CN1141399C - Twin introducer used for homogeneous fluorescent PCR testing - Google Patents
Twin introducer used for homogeneous fluorescent PCR testing Download PDFInfo
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Abstract
The present invention provides a twin primer which is a double-chain primer for preventing non-specificity amplification and homogeneous phase fluorescent PCR detection. The twin primer is composed of a positive primer and a negative primer which are hybridized, wherein the positive primer is used for amplifying an upstream primer and a downstream primer; the negative primer is complementary to the positive primer and passes through a modified sequence. The present invention comprises a marked type and a non-marked type. The 5' end of the positive primer is labelled or is not marked; the extension capability at the 3' end of the negative primer is lost by modification. The 5' end of the positive primer is marked or is not marked; the extension capability at the 3' end of the negative primer is lost by decoration. The twin primer with the duplex structure can effectively prevent non-specificity amplification in the PCR process, and the twin primer with simple preparation can be flexibly applied to various PCR detection technologies.
Description
(1) technical field
The present invention relates to a kind of amplification and detection of gene.
(2) background technology
Polymerase chain reaction (PCR) has been widely used in each research field based on nucleic acid, and the application aspect clinical diagnosis also increases day by day, and homogeneous fluorescent PCR is the novel detection technique that grew up in recent years.The present existing multiple mode of homogeneous fluorescent PCR detection technique can be divided into non-sonde-type and sonde-type two classes according to whether possessing the segmental ability of identification specific amplified.Non-sonde-type has fluorescence intercalator method and Sunrise Primer (claiming Amplifluor again), and sonde-type comprises 5 '-exonuclease technology (TaqMan probe), molecular beacon (molecular beacon) technology, the two probe techniques that are applied to Lightcycler, scorpion primer (Scorpions primer) and AmpliSensor etc.Last type design is simple, but owing to can't discern the non-specific amplification product, is very limited in actual applications.The reliability of measuring owing to increased the probe identification step, has been improved in one type of back, but the experimental design complexity, price is high.This two classes technical disadvantages have its source in that they all lack the mechanism of effective prevention non-specific amplification.
Prevention non-specific amplification technology mainly contains solid paraffin method and warm start method (hot-start) at present.The solid paraffin method is in advance archaeal dna polymerase to be separated with solid paraffin and other reacted constituent, like this, even have non-specific combination, owing to there is not the participation of polysaccharase also just can't form extension products at low thermophase.Along with temperature raises, low-melting combination is all dissociated, and this moment, polysaccharase just contacted with reacted constituent by the paraffin that melts, and amplified reaction takes place.This method has been eliminated the non-specific amplification that causes to this stage of heating up before the reaction beginning.Obviously, because paraffin can only be brought into play disposable effect, powerless to the contingent non-specific amplification of annealing stage in the later circulation.Because the operation of solid paraffin embrane method is loaded down with trivial details, easily introduce and pollute, use seldom at present.Another kind of method commonly used is the warm start technology, by polysaccharase is handled, as the polysaccharase antibody technique, polysaccharase is manually modified etc., make polysaccharase not have an activity, actively during high temperature just brought into play, as the famous AmpliTaq Gold of PE company etc. at low thermophase.Treated polysaccharase can mix with reactive component in advance, and its mechanism of action is similar to the solid paraffin method, equally can only be before reaction beginning brings into play disposable effect to this stage of heating up.The warm start technology is used wider at present, and existing many companies have released different modifying enzymes, but prevailing price is high.More than these technology all can't play a role in the whole process of amplification, thereby can not thoroughly eliminate non-specific amplification.
(3) summary of the invention
Purpose of the present invention aims to provide a kind of double-chain primer---twin introducer non-specific amplification, that be used for the homogeneous fluorescent PCR detection that prevents.
Twin introducer is the duplex structure that positive primer and negative primer hybridization form, and positive primer is the upstream and downstream primer that is used for pcr amplification, and negative primer is and positive primer complementary and modified sequence.Twin introducer comprises two kinds on border note type and non-marked type.
The marking type twin introducer is at 5 of positive primer ' end mark fluorescent dyestuff, 3 ' mark fluorescent quencher of negative primer, this modification makes negative primer not possess the extension ability, in the twin introducer of this structure, fluorescence dye and the fluorescent quenching agent end generation fluorescent quenching that exists together forms the twin introducer of non-fluorescence.The preparation method of marking type twin introducer is a fluorescence dye on 5 of positive primer ' end mark at first, fluorescent quenching agent on 3 of negative primer ' end mark, with the positive and negative primer behind the mark with 1:(1.0～2.0) mixed, at 1.0～2.0mM Mg
2+In the system, hybridization forms double-chain primer under 20～95 ℃.
