CN103882128A - Method for signal amplification and detection on target deoxyribonucleic acid (DNA) sequence at normal temperature - Google Patents

Method for signal amplification and detection on target deoxyribonucleic acid (DNA) sequence at normal temperature Download PDF

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CN103882128A
CN103882128A CN201410093830.3A CN201410093830A CN103882128A CN 103882128 A CN103882128 A CN 103882128A CN 201410093830 A CN201410093830 A CN 201410093830A CN 103882128 A CN103882128 A CN 103882128A
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赵美萍
吴曈勃
肖先金
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Peking University
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Abstract

The invention discloses a method for signal amplification and detection on a target deoxyribonucleic acid (DNA) sequence at normal temperature. A DNA signal amplification system is established by using Lambda excision enzyme and a double-tagging fluorescence nucleic acid probe, and the reaction velocity of the Lambda excision enzyme is improved by virtue of the synergistic effect of a fluorescence tagging radial and a mispairing basic group coexisting in a 5' ortho-position. By adopting the method, the target DNA sequence is rapidly, sensitively and specially detected at normal temperature, and convenience is brought for further large-scale and automatic detection. Besides, the method can be independently applied to signal amplification detection, and can be also used with the combination of other template amplification based DNA amplification techniques, such as rolling circle amplification (RCA) and selective polymerase chain reaction (PCR), and various requirements of DNA detection are met.

Description

Under normal temperature, target dna sequence is carried out the method for signal amplification and detection
Technical field
The present invention relates to DNA sequence dna detection field, comprise the technical fields such as detection by quantitative, single nucleotide polymorphism (SNP) somatotype and the low abundance point mutation detection of minim DNA.Specifically, the present invention utilizes Lambda excision enzyme and double-tagging Fluorescent Nucleic Acid Probe to set up DNA signal amplification system, under normal temperature condition, quick, highly sensitive, with high specificity target dna sequence is detected.
Background technology
DNA signal amplifies, and refers to by biochemical reaction and makes single target DNA sequence dna produce the signal of many times, thereby improve the detection sensitivity to target dna sequence.In the time that signal amplification system can carry out selectivity differentiation with the interference DNA sequence dna that has with it single base difference to target dna sequence and make the two signal amplification factor different, can realize the amplification of the two signal difference, thereby realize the detection of SNP somatotype and low abundance point mutation.
Develop take restriction enzyme, exonuclease III, endonuclease IV, DNA enzyme etc. as basic DNA signal amplification system at present, these systems are all used DNA as probe, utilize the base complementrity pairing effect of DNA double chain to identify specifically target dna sequence.
DNA signal amplification system based on restriction enzyme, detectability can reach 6.5pM(200 μ L system) (Li, J.J., Chu, Y., Lee, B.Y., andXie, X.S..Enzymatic signal amplification of molecular beaconsfor sensitive DNA detection.Nucleic Acids Res. (2008), 36:e36.).But the method is to requiring to exist in target dna sequence the cleavage site of restriction enzyme.Due to the restriction of restriction enzyme enzymic activity, and for the short piece that cuts rear generation is more easily left away, reaction needed is carried out at 55 ℃.This system for target dna sequence and have with it the discrimination of the interference DNA sequence dna of single base difference be 3-76 doubly, wherein the discrimination of high multiple requires the recognition site of the contiguous restricted property restriction endonuclease in base mispairing position.
DNA signal amplification system based on exonuclease III reacts 30min at 37 ℃, and detectability can reach 20pM(50 μ L system); Under 4 ℃ of conditions, react 24h, its detectability can reach 20aM(Zuo, X., Xia, F., Xiao, Y., and Plaxco, K.W..Sensitive and Selective Amplified Fluorescence DNA Detection Based onExonuclease III-Aided Target Recycling.J.Am.Chem.Soc. (2010), 132:1816-1818.).But exonuclease III amplification system is 1.5 times of less thaies for identification coupling with the discrimination of single base mismatch DNA sequence dna.
DNA signal amplification system based on DNA enzyme, can realize the reaction at 25 ℃, and its detection is limited to 1nM.By the design to molecular beacon sequence, can make system realize index and amplify, make detectability reach 1pM.But the whole reaction times reaches 14h(Wang, F., Elbaz, J., Teller, C., and Willner, I..Amplified Detection of DNA through an Autocatalytic and CatabolicDNAzyme-Mediated Process.Angew.Chem.Int.Ed. (2011), 50:295-299.).
DNA signal amplification system based on endonuclease IV, detectability can reach 60pM(50 μ L system, detection time 50min) (Xiao, X., Song, C., Zhang, C., Su, X., andZhaoM..Ultra-selective and sensitive DNA detection by a universalapurinic/apyrimidinic probe-based endonuclease IV signalamplification system.Chem.Commun. (2012), 48:1964-1966.).When introducing the recognition reaction of restriction endonuclease IV to cleavage site both sides base mispairing, and in conjunction with temperature during on the affecting of matched chain and mispairing chain stability, can reach 99-860 doubly for mating completely with the separating capacity of single base mismatch DNA sequence dna, detection sensitivity for point mutation can reach 0.01%-0.1%(Xiao, X., Liu, Y., andZhaoM..Endonuclease IV discriminates mismatches next to theapurinic/apyrimidinic site in DNA strands:constructingDNA sensing platforms with extremely highselectivity.Chem.Commun., 2013, 49, 2819-2821.).But this amplification system need to be optimized accurately and control temperature, and need under comparatively high temps, (>50 ℃) react, for having relatively high expectations of analytical instrument.When the impact of the temperature that is not coupled, while only relying on the character of restriction endonuclease IV identification base mismatch itself, except C:C mispairing discrimination can reach 179 times, the base mispairing discrimination of other types is only 3.11-18.9 times.
