTG GGT GAT ACA TAC A-3 ' (19 aggressiveness) (Seq.Id.No:6)
3 '-fluorescein HybProbe 1:
5 ' GAA ATT CTT TGT TTG TAA TAT ACT GCT CTC TC-Fluos-3 ' (32 aggressiveness)
(Sep.Id.No：7)
5’-Red6 10 HybProbe 1a：
5 ' Red610-TGA TTT GGT CCA CGT ACC-3 ' (18 aggressiveness)
(Sep.Id.No：8)
5’-Red640 HybProbe 1b：
5 ' Red640-TGA TTT GGT CCA AGTACC C-3 ' (19 aggressiveness)
(Sep.Id.No：9)
3 '-fluorescein HybProbe 2:
5 ' GTT GGA GAC GTC TGC AGG TAT GTA TTC ATA GAC TCA-Fluos-3 ' (37 aggressiveness)
(Seq.Id.No：10)
5’-Red705 HybProbe 2：
5 ' Red705-ATC TTC AAT TGT TCG AGG TT-3 ' (20 aggressiveness)
(Seq.Id.No：11)
3 '-fluorescein HybProbe 3:
5 ' ATT TCC TTG GGG AGA AAT CTC GTG CCC A-Fluos-3 ' (28 aggressiveness)
(Seq.Id.No：12)
5’-Red5 HybProbe 3：
5‘-Cy5-ACC TGG TGA TGA ATC CCTTAC TAT TTA GAA TAA GGA AC-3‘
(37 aggressiveness)
(Seq.Id.No：13)
According to following thermal cycling scheme, in above-mentioned disclosed LightCycler instrument, increase and curve analysis subsequently as best way of the present invention:
| Temperature [℃] | Time [second] | Climbing speed [℃/second] | Obtain thing | Circulation | |
| The cooling of sex change amplification melting curve | 95 95 55 72 95 40 80 40 | 600 0 1 20 60 60 0 30 | 20.0 20.0 20.0 3.0 20.0 20.0 0.1 20.0 | Not having the no successive of no single nothing does not have | 1 45 1 1 |
The results are shown among Fig. 7.As can from figure, inferring, can use suitable color compensating method, use hybridization probe to carry out 4 look curve analysis with 4 kinds of different acceptor dyes.Can detect the hybridization probe specificity peak that unwinds then, and not be subjected to any significant disturbing influence mutually.
Double-colored TaqMan detects
The LightCycler instrument of best way is provided for all methods of the Sensitive Detection of double-colored TaqMan mensuration according to the present invention.Excite generally acknowledged TaqMan dyestuff FAM and HEXs fully with 470nm LED, and after the color compensating function of using this instrument, can in having highly sensitive 530nm and 560nm sense channel, carry out difference ground and identify.By in the condition determination of determining, replenishing BSA, simply double-colored TaqMan mensuration scheme is transferred to according in the instrument of the present invention.
In Fig. 8, shown the different instances of successfully carrying out double-colored amplification and detection.Fig. 8 a is presented in the monochrome experiment of using FAM or HEX, has obtained the detection sensitivity of about 1 copy/μ l of double-colored TaqMan mensuration.(after the PCR reaction, only negative control does not produce fluorescence significantly increases).Fig. 8 b shows double-colored experiment, wherein always is to use 500 copies of first target DNA of TaqMan probe in detecting of Hex mark, and uses the different amounts (10 of TaqMan probe in detecting of FAM mark in same reaction
0-10
6Copy) second target DNA.