Non-marked type twin introducer is that positive primer is unmarked, and 3 of negative primer ' hold phosphorylation or add dideoxy nucleotide makes it lose the extension ability by modification.Being prepared as of non-marked type twin introducer at first will be born 3 of primer ' end phosphorylation or add dideoxy nucleotide, then with positive and negative primer with 1:(1.0～2.0) mixed after, at 1.0～2.0mM Mg
2+In the system, hybridization forms double-chain primer under 20～95 ℃.
The principle that the marking type twin introducer is used for the homogeneous fluorescent PCR detection is as follows:
(see figure 1) in the PCR reaction mixture of forming by the mark twin introducer, with twin introducer 1 and twin introducer 2 respectively as the upstream and downstream primer, all fluorescence dye (representing with zero) on 5 of positive primer ' end mark is born quencher on 3 of primer ' end mark (using ● expression).Combination or the positive non-specific combination of primer on template between the positive primer owing to do not exist free to possess the positive primer of extension ability, can not take place in the low thermophase before the reaction beginning, can not generate primer dimer or non-specific amplification product yet; The superfluous negative primer of free promptly allows to take place non-specific combination, because of not possessing the extension ability, also can't form amplified production.That is to say, adopt the pcr amplification system of twin introducer, before entering the sex change stage, effectively prevented the generation of non-specific amplification.Enter the thermally denature stage, sex change all takes place in twin introducer and template, and positive primer is released from twin introducer, and photoluminescent property is recovered.When temperature was reduced to annealing temperature, the positive primer of part combined with template, and the positive primer of part is then for the negative primer annealing of surplus becomes non-blooming twin introducer again, and simultaneously, negative primer also is bonded to template, but does not cause extension.These processes have been stopped the chance that non-specific combination takes place positive primer, that is to say, the round-robin annealing stage in other words full cycle stage (because of other stage does not anneal) twin introducer brought into play the effect of prevention non-specific amplification equally.The fluorescence of this elementary reaction system comes from the annealing and the extension of the positive primer that discharges.When temperature raise (as 72 ℃) enter extension during the stage, the positive primer of annealed will guide the synthetic of DNA, the amplified production of generation tool fluorescence; At this moment, twin introducer meeting Yin Wendu is higher than its fusing point and unwinds, and those and the negative primer of template bonded also can disintegrate down, and does not influence the carrying out of extension.Afterwards each is taken turns in the temperature cycle, and positive primer all can consume along with the accumulation of amplified production, and after PCR finishes, the fluorescence intensity of whole reflection system also will be accumulated.From above amplification procedure as can be seen, twin introducer PCR can not only and can bring into play the effect of prevention non-specific amplification before the reaction beginning in whole amplification cycles process.It can also be seen that marking type twin introducer PCR can adopt end assay method to obtain qualitative or semi-quantitative results, also can realize detection by quantitative by detecting the fluorescence intensity in each cycle annealing stage in real time.
The mark twin introducer is used for homogeneous fluorescent PCR and detects, and prevention non-specific amplification and the detection of realization homogeneous fluorescent are finished simultaneously by the twin introducer of mark.With other homogeneous phase PCR detection technique of same employing fluorescent quenching principle, compare as Sunriseprimer, molecular beacon, scorpion primer and TaqMan probe, in theory, the mark twin introducer has higher sensitivity for analysis.Because the former three fluorescent signal comes from switching poor of hair clip type primer or probe, or perhaps change to an increase degree that fluorescence after limit for length's distance arranged from zero distance between fluorescent substance and the quencher thereof; The fluorescent signal of TaqMan probe technique then is to come from probe poor before and after dissociating, or perhaps between fluorescent substance and the quencher thereof from an increase degree that limit for length's variable in distance fluorescence after the endless distance arranged.The fluorescent signal of marking type twin introducer PCR comes from the release of positive primer in the twin introducer, and it is to be to change to the endless distance increase degree of fluorescence afterwards from zero distance between fluorescent substance and the quencher thereof.The obvious latter's variable in distance maximum, thereby under the situation that adopts identical fluorescent substance and quencher, the latter's fluorescent signal is also maximum.