Based on the DNA signal amplification system of endonuclease IV/Lambda excision enzyme coupling, can at 37 ℃, react, detectability can reach 20pM(50 μ L system), it is 14-18 times for coupling and the separating capacity of single base mismatch DNA sequence dna, detection optimum sensitivity for point mutation can reach 0.5%(Xiao, X., Zhang, C., Su, X., Song, C., andZhaoM..A universal mismatch-directed signal amplification platform for ultra-selectiveand sensitive DNA detection under mild isothermal conditions.Chem.Sci. (2012), 3:2257-2261.).But the method needs the cooperation of two kinds of enzymes, system is comparatively complicated.In probe, need to design in addition abasic site, make on the one hand the stability decreases of probe self, make on the other hand cost improve.What is more important, the present invention finds under study for action, in the time that 5 ' ortho position of abasic site is G base, probe itself can slowly be cut by restriction endonuclease IV, makes fluorescence background signal increase, the sensitivity that impact detects.
In sum, the various DNA signal amplification system great majority based on nuclease of having reported are at present all comparatively complicated, need to just can reach higher sensitivity and the separating capacity to single base interference DNA sequence dna by accurate temperature controlling or other amplifying techniques that are coupled.In addition, also need analysis time to shorten, testing cost also needs further to be reduced, and makes method can meet the demand of disease early diagnosis and relevant life science, and becoming can widely used conventional sense means.
Summary of the invention
For the problems referred to above, the object of this invention is to provide a kind of can be under normal temperature condition to the method that target dna sequence carries out fast, highly selective signal amplifies, for quantitative analysis, single nucleotide polymorphism (SNP) somatotype and the low abundance point mutation detection etc. of minim DNA.
Lambda excision enzyme can be identified 5 ' of double-stranded DNA-PO specifically 4end, and from 5 '-PO 4end starts, and its place strand of continuity ground cutting, discharges 5 '-mononucleotide.For containing 5 '-PO 4the single stranded DNA of end or the unphosphorylated strand of 5 ' end or double-stranded DNA, the hydrolysis efficiency of Lambda excision enzyme is all very low.The present invention is further discovery under study for action, and in the time there is base mismatch in double-stranded DNA, Lambda excision enzyme can carry out selectivity identification to it, shows as cutting speed and changes regularly with the difference of mispairing position.In the time that there is labelling groups at the ortho position of base mismatch, the variation of cutting speed is more obvious.
Utilize above-mentioned character, the present invention design, for the specificity double-tagging fluorescent probe of target dna sequence, adopts following technical scheme, at normal temperatures to target dna sequence carry out fast, highly selective signal amplifies and detection.Specifically comprise the following steps:
1) the synthetic 5 '-PO that contains of design 4the double-tagging strand Fluorescent Nucleic Acid Probe of end, builds the signal amplification system based on Lambda excision enzyme.Probe mark fluorophor (F) and quencher group (Q), wherein fluorophor is near 5 ' end, and with 5 ' end at least one Nucleotide of being separated by, with the distance of 3 ' end at ten more than Nucleotide; Quencher group can be marked near 3 ' end and with fluorophor seven optional positions more than Nucleotide that are separated by.Owing to there being FRET (fluorescence resonance energy transfer) (FRET) effect between fluorophor and quencher group, make this probe quenching of fluorescence under original state.
2) after above-mentioned probe is mixed with containing the system to be measured of target dna sequence, add Lambda excision enzyme, lower reaction of normal temperature (between 25 ℃-37 ℃ all can).Wherein, the relative target dna sequence of probe should be greatly excessive, to realize the amplification of signal; The add-on of Lambda excision enzyme does not have clear and definite restriction, considers reaction times and cost, and recommending to make its final concentration in system is 0.01-0.2U/ μ L.The double-stranded DNA that probe and this target dna sequence form is identified by Lambda excision enzyme, contains 5 '-PO 4the probe strand of end is progressively hydrolyzed by Lambda excision enzyme, and when cut the leaving after probe of Nucleotide of mark fluorescent group, fluorophor is away from quencher group, and FRET (fluorescence resonance energy transfer) system is destroyed, discharges fluorescent signal.Due to after probe is hydrolyzed, it is complete that target dna sequence still keeps, and can be combined with new probe molecule to form double-strandedly, continue to be identified and cut by Lambda excision enzyme, thereby this process constantly circulation realize the amplification of fluorescent signal.The principle of whole signal amplification process is referring to shown in accompanying drawing 1.
Further, the present invention has found the character of Lambda excision enzyme series of novel in research process.Accompanying drawing 2a) show the Basic Design of testing several main probes used.Hereinafter N3-F15 probe span 5 '-PO 4the 3rd upper unmarked group (N) of end, mark fluorophor (F) on the 15th, at 3 ' end mark quencher group (Q); D3-F15 probe span 5 '-PO 4the 3rd upper mark digoxin (D) of end, the 15th upper mark fluorophor (F), 3 ' end mark quencher group (Q); F3-N15 probe span 5 '-PO 4the 3rd upper mark fluorophor (F) of end, the 15th upper unmarked group (N), 3 ' end mark quencher group (Q).Under normal circumstances, in the time there is base mismatch in DNA substrate sequence, the hydrolysis rate of the Lambda excision enzyme (LeeG. that can slow down, YooJ., LeslieB.J.andHaT., Single-molecule analysis reveals three phases of DNAdegradation by an exonuclease.Nat.Chem.Biol. (2011), 7:367-374).But in experiment of the present invention, find, as 5 '-PO in DNA substrate 4while having labelling groups (as fluorophor, digoxin group etc.) and its 5 ' ortho position to be base mismatch in the strand of end place, the hydrolysis rate of Lambda excision enzyme does not only slow down, and obviously accelerates on the contrary.Concrete data and being analyzed as follows:
(1) referring to Fig. 3 a) shown in, probe N3-F15 and target dna chain when hybridization that has single base mismatch at different positions, the variation of Lambda excision enzyme hydrolysis reaction fluorescence climbing speed.Can see, when the fluorophor in probe is marked at apart from 5 '-PO 4in the base that end is the 15th time, compared with the substrate two strands (No. 0 sample) of mating completely, in the time having single base mismatch for 14, fluorescence climbing speed is significantly accelerated.