Dynamicrange (being used to indicate the relative different of two target levels that will detect) is about 10
3
The reference tabulation
Bernard P.S.，et al.，Analytical Biochemistry255(1998)101-107
Higucbi R，C Fockler G Dollinger and R Watson，Kinetic PCR analysis：real：real time monitoring of DNA amplification reactions，Bio/Technology 11(1993) 1026-1030
Higuchi R，G Dollinger，PS Walsh and R.Griffith，Simultaneous amplification and detection of specific DNA sequences，Bio/Technology 10(1992)413- 417
Matthews，J.A.，and Kricka，L.J.，Analytical Biochemistry169(1988)1-25Pedersen，S.，Bioradiations 107(2001)10-11
Review：Resonance Energy Trangfer Editors：Meer，B Wieb van der et al，VCH Publishers INC.，1994
US 5,118,801
US 5,538,848
US 5,750,409
US 6,015,674
US 6,174,670
US 6,197,520
US 6,369,893
WO 02/14555
WO 97/46707
WO 97/46712
WO 98/46714
Sequence table
<110>Roche Diagnostics GmbH
F.Hoffmann-La Roche AG
＜120〉Gai Liang system for multicolor real time PCR
<130>21810 EP
<150>EP 03007458.7
<151>2003-04-04
<150>EP 03014929.8
<151>2003-08-07
<160>13
<170>PatentIn version 3.1
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Claims (30)
1. a composition or reaction mixture that carries out multicolor real time PCR, it comprises at least 3 pairs of FRET hybridization probes, the every pair of hybridization probe by the FRET donor probe that carries FRET donor part and carry have 550 and 710nm between the FRET acceptor probe of FRET acceptor portion of emission maximum form, one of them FRET acceptor portion is selected from Rh6G and TAMRA.
2. according to the composition or the reaction mixture of claim 1, it comprises that 4-5 is to the FRET hybridization probe.
3. according to the composition or the reaction mixture of claim 1, it comprises 4 pairs of FRET hybridization probes definitely.
4. according to the composition or the reaction mixture of claim 1, wherein at least 3 FRET donor parts are identical.
5. according to the composition or the reaction mixture of claim 4, wherein said at least 3 FRET donors partly are fluoresceins.
6. according to the composition or the reaction mixture of claim 2, wherein at least 4 FRET donor parts are identical.
7. according to the composition or the reaction mixture of claim 6, wherein said at least 4 FRET donors partly are fluoresceins.
8. according to the composition or the reaction mixture of claim 3, wherein 4 FRET donor parts are identical definitely.
9. composition according to Claim 8 or reaction mixture, wherein said 4 FRET donors definitely partly are fluoresceins.
10. according to the composition or the reaction mixture of claim 1, wherein all FRET donor parts are all identical.
11. according to the composition or the reaction mixture of claim 10, wherein said all FRET donor parts all are fluoresceins.
12. according to each composition or reaction mixture among the claim 1-11, wherein at least one other FRET donor partly is selected from Atto425 and WI343.
13. according to each composition or reaction mixture among the claim 1-11, one of them FRET acceptor portion is selected from LC-Red 705, Cy5.5 and JA286.
14. according to each composition or reaction mixture among the claim 1-11, wherein at least one, two or three FRET acceptor portions are selected from Cy5, LC-Red 640 and LC-Ked610.
15. a system that carries out multicolor real time PCR, it comprises
The PCR in real time instrument and
According to each composition or reaction mixture among the claim 1-14.
16. according to the system of claim 15, feature is that described PCR in real time instrument comprises
At least one light source
At least 4 fluorimetric detector entities, described each entity have the center that differs 25nm at least each other and detect wavelength
Feature is that described detector entity can
Detect at least 3 not right maximum fluorescence emission of FRET hybridization probe of isolabeling simultaneously
Detect simultaneously at least 2 not the TaqMan hybridization probe of isolabeling maximum fluorescence emission and
Detect the maximum fluorescence emission of SybrGreenI
The device of heating and cooling
The a plurality of reaction vessels that hold reaction mixture.
17. according to the system of claim 16, wherein said light source is LED.
18. according to the system of claim 16, wherein said PCR in real time instrument comprises 5-6 fluorimetric detector entity.
19. according to the system of claim 16, wherein said each entity has the center that differs 30nm at least each other and detects wavelength.
20. according to the system of claim 16, wherein said detector entity can detect 4 not right maximum fluorescence emission of FRET hybridization probe of isolabeling simultaneously.