Compare from the angle of preparation, the marking type twin introducer is the simplest.At first, twin introducer can directly be finished design according to primer sequence; Secondly, the mark of twin introducer is the single-ended modification of oligonucleotide, can directly prepare by solid phase synthesis, and one step of purifying finishes, so productive rate is also high.Other probe or primer are all two modification the (both-end is modified or inner the insertion modified), and must carry out special processing according to rule each other, because these rules all belong to experience character, usually needing to attempt for several times could be successful, and individual cases then need special synthetic merchant to finish as the scorpion primer.Two modification purifying are loaded down with trivial details, and productive rate is low, so price is generally high.For sonde-type, another problem of existence is, owing to need design the probe that can detect special amplified production, comprises sequence or unknown or be the situation in alterable height zone for those primers, and the difficulty of probe design will increase greatly.
Marking type twin introducer PCR homogeneous fluorescent detection technique itself has very big handiness, according to above-mentioned principle, possesses certain empirical people and can design various modes.Such as, in the PCR reaction, can a side adopt the mark twin introducer, opposite side adopts positive primer or non-marked twin introducer.Twin introducer wants the negate primer not possess the extension ability, according to this requirement, except adopting dideoxy nucleotide and phosphorylation approach, possess certain empirical people and can design multiple mode, do not possess extension ability peptide nucleic acid(PNA) etc. such as negative primer employing, these new ways also may be given double-chain primer with new advantage, as insensitive to magnesium ion in the reaction system, and anti-degradation capability etc.Measure sensitivity in order further to improve, the mark quantity of fluorescent substance in the marking type twin introducer and quencher can be not limited to an end, can further in the middle of chain, insert the base of a plurality of modifications, under the situation of not disturbing amplification, sensitivity is greatly improved.
Marking type twin introducer PCR homogeneous fluorescent detection technique can have compatibility with prior art, that is to say, original most homogeneous fluorescent PCR detection techniques can adopt the mark twin introducer, and may increase some new functions.Such as Sunrise primer (claiming Amplifluor again) for non-sonde-type, when a side primer is Sunrise primer, another primer just can adopt the mark twin introducer, and the benefit of doing like this is to avoid the non-specific amplification that dimeric formation causes between primer.Molecular beacon for sonde-type, use has identical fluorophor and quencher (refers to have the fluorophor and the quencher of same effect here, and not necessarily duplicate material, as follows) the mark double-chain primer, just may further improve the sensitivity of mensuration, because molecular beacon possesses the ability of identification specific amplified, but do not possess prevention non-specific amplification function.After adopting the mark double-chain primer, the consumption of having avoided non-specific amplification to cause helps to improve the specific amplified productive rate on the one hand, and on the other hand, fluorescent signal has also been contributed in the amplification of mark double-chain primer itself.Similarly, for other sonde-type such as TaqMan and Amplisensor,, also can reach similar effect as long as the marking type double-chain primer uses identical fluorophor and quencher.For the scorpion primer, can a side adopt the mark double-chain primer, for the two probes that are used for Lightcycler, because the latter adopts energy transfer principles but not the fluorescent quenching approach, the mark double-chain primer can not directly use, and still, is understandable that, if both one of in addition conversion on fluorescence dye is selected, just can reach the purpose of compatibility.
Mark design in the marking type twin introducer also can be used for, in the PCR reaction, mark is to prevent false negative and realize the effective way that theorem detects in adding.Interior target traditional concept is to use and the identical primer of target sequence, and its sequence is close with target sequence again, so that the two amplification efficiency is approaching as far as possible, but can distinguish them in order to detect, and the two need keep certain difference again.This point becomes target principal contradiction in the design.Twin introducer PCR provides new approaches for mark in designing.The design philosophy of mark PCR is in the twin introducer, the length of interior label sequence is consistent with template, part adopts identical sequence between templa-primer between the primer of interior label sequence, and two interior label primers or one of them are different with the templa-primer sequence, but its fusing point is consistent with templa-primer with annealing efficiency.Interior label primer adopts different fluorescent marks so that fluorometric assay with templa-primer.To adopt primer to comprise sequence different with traditional design, it is identical that the interior label primer that adopts the mark twin introducer to design comprises sequence, but both amplimer sequence differences, but the primer of target sequence and interior target primer be in annealing temperature, and aspects such as efficient should be consistent as far as possible.The method of distinguishing them when being used to detect then is to utilize different mark fluorescent dyestuffs.Because, the sequence amplification part of interior mark and target sequence amplification is identical, the influence factor that is decided by the two efficient just has only primer sequence, obviously, in the extension stage, primer sequence is very little to the influence of amplification, therefore, this design itself is through having dwindled the difference between the amplification of target sequence and interior mark greatly, and different fluorescence dyes makes a distinction them when detecting at an easy rate.On the other hand, it is also simple relatively to design the identical primer of annealing efficiency on target sequence primer basis.Interior target generates can be by many technology, as realizations such as PCR mutating technologies.For certain empirical people is arranged, can adopt multiple approach flexibly to realize this point.