(2) as Fig. 3 b) as shown in the measurement result of probe F3-N15, fluorophor mark is changed into apart from 5 '-PO 4in the base that end is the 3rd, in the time having single base mismatch for 2, fluorescence climbing speed is significantly accelerated.
Above two groups of experimental results tentatively show, in the time that there is single base mismatch at labelling groups 5 ' ortho position, fluorescence climbing speed be 5 ' ortho position coupling probe 1.8-2.0 doubly.
(3) as Fig. 3 c) as shown in the measurement result of probe D3-F15, this probe only than N3-F15 probe apart from 5 '-PO 4in the base that end is the 3rd multiple labeling the structure of a digoxin, all the other marks and sequence are in full accord.Contrast probe N3-F15 and D3-F15 and can see with the speed of reaction of entirely mating after target sequence (No. 0 sample) combination, 3 mark digoxin groups itself do not make Lambda excision enzyme hydrolysis reaction accelerate, and slightly slow down on the contrary.In the time that its 5 ' ortho position (2) are base mismatch, speed of reaction is accelerated to some extent than the sequence of full coupling.And in the time that 14 of target sequences are base mismatch, add the speed of reaction of D3-F15 probe system significantly to accelerate than full matching sequence.
Above result shows, the synergistic result of base mismatch that fluorescent mark group and 5 ' ortho position thereof coexist can make the speed of reaction of Lambda excision enzyme significantly accelerate.
According to above character, we can be by mark fluorophor in probe the reaction process in the time that the structure design (referring to Fig. 2 b)) of its 5 ' ortho position introducing base mismatch is accelerated Lambda excision enzyme detection object chain, thereby the signal of realizing in the short period of time higher multiple amplifies, and improves the sensitivity that object chain is detected.
Further, the present invention studies discovery, and for probe F3-N15, in the time that 2 of target sequences are base mismatch, if be also base mismatch at 4 or 5 simultaneously, the former obviously declines at speed of reaction, and the latter becomes hardly.And in the time that 2,4,5 three positions are base mismatch, the hydrolysis reaction of Lambda excision enzyme almost cannot carry out simultaneously.Utilize this characteristic, we can design the probe sequence with the two mispairing of target dna sequence, realize two differentiations of only having the DNA sequence dna of single base difference.As Fig. 2 c) as shown in, 4 of probe and couplings corresponding with base to be distinguished in target dna sequence, are marked at 3 by fluorophor, the base of 2,5 is all designed to and corresponding base mispairing in target sequence.The iodine of target dna sequence will obviously be accelerated like this, and have with it the interference sequence of single base difference because of 2,4,5 three while base mispairings, reaction will be suppressed significantly, by Lambda excision enzyme identification and hydrolytic action specifically, makes target sequence obtain selectivity amplification detection thus, and that the impact of interference sequence drops to is minimum, this is all very favourable for selectivity and sensitivity of improving SNP somatotype and point mutation detection.
To sum up, we have designed two kinds of different signal amplification detection systems, are respectively used to quantitative amplification analysis and single nucleotide polymorphism (SNP) somatotype and the low abundance point mutation detection etc. of minim DNA.Concrete technical scheme is as follows:
1. the signal of the minim DNA based on single mispairing double-tagging probe amplifies analytical procedure.When target dna sequence content in testing sample very low (sample of DNA absolute content below 1.0pmol, minimum can measure 0.1fmol), while coexisting without other similar DNA sequence dnas again simultaneously, the synthetic 5 '-PO that contains of design 4the double-tagging strand Fluorescent Nucleic Acid Probe of end, fluorophor (F) is marked on apart from 5 ' end is near but distance is no less than the position of 1 Nucleotide, and with the distance of 3 ' end at ten more than Nucleotide, quencher group (Q) can be marked at and fluorophor seven optional positions more than Nucleotide that are separated by.In the base at design labelling groups 5 ' ortho position and target dna sequence, the base of correspondence position is base mismatch, and other bases are coupling base.
With aforementioned common double the mark 5 '-PO that does not contain base mismatch 4end single-stranded probe is compared, Lambda excision enzyme is accelerated greatly to be combined the hydrolysis rate of product with object chain containing the new probe of single base mismatch, thereby significantly improves the sensitivity (can reach 2pM-20nM(50 μ L system) of method for amplifying signal detection minim DNA content).
2. the highly selective DNA signal amplification detecting process based on two mispairing double-tagging probes.In the time carrying out single nucleotide polymorphism (SNP) somatotype and low abundance point mutation detection, the technological difficulties of signal amplification system are the signal of How to choose ground amplification target DNA sequence dna, and other have the interference sequence of single base difference not substantially to be exaggerated.According to aforesaid properties, for the synthetic 5 '-PO that contains of base to be distinguished position design 4the double-tagging strand Fluorescent Nucleic Acid Probe of end, on probe sequence, find the base with this target dna sequence base complementrity contraposition, 5 ' the ortho position mark fluorescent group (F) at this to bit base, 3 ' ortho position is designed to base mismatch, and 5 ' ortho position of fluorophor is designed to base mismatch, other bases are coupling base.Fluorophor and 5 ' end at least one Nucleotide of being separated by, and with the distance of 3 ' end at ten more than Nucleotide, quencher group (Q) can be marked at and fluorophor seven optional positions more than Nucleotide that are separated by.
