CN102296116A - Method for signal amplification and detection of target sequence of DNA - Google Patents
Method for signal amplification and detection of target sequence of DNA Download PDFInfo
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- CN102296116A CN102296116A CN201110259113XA CN201110259113A CN102296116A CN 102296116 A CN102296116 A CN 102296116A CN 201110259113X A CN201110259113X A CN 201110259113XA CN 201110259113 A CN201110259113 A CN 201110259113A CN 102296116 A CN102296116 A CN 102296116A
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Abstract
The invention discloses a method for THE signal amplification and detection of the target sequence of a DNA, which comprises: preparing a single-abasic-site dual-labeled fluorescent probe, wherein one end of the probe is labeled with a fluorescent group, the other end of the probe is labeled with a quenching group, and except for an abasic site, the sequence of the probe is completely matched with the target sequence of the DNA; and at a certain detection temperature, specifically bonding the probe and the target sequence to form a double-stranded vacant site, identifying and digesting the double-stranded vacant site by endonucleases IV, dissociating the cut probe pieces from the target sequence, producing a fluorescent signal, bonding the target sequence released by dissociation with another probe, repeating the processes, and thus, accomplishing the signal amplification and detection of the target sequence. When the method disclosed by the invention is used, high-selectivity and high-sensitivity low-abundance gene mutation detection can be realized.
Description
Technical field
The present invention relates to the detection in Gene Mutation technical field.Specifically, relate to a kind of amplification system, utilize this amplification system to realize highly selective, highly sensitive low abundance detection in Gene Mutation based on single dealkalize base fluorescent probe and restriction endonuclease IV.
Background technology
Transgenation is closely related with human cancer, and some important sudden changes can be used as the biomarker of cancer diagnosis, treatment and prognostic evaluation, so the detection of transgenation has great clinical meaning.But owing to the influence of tumor site in factors such as different times, far-end transfer and tumour cell and normal cell fusions, transgenation is usually expressed as low abundance.Thereby require detection method both to have highly sensitive (detecting very low absolute magnitude), have highly selective (in numerous wild-type chains, selecting the sudden change chain) again.
The method of the most frequently used detection transgenation is order-checking, comprise Sanger sequencing and Pyrosequencing, sequence measurement can provide concrete sequence to form, but the selectivity of this method is not enough, detectability can only reach 5%-20%, and the pre-treatment process of sample is very complicated very.The sequence-specific fluorescent probe method (real-time fluorescence PCR) of PCR-based has overcome the low problem of detectability, have highly sensitive and highly selective concurrently, but the design that detects probe in real time in the PCR process has a lot of restrictive requirements, comprise: probe 3 ' end can not match with primer, and the distance of probe and primer is subject to certain restrictions or the like.
In recent years, some method for amplifying signal have been developed based on the sequence-specific probe.Mainly comprise: restriction enzyme amplification system and excision enzyme III signal amplification system.These two kinds of methods all have very high sensitivity, can detect the coupling dna sequence dna of 1fmol.But they also have some defectives: the restriction enzyme amplification system is a restriction enzyme owing to what use, thereby object chain to be measured is had certain sequence requirement, thereby has limited this method range of application in practice greatly.And the excision enzyme III in the excision enzyme III amplification system can't be in comparatively high temps (more than 50 ℃) work, and the detected temperatures of system is lower than mispairing and the double-stranded melting temperature (Tm) of coupling, thereby the separating capacity of probe is lost greatly.
Therefore, press for and develop the amplification system that the energy selectivity is amplified jump signal, be used for highly selective, highly sensitive low abundance sudden change detection.
Summary of the invention
The present invention seeks to set up a kind of alternative amplification system that amplifies jump signal, realize highly selective, highly sensitive low abundance detection in Gene Mutation.
Technical scheme of the present invention is, utilizes the differentiation effect of single dealkalize base probe, in conjunction with the cutting of restriction endonuclease IV, reaches the purpose that signal amplifies, thereby detects the DNA target sequence, especially contains the target sequence in specificity site (as the mutational site).