The marking type twin introducer also can be used for multiple fluorescence PCR, and in such reaction system, mutually different fluorescent marker dyes is adopted in each reaction.After reaction,, can reach the purpose that detects each reaction simultaneously by detecting the fluorescent reaction of different dyes.Compare with general multiplex PCR, the twin introducer multiple fluorescence PCR has two clear superiorities: the one, can realize that homogeneous fluorescent detects simultaneously, and the 2nd, double-chain primer helps to prevent the non-specific combination between a plurality of primers in the primer multiplex PCR.
A kind of typical fluorescence indication mechanism that non-marked type twin introducer is used for homogeneous fluorescent PCR detection employing is exactly the fluorescence intercalator.The fluorescence intercalator is that a class can embed double-stranded DNA and sends the fluorescence dye of characteristic fluorescence.In homogeneous fluorescent PCR detected, that the most frequently used was SYBR Green I at present.SYBR Green I can be directly used in the conventional PCR reaction, is the simple pathway that realizes that homogeneous fluorescent measures, and still, because the fluorescence intercalator does not possess identification non-specific amplification ability, the application in homogeneous fluorescent PCR detects is very limited.After adopting the non-marked double-chain primer, this situation is able to radical change, and double-chain primer possesses the effect at the omnidistance performance prevention of PCR non-specific amplification, if intercalator is only indicated the DNA of amplification, just can realize special homogeneous fluorescent mensuration.
In the PCR reaction mixture (see figure 2) of forming by non-marked twin introducer and fluorescence intercalator, be the upstream and downstream primer with twin introducer 3 and twin introducer 4, all modified blind end (representing) that forms of 3 of its negative primer ' end with X.Combination or the positive non-specific combination of primer on template between the positive primer owing to do not exist free to possess the positive primer of extension ability, can not take place in the low thermophase before the reaction beginning, can not generate primer dimer or non-specific amplification product yet; The superfluous negative primer of free promptly allows to produce non-specific combination, because of it does not possess the extension ability, also can't form amplified production.That is to say, adopt the pcr amplification system of twin introducer, before entering the sex change stage, effectively prevented the generation of non-specific amplification.The existence of fluorescence intercalator and double-stranded DNA, making the reaction solution that is in this stage is tool fluorescence.In the process that enters the thermally denature stage, after the arrival certain temperature, sex change all takes place in twin introducer and template, and positive primer is released from twin introducer.Because do not have double-stranded DNA and combine with the fluorescence intercalator this moment, this moment, the fluorescence of reaction system was minimum.After temperature is reduced to annealing temperature, positive primer of part and template annealing, the positive primer of part is generated twin introducer again by excessive negative primer annealing, and simultaneously, negative primer also is bonded to template, but can not cause extension.These processes have been stopped the chance that non-specific combination takes place positive primer, that is to say, the round-robin annealing stage in other words full cycle stage (because of other stage does not anneal) twin introducer brought into play the effect of prevention non-specific amplification equally.The fluorescence of this elementary reaction system comes from combining of all double-stranded DNAs that intercalator and annealing forms.When temperature raise (as 72 ℃) enter extension during the stage, the positive primer guiding of annealed DNA's is synthetic, intercalator will combine with double-stranded DNA and send fluorescence, and, along with the prolongation of DNA chain, have more intercalator combinations, so the fluorescence of system is stronger in the later stage of extending.Be in the twin introducer in extension stage and can Yin Wendu be higher than its fusing point and unwind, those be born primer with the template bonded and disintegrate down too.That is to say that unique source of the fluorescence of system is exactly the double-stranded DNA of positive primer annealing and extension at this moment.It has represented the generation of specific amplified product.In the temperature cycle afterwards, positive primer will be consumed (negative primer no consumption) because of generating amplified production.Fluorescence intensity in that the extension stage is measured can reflect the generation of amplified production strictly according to the facts.Being chosen in this stage measures each round-robin fluorescence intensity and has just realized The real time measure, as can be seen, twin introducer PCR can not only and can bring into play the effect that prevents non-specific amplification before the reflection beginning in entire reaction course, guaranteed the specificity of measurement result.