After this probe is combined with target sequence, due to the existence of labelling groups both sides base mismatch, the reaction that target sequence is exaggerated is accelerated greatly.And for the interference sequence that has single base difference, owing to there being three base mismatch to exist in labelling groups both sides, speed of reaction obviously declines.The impact of comprehensive above two aspects, designed probe is significantly enlarged the difference of target sequence and interference sequence, thereby the selectivity detecting is improved greatly.
Above method can be for the somatotype of single nucleotide polymorphism (SNP) and the detection of point mutation.In SNP somatotype, in sample, only contain a kind of genotype, it is comparatively easy to detect; And in point mutation, in sample, there is mutant DNA sequence and wild-type DNA sequence dna simultaneously, and at the disease initial stage, the abundance of mutant DNA sequence is conventionally very low, and the signal of the wild-type sequence that when detection, signal is often coexisted is in a large number covered.Utilize aforesaid method, low-abundance mutant DNA sequence is considered as to target dna sequence, wild-type DNA sequence dna is considered as to the interference DNA sequence dna of single base difference, designing probe makes it have two site mispairing (to lay respectively at 3 ' ortho position and 5 ' ortho position+1 of mutational site contraposition with mutant DNA sequence, fluorophor is marked to 5 ' ortho position of mutational site contraposition, quencher group is marked at fluorophor is separated by near seven above probe 3 ' ends of Nucleotide), wild-type DNA sequence dna will have three site mispairing with probe.Like this, in the time there is mutant DNA sequence and wild-type DNA sequence dna in system simultaneously, the signal of mutant DNA sequence will be amplified by selectivity, and the signal of wild-type DNA sequence dna is effectively suppressed, thereby realize the highly selective of low abundance point mutation, highly sensitive detection.
Probe length of the present invention is traditionally arranged to be 15-68 Nucleotide.For guaranteeing the stability of probe and target dna sequence hybridization, probe length should be at 15 more than Nucleotide.And the long meeting of probe is slowed down the signal generation speed of amplification system, also can make method cost improve, therefore general control is in 68 Nucleotide.
Fluorophor on described probe and quencher group are selected from conventional FRET group pair, as fluorophor FAM and quencher group B HQ1, and fluorophor FAM and quencher group TAMRA, fluorophor ROX and quencher group B HQ2 etc.
The present invention also provides the test kit that target dna sequence is carried out to specific signals amplification and detection.This test kit comprises: double-tagging fluorescent probe, Lambda excision enzyme and Lambda excision enzyme reaction buffered soln.Described double-tagging fluorescent probe is 5 '-PO 4the strand of end, simultaneously mark fluorescent group and quencher group; 5 ' end of described fluorophor and described strand at least one Nucleotide of being separated by, and with the distance of 3 ' end of described strand at ten more than Nucleotide, described quencher group is marked at and fluorophor seven optional positions more than Nucleotide that are separated by.
Described Lambda excision enzyme reaction buffered soln is to make Lambda excision enzyme normally bring into play active buffered soln, conventionally contain buffer ions to and salt (the two total concn is less than 200mM), Mg 2+ion (concentration is greater than 0.1mM), tensio-active agent, pH8.8-9.4, preferably 5-20mMTris-HCl+5-30mM (NH 4) 2sO 4+ 5-30mMKCl+1-6mMMgSO 4+ 0.05-0.3%TritonX-100,25 ℃ of pH8.8-9.4@.
Conventional Lambda excision enzyme reaction buffered soln is as ThermoPol buffered soln, and the Lambda excision enzyme that NEB company provides reacts buffered soln (67mMGlycine-KOH+2.5mMMgCl 2+ 50 μ g/mlBSA, 25 ℃ of pH9.4@), the Lambda excision enzyme reaction buffered soln (67mMGlycine-KOH+2.5mMMgCl that provides of Fermentas company 2+ 0.01% (v/v) TritonX-100,25 ℃ of pH9.4@) etc.Preferably ThermoPol buffered soln.
According to the difference of system to be detected, be specifically divided into following two kinds:
1. minim DNA quantitatively amplifies detection kit.Be applicable to only contain target dna sequence to be measured, the sample system coexisting without other similar DNA sequence dnas.Test kit comprises: single mispairing double-tagging fluorescent probe, Lambda excision enzyme and Lambda excision enzyme reaction buffered soln (10mMTris-HCl+10mM (NH 4) 2sO 4+ 10mMKCl+2mMMgSO 4+ 0.1%Triton X-100,25 ℃ of pH8.8@or other can make Lambda excision enzyme normally bring into play active buffered soln, preferably final concentration is 1 × ThermoPol buffered soln).
Wherein single mispairing double-tagging fluorescent probe is 5 '-PO 4the strand of end, simultaneously mark fluorescent group and quencher group.Fluorophor and 5 ' end at least one Nucleotide of being separated by, and with the distance of 3 ' end at ten more than Nucleotide, quencher group can be marked at and fluorophor seven optional positions more than Nucleotide that are separated by.The base mispairing of 5 ' of fluorophor ortho position base and target dna sequence in probe, other Mismatchings.
The probe of mentioned reagent box adds Lambda excision enzyme after mixing with system to be measured, and Lambda excision enzyme can be identified the two strands that probe and target dna sequence form, cutting probe and produce fluorescent signal.After probe is cut, new probe can continue to be combined with target dna sequence and be cut, and amplifies thereby realize signal.