21. according to the system of claim 16, wherein said detector entity can detect 5 not right maximum fluorescence emission of FRET hybridization probe of isolabeling simultaneously.
22. according to each system among the claim 16-21, feature is that described PCR in real time instrument comprises a light source definitely.
23. a method that increases and detect a plurality of target DNA sequences, it comprises
A) provide composition or reaction mixture according to claim 1-14,
B) make described reaction mixture carry out the thermal cycling scheme, so that the amplification of described a plurality of target sequences can take place,
C) after many amplification cycles, at least once monitor the hybridization of described every pair of FRET hybridization probe.
24. according to the method for claim 23, the wherein hybridization of at least monitoring temperature dependency mode.
25. a PCR in real time instrument, it comprises
Light source definitely
5-6 fluorescence monitor entity, described each entity have the center that differs 25nm at least each other and detect wavelength
Feature is that described detector entity can
Detect at least 3 not right maximum fluorescence emission of FRET hybridization probe of isolabeling simultaneously,
Detect simultaneously at least 2 not the TaqMan hybridization probe of isolabeling maximum fluorescence emission and
Detect the maximum fluorescence emission of SybrGreenI
The device of heating and cooling
The a plurality of reaction vessels that hold reaction mixture.
26. according to the PCR in real time instrument of claim 25, wherein said light source is LED.
27. according to the PCR in real time instrument of claim 25, wherein said each entity has the center that differs 30nm at least each other and detects wavelength.
28. according to the PCR in real time instrument of claim 25, wherein said detector entity can detect 4 not right maximum fluorescence emission of FRET hybridization probe of isolabeling simultaneously.
29. according to the PCR in real time instrument of claim 25, wherein said detector entity can detect 5 not right maximum fluorescence emission of FRET hybridization probe of isolabeling simultaneously.
30. according to each instrument among the claim 25-29, feature is that the wavelength region that wavelength is selected from 520-540nm, 545-565nm, 570-590nm, 600-620nm, 630-650nm, 660-680nm and 700-720nm is detected at described center.
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| US8404438B2 (en) * | 2006-11-30 | 2013-03-26 | Arkray, Inc. | Probes for detection of SULT1A1 gene, reagent containing the same, and the uses thereof |
| US8383068B2 (en) * | 2009-05-14 | 2013-02-26 | Icubate, Inc. | Apparatus for performing amplicon rescue multiplex PCR |
| EP2463661B1 (en) * | 2010-11-15 | 2014-01-08 | F. Hoffmann-La Roche AG | Instrument and method for the automated thermal treatment of liquid samples |
| CN105866090A (en) * | 2016-06-03 | 2016-08-17 | 苏州百源基因技术有限公司 | Ultraviolet visible light fluorescence detection system |
| CN105973855A (en) * | 2016-06-03 | 2016-09-28 | 苏州百源基因技术有限公司 | Infrared visible light fluorescence detection system |
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|---|---|---|---|---|
| US5869255A (en) * | 1994-02-01 | 1999-02-09 | The Regents Of The University Of California | Probes labeled with energy transfer couples dyes exemplified with DNA fragment analysis |
| US6150107A (en) * | 1996-10-04 | 2000-11-21 | The Regents Of The University Of California | Methods of sequencing and detection using energy transfer labels with cyanine dyes as donor chromophores |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5869255A (en) * | 1994-02-01 | 1999-02-09 | The Regents Of The University Of California | Probes labeled with energy transfer couples dyes exemplified with DNA fragment analysis |
| US6177247B1 (en) * | 1994-02-01 | 2001-01-23 | The Regents Of The University Of California | Detection methods using probes labeled with energy transfer coupled dyes for DNA fragment analysis |
| US6150107A (en) * | 1996-10-04 | 2000-11-21 | The Regents Of The University Of California | Methods of sequencing and detection using energy transfer labels with cyanine dyes as donor chromophores |
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