In practical measurement, it is identical with conventional fluorescence intercalator The real time measure mode that non-marked twin introducer PCR measures mode, promptly at first the fluorescence intensity in assaying reaction warm start stage (this moment fluorescence intensity minimum) as background, measure the fluorescence intensity in each circulation extension later stage in stage later on, the difference of the two is as this round-robin fluorescence intensity.It is pointed out that non-marked twin introducer PCR is not suitable for adopting end assay method to carry out qualitative or semiquantitative determination.Reason is, a large amount of existence before and after the twin introducer reaction make reaction system all be fluorescence, the fluorescence intensity of amplification afterreaction system changes the factor decision by---fluorescence intensity that the fluorescence intensity that twin introducer consumption causes reduces and the amplified production accumulation causes increases---two mutual growth and decline, and its net result is difficult to reflect the difference of primary template amount.
Non-marked type twin introducer homogeneous fluorescent PCR is more simple than marking type in design.
The non-marked twin introducer provides prevention non-specific amplification ability, and is then unfettered on selection fluorescence indication mechanism.Adopting the fluorescence intercalator is a kind of the easiest approach.In fact, existing most homogeneous fluorescent PCR detection technique can directly adopt the non-marked twin introducer.Such as the Sunrise primer for non-sonde-type, when a side primer was Sunrise primer, another primer just can adopt the non-marked twin introducer, and the benefit of doing like this is, can avoid that dimeric formation causes non-specific amplification between primer.Molecular beacon for sonde-type, use the non-marked twin introducer, can further improve the sensitivity of mensuration on the one hand---because molecular beacon possesses the ability of identification specific amplified, but do not possess prevention non-specific amplification function, the consumption that double-chain primer has avoided non-specific amplification to cause helps to improve the specific amplified productive rate.On the other hand, the specific amplified ability that twin introducer PCR possesses adds the special indication ability of probe, makes this be combined in theory reliable detection system the most.For two probes of other sonde-type such as TaqMan, Amplisensor and Lightcycler, adopt the non-marked twin introducer, also can obtain similar effect.For the scorpion primer, can a side adopt the scorpion primer, opposite side adopts the non-marked twin introducer.Different with the mark twin introducer, the non-marked type can directly adopt these fluorescence indication mechanisms, and need not consider the apolegamy of fluorophor and quencher.The non-marked twin introducer combines with various fluorescence indication mechanisms, makes that also the mensuration mode is more flexible.In fact, adopt cold twin introducer, the reason that does not change these fluorometric assay systems is measured mode.As combining with the molecular beacon of sonde-type, both can carry out endpoint determination, can carry out The real time measure at the round-robin annealing stage again, because molecular beacon itself just possesses these mensuration modes.With the combination of two probes, scorpion primer and the Amplisensor etc. of Sunrise primer, TaqMan, Lightcycler, also be analogue.
In some PCR reaction, the non-marked twin introducer also can adopt a side to use twin introducer and opposite side uses the mode of positive primer.The non-marked twin introducer also wants the negate primer not possess the extension ability.Negative primer can adopt multiple Design Mode, except adopting dideoxy nucleotide and phosphorylation approach, possess certain empirical people and can design multiple mode, do not possess the peptide nucleic acid(PNA) of extension ability etc. such as employing, these new ways also may be given double-chain primer with new advantage, as insensitive to magnesium ion in the reaction system, anti-degradation capability etc.
(4) description of drawings
Fig. 1 is the PCR principle schematic of marking type twin introducer.
Fig. 2 is the PCR principle schematic of non-marked type twin introducer.
Fig. 3 is marking type twin introducer and traditional primer PCR agarose gel electrophoresis contrast figure.
Fig. 4 is marking type twin introducer PCR fluoroscopic examination result.
Fig. 5 is used for the fluoroscopic examination result of human telomerase for one-sided marking type twin introducer PCR.