2. highly selective DNA signal amplification detection test kit.Be applicable to exist the sample system of the target dna sequence to be measured interference DNA sequence dna different with only having with it base simultaneously.Test kit comprises: two mispairing double-tagging fluorescent probes, Lambda excision enzyme and Lambda excision enzyme reaction buffered soln (10mMTris-HCl+30mM (NH 4) 2sO 4+ 30mMKCl+2mMMgSO 4+ 0.1%TritonX-100,25 ℃ of pH8.8, or other can make Lambda excision enzyme normally bring into play active buffered soln, and preferably final concentration is 1 × ThermoPol buffered soln+20mMKCl+20mM (NH 4) 2sO 4).Wherein two mispairing double-tagging fluorescent probes are 5 '-PO 4the strand of end, simultaneously mark fluorescent group and quencher group.Fluorophor should be marked in probe with target sequence in base complementrity to be distinguished in 5 ' ortho position base of bit base, and with 5 ' end at least one Nucleotide of being separated by, at ten more than Nucleotide, quencher group can be marked at and fluorophor seven optional positions more than Nucleotide that are separated by with the distance of 3 ' end.Meanwhile, in designing probe, 5 ' of fluorophor ortho position and 3 ' ortho position+1 are the base mismatch of target dna sequence, other Mismatchings.
The probe of mentioned reagent box adds Lambda excision enzyme after mixing with system to be measured.Lambda excision enzyme is far smaller than the double-stranded hydrolysis rate to target dna sequence and probe formation to the double-stranded hydrolysis rate of interference sequence and probe formation, thereby optionally make the signal of target dna sequence amplify, and the signal of interference sequence is effectively suppressed.
In test kit of the present invention, described double-tagging fluorescent probe length is traditionally arranged to be 15-68 Nucleotide.
Fluorophor on described double-tagging fluorescent probe and quencher group are selected from conventional FRET group pair, as fluorophor FAM and quencher group B HQ1, and fluorophor FAM and quencher group TAMRA, fluorophor ROX and quencher group B HQ2 etc.
Than other signal amplification techniques, the present invention has following significant advantages:
1. universality is strong.The reaction system of amplifying based on Lambda excision enzyme cutting to target dna sequence without particular requirement.The length of probe, the kind of labelling groups and position thereof, the interval of being combined with target dna sequence can be selected flexibly.Meanwhile, the present invention measures the selectivity of six kinds of dissimilar base mispairing, and result shows can be for the detection of all types of single base mismatches.Therefore, present method is applicable to the detection of all kinds of target dna sequences and all kinds of single base mutation types.
2. highly sensitive, selectivity is strong.Present method has been utilized the molecular recognition characteristic of Lambda excision enzyme to modification group on DNA probe and adjacent base mismatch composite structure, can reach 2pM(50 μ L system for the detection sensitivity of target dna), and reaction can complete in 20min, utilize the method that nuclease carries out DNA amplification detection aspect sensitivity and detection rates two, all having significantly raising than existing.And the characteristic of utilizing Lambda excision enzyme to distinguish the multiple base mismatch in labelling groups both sides, without optimizing temperature condition, reaction at normal temperatures, to target dna sequence with have single base difference interference sequence discrimination up to 7.2-320 doubly, be greatly better than existing system.
3. system is simple, and mild condition is applied widely.In the signal amplification system building in the present invention, only need a kind of enzyme of Lambda excision enzyme, and base in probe is natural deoxyribonucleotide and do not need special synthetic base, easily system is easy to get.It is worth emphasizing that especially, present method can realize under normal temperature condition, without complicated temperature regulating device, detects and provides convenience for further realizing mass-producing, automatization.Meanwhile, present method is carried out signal amplification detection except using separately, can also the DNA amplifying technique based on template amplification combine use as rolling circle amplification (RCA) and selective PCR etc. with other, meets the demand of various DNA detection.
Accompanying drawing explanation
Fig. 1 is that the present invention utilizes Lambda excision enzyme and 5 '-PO 4the double-tagging strand Fluorescent Nucleic Acid Probe of end carries out the principle schematic of signal amplification detection to DNA object chain.
Fig. 2 is the structural representation of several main probes of the designed use of the present invention.(a) at distance 5 '-PO 4the 3rd or the 15th not isoplastic probe structure schematic diagram of mark of end; (b) fluorophor is marked in 3, single mispairing double-tagging probe structure schematic diagram (wherein X represents base mismatch) that its 5 ' ortho position is base mismatch; (c) fluorophor is marked in 3, and its 5 ' ortho position and 3 ' ortho position+1 are two mispairing double-tagging probe structure schematic diagram (wherein X, Y represent base mismatch, and N is the coupling base according to object chain sequences Design) of base mismatch.
Fig. 3 is three kinds of different probes and has after the target dna chain combination of single base mismatch at different positions, the measurement result of the fluorescence climbing speed being hydrolyzed by Lambda excision enzyme.Wherein No. 0 expression probe mates completely with target dna chain, represents respectively that probe and target dna chain have the situation of single base mismatch in 1-14 position for No. 1-14.(a) N3-F15 probe; (b) D3-F15 probe; (c) F3-N15 probe.
Fig. 4 is that fluorophor is marked in 3, when its both sides and target dna sequence have two or three base mismatch, and the measurement result of the fluorescence climbing speed being hydrolyzed by Lambda excision enzyme.Wherein coupling represents that probe mates completely with target dna chain.
Fig. 5 is that embodiment 1 utilizes fluorophor to be marked in 3, the result that single mispairing double-tagging probe that its 5 ' ortho position is base mismatch detects lower concentration DNA target sequence.(a) the fluorescent value change curve of single mispairing double-tagging probe assay different concns DNA target sequence; (b) with adopt the contrasting of common full coupling double-tagging fluorescent probe measurement result.Measurement result when its empty represents in sample solution containing target dna sequence.
Fig. 6 is that embodiment 2 utilizes fluorophor to be marked in 3, and its 5 ' ortho position and 3 ' ortho position+1 are the fluorescent value change curve that two mispairing double-tagging probes of base mismatch detect dissimilar low abundance point mutation.(a) C:C mispairing; (b) C:A mispairing; (c) C:T mispairing.
Embodiment
Below in conjunction with accompanying drawing, further set forth the present invention by specific embodiment.It will be understood by those of skill in the art that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
The detection > of embodiment 1< lower concentration target dna sequence
As Fig. 2 b) as shown in, in this embodiment, target compound is DNA single chain.5 '-PO 4fluorophor FAM in the strand double-tagging Fluorescent Nucleic Acid Probe of end is marked in the base of the 3rd Nucleotide of 5 ' end, and quencher group B HQ1 is marked at 3 ' end.Prepare respectively the target dna solution of different concns.Detection concrete steps are as follows:
At 1 × ThermoPol buffered soln (20mMTris-HCl+10mM (NH 4) 2sO 4+ 10mMKCl+2mMMgSO 4, 25 ℃ of pH8.8@) in, probe is mixed with the target dna of different concns, after the annealing that heats up (85 ℃ of 90s of hot program, 65 ℃ of 90s, 50 ℃ of 90s, 37 ℃ of 180s), add Lambda excision enzyme, rapid test solution fluorescent value is over time.
In this embodiment, the probe sequence of design is as follows:
5’-PO 4-TCT(-FAM)CCACAGACACATACTCCA-BHQ1-3’（SEQ?ID?No.1）
Mark fluorophor FAM on the 3rd, 5 ' the end downstream nucleotide base of this probe, 3 ' end mark quencher group B HQ1.
Target dna sequence is as follows:
5 '-GTTTTAAATTA tGGAGTATGTGTCTGTGGAa acGAGAGTAAG-3 ' (SEQ ID No.2) (underscore part is mated with probe is complementary, overstriking base and probe mispairing)
For comparing, synthesize in addition the DNA sequence dna that contrasts mating completely with probe
5 '-GTTTTAAATTA tGGAGTATGTGTCTGTGGAGAcGAGAGTAAG-3 ' (SEQ ID No.3) (underscore part is mated with probe is complementary)
In 50 μ L reaction systems of different concns target sequence: probe amount: 5.0pmol; Target sequence SEQ IDN amount o.2: 1.0pmol, 100fmol, 10fmol, 1.0fmol, 100amol, 10amol, all adds the Lambda excision enzyme of 1.25U in each reaction soln.
Blank system: probe 5.0pmol, target or contrast DNA sequence dna content: 0.
Hot program: 37 ℃ of 1200s, every 5s measures first order fluorescence value.
Fluorometric assay instrument is real-time fluorescence PCR instrument rotor-gene6000, and when detection, the sensitivity of detector (gainlevel) is: 10.
Detected result: fluorescent value change curve as shown in Figure 5.What wherein Fig. 5 a) represented is in reaction system, to add 1.0pmol, 100fmol, 10fmol, 1.0fmol, 100amol, the real-time fluorescence change curve of solution when 10amol and 0 target sequence SEQIDNo.2.As seen from the figure, in the time that the amount of target sequence SEQIDNo.2 is more than or equal to 100amol, fluorescent signal can be distinguished with blank system, detects and is limited to 100amol.What Fig. 5 b) represented is in reaction system, to add 1fmol, 100amol target sequence SEQIDNo.3 with add 100amol, 10amol target sequence SEQIDNo.2 and containing the result of target sequence blank sample contrast, the design of visible fluorescence group 5 ' ortho position base mismatch makes for the detectability of target sequence than the common full coupling double-tagging probe approximately order of magnitude that declined.
The detection > of the low abundance point mutation of embodiment 2<
In this embodiment, target compound is DNA single chain.5 '-PO 4fluorophor FAM in the strand double-tagging Fluorescent Nucleic Acid Probe of end is marked in the base of the 3rd Nucleotide of 5 ' end, and quencher group B HQ1 is marked at 3 ' end.Probe distance 5 ' end the 2nd, 5 and the mispairing of target dna strand, all the other bases are all mated.In sample, have the interference chain that has single base difference with target dna chain, the corresponding probe in position of this list base, apart from the 4th Nucleotide of 5 ' end, therefore disturbs chain and probe the 2nd simultaneously, and 4,5 all form mispairing.
This embodiment comprises two experiments:
(1) discrimination of dissimilar mispairing
By changing the base type of probe apart from the 4th Nucleotide of 5 ' end, measure the discrimination of dissimilar mispairing.Detection concrete steps are as follows:
Design and synthesize mark fluorophor FAM on 5 ' the 3rd, downstream of end nucleotide base, 3 ' end mark quencher group B HQ1, the 4th Nucleotide is respectively the double-tagging fluorescent probe of C, T, tri-kinds of different bases of A.1 × ThermoPol buffered soln+20mM (NH 4) 2sO 4in the buffered soln of+20mMKCl composition, by three kinds of different probes respectively with dissimilar 2, the interference DNA sequence dna of 5 bit mismatch object chains or 2,4,5 bit mismatch mixes, annealing (85 ℃ of 90s of hot program heat up, 65 ℃ of 90s, 50 ℃ of 90s, 25 ℃ of 180s) after, add Lambda excision enzyme to mix, rapid test solution fluorescent value over time.