(5) embodiment
The invention will be further described below by embodiment.
The pcr amplification of human beta-globin is the PCR program of a standard, and we illustrate that with it the marking type twin introducer is used for the principle of homogeneous fluorescent PCR.
The preparation of mark twin introducer: positive primer is to modify at 5 of traditional primer ' introducing fluorescence, and this example adopts FAM.Specific design is: 5 '-FAM-GAA GAG CCA AGG ACA GGT AC-3 ' and 5 '-FAM-CAA CTT CAT CCACGT TCA CC-3 '; The sequence of two negative primers can directly adopt the complementary sequence of its positive primer, also can improve the dissociation efficiency of positive primer in the some bases of its 5 ' shortening.Its 3 ' hold then mark quencher DABCYL.In this example, base of 5 of negative primer ' shorten, that is: 5 '-TAC CTG TCC TTG GCT CCT C-Dabcyl-3 ' and 5 '-GTG AAC GTG GATGAA GTT G-Dabcyl-3 '.
With 1: 1.5 mixed, hybridization solution contained 1.5mM Mg with positive primer and negative primer
2+94 ℃ of 5min of hybridization conditions, 55 ℃ of 2min.Cooling, freezing preservation is as the primer of PCR reaction.
Normal people's peripheral blood is provided by the laboratory volunteer.With lymphocyte liquid separating method separating blood white corpuscle with the extracting of conventional phenol chloroform, ethanol precipitation extracts white corpuscle DNA.
The PCR reaction: PCR reaction cumulative volume is 25 μ L, includes 10mM Tris-HCl, pH8.3,50mM KCl, 1.0UTaq enzyme, 200 μ M dNTP, 2.0mM MgCl
2, the twin introducer of the positive primer of 0.4 μ M, 5 μ L templates, 50 μ L paraffin oils, through 95 ℃, the pre-sex change of 5min, 95 ℃ again, 30s, 55 ℃, 35s, 72 ℃, 1min, after 40 circulations, 72 ℃ are extended 5min.Directly carrying out fluorescent value behind the pcr amplification measures.The results are shown in Figure 3,4.Can find out obviously that from Fig. 3 marking type twin introducer PCR can effectively prevent non-specific amplification (b represents blank).Among Fig. 4, F represents fluorescence intensity, and s is a sample, and as seen from the figure, the concentration of template is directly proportional with fluorescence intensity.
Telomerase is a kind of tumor markers of wide spectrum, and the measuring method of Telomerase is loaded down with trivial details at present, and the explanation of this example adopts one-sided mark twin introducer can realize that homogeneous fluorescent PCR detects fast, and the TS primer is: 5 '-AAT CCG TCG AGC AGAGTT-3 '.
The preparation of twin introducer: positive primer is on the TX of Telomerase primer basis, at 5 ' end flag F AM, that is: 5 '-FAM-CCC TTACCC TTA CCC TTA CCC TAA, negative primer is being selected its negative complementary series, wherein 5 ' end shortens a base, 3 ' end is then used the quencher mark, that is: 5 '-TAG GGT AAG GGT AAG GGT GGT AAG GG-Dabcyl-3 '.With 1: 1.5 mixed, hybridization solution contained 2.0mM Mg with positive and negative primer
2+Hybridization conditions is 94 ℃ of 5min, 55 ℃ of 2min.Cooling, freezing preservation is as the primer of PCR reaction.
Telomerase extracts from gastric carcinoma cells MGc80-3.
The extension system 25 μ L of Telomerase include 20mM Tris-HCl, pH8.3,200 μ M dNTP, 0.4 μ M TS primer, 5 μ g CHAPS cell extracts, 50 μ L paraffin oils.Through 22 ℃, after the 10min reaction, 95 ℃ again, the 15min thermally denature adds 0.4 μ M downstream twin introducer (containing 2U Taq enzyme) and carries out pcr amplification.The PCR reaction conditions is: through 94 ℃ of pre-sex change 60s, and 94 ℃ of 30s again, 50 ℃ of 30s, 72 ℃ of 1.5min, 30 circulations are extended 5min for back 72 ℃.The PCR product is directly carried out fluorescence measurement.The results are shown in Figure 5.
Adopt non-marked type twin introducer to carry out homogeneous fluorescent PCR and detect, need to adopt corresponding fluorescence indication mechanism, this example is utilized the fluorescence intercalator, and the design and the detection method of non-marked twin introducer is described.