In this experiment, the sequences Design of three kinds of different probes is as follows:
5’-TCT(-FAM)CCACAGACACATACTCCA-BHQ1-3’（SEQ?ID?No.1）
5’-TCT(-FAM)TCACAGACACATACTCCA-BHQ1-3’（SEQ?ID?No.4）
5’-TCT(-FAM)ACACAGACACATACTCCA-BHQ1-3’（SEQ?ID?No.5）
Target dna sequence is as follows:
5 '-GTT tTAAATTATGGAGTATGTGTCTGTtG aa acGAGAGTAAG-3 ' (SEQ ID No.6) (underscore part and three kinds of probes are complementary coupling all, overstriking base and three kinds of equal mispairing of probe, italic base and SEQ ID No.1 probe matching, with other two kinds of equal mispairing of probe)
5 '-GT tTTAAATTATGGAGTATGTGTCTGTtC aa acGAGAGTAAG-3 ' (SEQ ID No.7) (underscore part and all complementary couplings of three kinds of probes, overstriking base and three kinds of equal mispairing of probe)
5 '-GTT tTAAATTATGGAGTATGTGTCTGTtA aa acGAGAGTAAG-3 ' (SEQ ID No.8) (underscore part and three kinds of probes are complementary coupling all, overstriking base and three kinds of equal mispairing of probe, italic base and SEQ ID No.4 probe matching, with other two kinds of equal mispairing of probe)
5 '-GTT tTAAATTATGGAGTATGTGTCTGTtT aa acGAGAGTAAG-3 ' (SEQ ID No.9) (underscore part and three kinds of probes are complementary coupling all, overstriking base and three kinds of equal mispairing of probe, italic base and SEQ ID No.5 probe matching, with other two kinds of equal mispairing of probe)
In 50 μ L reaction systems: probe amount: 10pmol; Target sequence amount: 5pmol.In each reaction system, add respectively the Lambda excision enzyme of 0.83U.
Hot program: 25 ° of C1200s, every 5s measures first order fluorescence value.Fluorometric assay instrument is real-time fluorescence PCR instrument rotor-gene6000, and when detection, the sensitivity of detector (gainlevel) is: 7.
Detected result: the discrimination of six kinds of mispairing types is as shown in table 1.
The discrimination of the dissimilar base mispairing of table 1
Figure BDA0000476751450000111
(2) detection of low abundance sudden change
System to be measured is the mixed system of wild chain, sudden change chain, and designing probe makes the 2nd of chain and its 5 ' end that suddenly change, 5 bit mismatch, wild chain and the 2nd, 4,5 bit mismatch.Fixing wild chain is constant with the total concn of sudden change chain, and change sudden change chain accounts for the ratio of total concn, and the mixing solutions of preparing a series of gradient dilutions detects.Detection concrete steps are as follows:
Design and synthesize double-tagging fluorescent probe, at 1 × ThermoPol buffered soln+20mM (NH 4) 2sO 4in the buffered soln of+20mM KCl composition, probe is mixed with the solution to be measured of the chain that suddenlys change containing different ratios, annealing (85 ℃ of 90s of the hot program of hot program heat up, 65 ℃ of 90s, 50 ℃ of 90s, 25 ℃ of 180s) after, add Lambda excision enzyme to mix, rapid test solution fluorescent value is over time.
In this experiment, the probe sequence of design is as follows:
5’-TCT(-FAM)CCACAGACACATACTCCA-BHQ1-3’（SEQ?ID?No.1）
Mark fluorophor FAM on the 3rd nucleotide base of 5 ' end of this probe, 3 ' end mark quencher group B HQ1.
Wild chain-ordering is as follows:
5 '-GTT tTAAATTATGGAGTATGTGTCTGTt gAa acGAGAGTAAG-3 ' (SEQ ID No.6) (underscore part is mated with probe is complementary, overstriking base and probe mispairing)
Three kinds of different sudden change chain-orderings are as follows:
5 '-GTT tTAAATTATGGAGTATGTGTCTGTtC aa acGAGAGTAAG-3 ' (SEQ ID No.7) (underscore part is mated with probe is complementary, overstriking base and probe mispairing)
5 '-GTT tTAAATTATGGAGTATGTGTCTGTtA aa acGAGAGTAAG-3 ' (SEQ ID No.8) (underscore part is mated with probe is complementary, overstriking base and probe mispairing)
5 '-GTT tTAAATTATGGAGTATGTGTCTGTtT aa acGAGAGTAAG-3 ' (SEQ ID No.9) (underscore part is mated with probe is complementary, overstriking base and probe mispairing)
The 50 μ L reaction systems containing different ratios sudden change chain-ordering: probe amount: 10pmol; Sudden change chain ratio: 100%, 10%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02% combination chain (sudden change chain and wild chain) total amount is 5.0pmol; In each reaction system, add the Lambda excision enzyme of 5U.
Blank group: probe amount: 10pmol; Wild chain 5.0pmol, sudden change chain 0pmol.
Hot program: 25 ℃ of 1200s, every 5s measures first order fluorescence value.
Fluorometric assay instrument is real-time fluorescence PCR instrument rotor-gene6000, and the sensitivity of detector when detection (gain level) is: 7.
Detected result: fluorescent value change curve as shown in Figure 6.Wherein scheme a), b), c) represent respectively the fluorescence real-time curve that sudden change chain detects under different mutant proportions while being SEQ ID No.7, No.8, No.9.Visible for best SEQ ID No.7(probe 5 ' end the 4th bit base C:C mispairing of effect), detectability can reach 0.05%.
Figure IDA0000476751540000011
Figure IDA0000476751540000021
Figure IDA0000476751540000031

Claims (12)

1. the method for under normal temperature, target dna sequence being carried out signal amplification and detection, comprises the following steps:
1) the synthetic 5 '-PO that contains of design 4the double-tagging strand Fluorescent Nucleic Acid Probe of end, probe mark fluorophor and quencher group, described fluorophor is near 5 ' end, and with 5 ' end at least one Nucleotide of being separated by, with the distance of 3 ' end at ten more than Nucleotide; Described quencher group be marked near 3 ' end and with fluorophor seven optional positions more than Nucleotide that are separated by;
2) after step 1) being designed to synthetic probe and mixing with containing the system to be measured of target dna sequence, add Lambda excision enzyme, react at normal temperatures.
2. the method for under normal temperature as claimed in claim 1, target dna sequence being carried out to signal amplification and detection, it is characterized in that, the base of correspondence position in the base at described fluorophor 5 ' ortho position and target dna sequence is designed to base mismatch, and other bases are coupling base.
3. the method for under normal temperature as claimed in claim 1, target dna sequence being carried out to signal amplification and detection, it is characterized in that, on described probe sequence, find the base with the contraposition of described target dna sequence base complementrity, 5 ' the ortho position mark fluorescent group at this to bit base, 3 ' ortho position is designed to base mismatch, and 5 ' ortho position of fluorophor is designed to base mismatch, other bases are coupling base.
4. the method for under the normal temperature as described in as arbitrary in claim 1-3, target dna sequence being carried out to signal amplification and detection, is characterized in that, described probe length is set to 15-68 Nucleotide.
5. the method for under the normal temperature as described in as arbitrary in claim 1-3, target dna sequence being carried out to signal amplification and detection, it is characterized in that, described fluorophor and quencher group are selected from FRET group pair, comprise fluorophor FAM and quencher group B HQ1, fluorophor FAM and quencher group TAMRA, fluorophor ROX and quencher group B HQ2.
6. the test kit that under normal temperature, target dna sequence is carried out signal amplification and detection, comprising: double-tagging fluorescent probe, Lambda excision enzyme and Lambda excision enzyme reaction buffered soln; Described double-tagging fluorescent probe is 5 '-PO 4the strand of end, simultaneously mark fluorescent group and quencher group; 5 ' end of described fluorophor and described strand at least one Nucleotide of being separated by, and with the distance of 3 ' end of described strand at ten more than Nucleotide, described quencher group is marked at and fluorophor seven optional positions more than Nucleotide that are separated by.
7. the test kit that under normal temperature as claimed in claim 6, target dna sequence is carried out to signal amplification and detection, is characterized in that, described Lambda excision enzyme reaction buffer contains buffer ions that total concn is less than 200mM to being greater than the Mg of 0.1mM with salt, concentration 2+ion, tensio-active agent, pH8.8-9.4.
8. the test kit that under normal temperature as claimed in claim 7, target dna sequence is carried out to signal amplification and detection, is characterized in that, described Lambda excision enzyme reaction buffer comprises 5-20mMTris-HCl+5-30mM (NH 4) 2sO 4+ 5-30mMKCl+1-6mMMgSO 4+ 0.05-0.3%TritonX-100,25 ℃ of pH8.8-9.4@.
9. the test kit that under normal temperature as claimed in claim 6, target dna sequence is carried out to signal amplification and detection, it is characterized in that, described double-tagging fluorescent probe is single mispairing double-tagging probe, 5 ' ortho position base of fluorophor and the base mispairing of target dna sequence in described single mispairing double-tagging probe, other Mismatchings.
10. the test kit that under normal temperature as claimed in claim 6, target dna sequence is carried out to signal amplification and detection, it is characterized in that, described double-tagging fluorescent probe is two mispairing double-tagging fluorescent probes, in described pair of mispairing double-tagging fluorescent probe, 5 ' ortho position of fluorophor and 3 ' ortho position+1 are the base mismatch of target dna sequence, other Mismatchings.
The test kit that under 11. normal temperature as described in as arbitrary in claim 6-10, target dna sequence is carried out to signal amplification and detection, is characterized in that, described double-tagging fluorescent probe length is set to 15-68 Nucleotide.
The test kit that under 12. normal temperature as described in as arbitrary in claim 6-10, target dna sequence is carried out to signal amplification and detection, it is characterized in that, described fluorophor and quencher group are selected from FRET group pair, comprise fluorophor FAM and quencher group B HQ1, fluorophor FAM and quencher group TAMRA, fluorophor ROX and quencher group B HQ2.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108998505A (en) * 2018-07-30 2018-12-14 苏州先达基因科技有限公司 A kind of gene polymorphism sites detection technique and its kit
CN109576352A (en) * 2018-11-25 2019-04-05 江苏宏微特斯医药科技有限公司 Single tube detects method, probe and its kit of multiple object to be measured nucleic acid sequences

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101506363A (en) * 2006-08-14 2009-08-12 索尼株式会社 Nucleic acid strand useful in detecting substance and method thereof
CN102660640A (en) * 2012-04-16 2012-09-12 北京大学 Method for specific signal amplification and detection of DNA targeted sequence

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101506363A (en) * 2006-08-14 2009-08-12 索尼株式会社 Nucleic acid strand useful in detecting substance and method thereof
CN102660640A (en) * 2012-04-16 2012-09-12 北京大学 Method for specific signal amplification and detection of DNA targeted sequence

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIN LIU ET AL: "A DNA machine for sensitive and homogeneous DNA detection via lambda exonuclease assisted amplification", 《TALANTA》, vol. 115, 4 July 2013 (2013-07-04), pages 819 - 822, XP028720624, DOI: 10.1016/j.talanta.2013.06.062 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108998505A (en) * 2018-07-30 2018-12-14 苏州先达基因科技有限公司 A kind of gene polymorphism sites detection technique and its kit
CN108998505B (en) * 2018-07-30 2022-03-15 苏州先达基因科技有限公司 Gene polymorphism site detection technology and kit thereof
CN109576352A (en) * 2018-11-25 2019-04-05 江苏宏微特斯医药科技有限公司 Single tube detects method, probe and its kit of multiple object to be measured nucleic acid sequences
CN109576352B (en) * 2018-11-25 2022-03-15 江苏宏微特斯医药科技有限公司 Method, probe and kit for detecting multiple target nucleic acid sequences to be detected through single tube

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