The preparation of non-marked twin introducer: the primer sequence of selecting amplification HBV: primer 1:5 '-CTT TAT AAG GATCAA TGT CCA TGC-3 ' and primer 2: 5 '-GAA ATA TTC CTA GTT ACA GGT ACG-3 '.The negative primer of primer 1 is: 5 '-GCA TGG ACA TTG ATC CTT ATA AAG-3 '-PHOR, the negative primer of primer 2 is: 5 '-CGT ACC TGT AAC TAG GAA TAT TTC-3 '-PHOR, wherein, PHOR is a phosphate, is used to seal the extension of negative primer.With 1: 1.5 mixed, hybridization solution contained 1.5mM Mg with positive and negative primer
2+, hybridization conditions is 94 ℃ of 5min, 55 ℃ of 2min.Cooling, freezing preservation is as the primer of PCR reaction.
Get clinical serum specimen, water-boiling method extracts DNA routinely.
The PCR reaction: PCR reaction cumulative volume is 25 μ L, includes 10mM Tris-HCl, pH8.3,50mM KCl, 1.0UTaq enzyme, 200 μ M dNTP, 1.5mM MgCl
2, the twin introducer of the positive primer of 0.4 μ M, 1: 10,000 SYBRGreen I (Molecular Probes Inc) and 5 μ L templates, Dropwise 50 μ L paraffin oil is through 95 ℃, the pre-sex change of 5min, 95 ℃ again, 30s, 55 ℃, 35s, 72 ℃, 1min, after 35 circulations, 72 ℃ are extended 5min.Carry out The real time measure by the fluorescence intensity of measuring the extension stage.
Claims (5)
1. be used for the twin introducer that homogeneous fluorescent PCR detects, it is characterized in that twin introducer is the duplex structure that positive primer and negative primer hybridization form, positive primer is the upstream and downstream primer that is used for pcr amplification, and negative primer is and positive primer complementary and modified sequence.
2. the twin introducer that is used for the homogeneous fluorescent PCR detection as claimed in claim 1 is characterized in that twin introducer is the marking type twin introducer, 5 of its positive primer ' end mark fluorescent dyestuff, 3 of negative primer ' end mark fluorescent quencher.
3. as claim 1 and the 2 described twin introducers that are used for the homogeneous fluorescent PCR detection, it is characterized in that it is prepared as at first fluorescence dye on 5 of positive primer ' end mark, fluorescent quenching agent on 3 of negative primer ' end mark, with the positive and negative primer behind the mark with 1:(1.0～2.0) mixed, at 1.0～2.0mM Mg
2+In the system, hybridization forms double-chain primer under 20～95 ℃.
4. the twin introducer that is used for the homogeneous fluorescent PCR detection as claimed in claim 1 is characterized in that twin introducer is a non-marked type twin introducer, and its positive primer is unmarked, 3 of negative primer ' hold phosphorylation or add dideoxy nucleotide.
5. as claim 1 and the 4 described twin introducers that are used for the homogeneous fluorescent PCR detection, it is characterized in that it is prepared as at first in 3 of negative primer ' end phosphorylation or adds dideoxy nucleotide, then with positive and negative primer with 1:(1.0～2.0) mixed after, at 1.0～2.0mM Mg
2+In the system, hybridization forms double-chain primer under 20～95 ℃.
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| KR100580639B1 (en) * | 2003-12-30 | 2006-05-16 | 삼성전자주식회사 | Apparatus for detecting fluorescence flow |
| EP2705159B1 (en) * | 2011-05-06 | 2018-03-21 | Qiagen GmbH | Methods for sequencing, amplification and detection of nucleic acids comprising internally labelled primer |
| CN102352411B (en) * | 2011-09-29 | 2013-06-12 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
| CN103205425B (en) * | 2012-01-17 | 2015-07-01 | 北京万达因生物医学技术有限责任公司 | Antisense interference oligonucleotide used for inhibiting primer non-specific amplification |
| CN103114131B (en) * | 2012-11-30 | 2018-10-02 | 珠海市坤元农业科技有限公司 | A kind of primer middle part sequence interferes round pcr |
| CN110699437B (en) * | 2019-11-11 | 2023-06-20 | 益善生物技术股份有限公司 | Human SDC2 gene methylation detection kit |
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