In one aspect of the invention, provide a kind of the DNA target sequence has been carried out the method that signal amplifies and detects, comprised the steps:
1) the double-tagging fluorescent probe of the single dealkalize base of preparation, this probe is a single stranded DNA, an end mark fluorescent group, the other end mark quencher group, except that abasic site, its sequence and DNA target sequence mate fully, and 5 of terminal and 3 ' ends of abasic site distance probes 5 ' are more than the nucleotide residue;
2) described probe is mixed with system to be measured, add restriction endonuclease IV then, carrying out real-time fluorescence under 47-60 ℃ detected temperatures measures, under detected temperatures, probe combines with the target sequence specificity, formed double-stranded room is discerned and is cut by restriction endonuclease IV, probe fragment after the cutting disintegrates down from target sequence, send fluorescence, and the target sequence that discharges continues to combine with another probe specificity, repeat said process, the signal of realizing target sequence amplifies and detection, and detected fluorescence intensity is high more to show that the content of target sequence in the system to be measured is high more, wherein, described detected temperatures is lower than the melting temperature (Tm) of the two strands of probe and target sequence formation, but is higher than the melting temperature (Tm) of the two strands that other sequences form in probe and the system to be measured.
Probe of the present invention is single dealkalize base dna probe, can directly prepare the dna single chain of single site dealkalize base (purine or pyrimidine) by chemical synthesis, also can obtain by other approach, for example: preparation earlier contains the double-tagging fluorescent probe in single uridylic (U) deoxynucleotide site, use uridylic-DNA-glycosylase (Uracil DNA-glycosylase then, UDG) handle, excision uridylic base, remaining ribodesose residue obtains single dealkalize base probe (AP-probe).
The DNA target sequence that aforesaid method detected typically refers to the dna sequence dna with specificity site (as mutational site), and the detection with transgenation is that example illustrates principle of the present invention below.
In the detection of transgenation, except that abasic site, single dealkalize base probe sequence and mutant mate fully, and therefore, with wild chain one or more bases (at single mutation or multi-mutant site) being arranged is mispairing.Because the influence of mispairing, (perfect-match duplex, (note is made T to melting temperature (Tm) PM) to the coupling two strands of probe and sudden change chain formation
PM) (mismatch duplex, (note is made T to melting temperature (Tm) MM) than the mispairing two strands of probe and wild chain formation
MM) height.So, when rising to detected temperatures (note is made T) between two melting temperature (Tm)s, i.e. T
MM＜T＜T
PM, the two strands of probe and wild chain formation can't stable existence and unwind, and probe but can be specifically in conjunction with the sudden change chain under this temperature.Consider separating capacity and non-specific binding, the length of probe is traditionally arranged to be the 20-30 nucleotide residue, and the relevant detection temperature generally can be at 50-60 ℃.
Restriction endonuclease IV is a kind of depurination/pyrimidine (AP) endonuclease, the complete AP site on the hydrolysis DNA, and first phosphodiester bond that cutting 5 ' end links to each other with AP site produces 3 ' hydroxyl and 5 ' deoxyribose phosphate end.The recommended work temperature of restriction endonuclease IV is 37 ℃, whether has activity so must probe into enzyme under comparatively high temps (50-60 ℃).Can realize that by testing signal amplification system of the present invention signal amplifies between 47-60 ℃, confirm that restriction endonuclease IV has very high activity between 47-56 ℃, have certain activity in the time of 60 ℃.Therefore, amplification system of the present invention can both works better between 47-60 ℃.
Under suitable temperature, single dealkalize base probe is optionally hybridized with the sudden change chain, forms double-stranded room.Restriction endonuclease IV has the character in the double-stranded room of cutting, and its cutting speed is much larger than the speed in cutting single-chain room.Therefore, probe is discerned and is cut by restriction endonuclease IV with the double-stranded room of sudden change chain formation.Probe fragment after the cutting because length is shorter, can't combine with object chain under detected temperatures, so disintegrate down from object chain.Before dissociating, the fluorophor at complete probe two ends and quencher group be owing to FRET (fluorescence resonance energy transfer) (FRET) is in the quencher state, after dissociating, two groups away from, fluorophor no longer by quencher, thereby send fluorescent signal.Simultaneously, the object chain that discharges that dissociates can continue to combine with another single dealkalize base probe, repeats said process, realizes the amplification of signal.
Can be used for fluorophor of the present invention and quencher group thereof, should to satisfy the absorption spectrum of the emmission spectrum of fluorophor and quencher group effectively overlapping, thereby high efficiency FRET takes place.Common FAM and the TAMRA of comprising; FAM and BHQ1; TET and BHQ2 or the like.
In another aspect of this invention, provide a kind of the DNA target sequence has been carried out the test kit that signal amplifies and detects, under certain detected temperatures, utilize restriction endonuclease IV that the double-tagging fluorescent probe that specificity is incorporated into the single dealkalize base on the DNA target sequence is cut, the probe fragment is disintegrated down from target sequence, produce fluorescent signal, simultaneously, target sequence after dissociating can combine with another single dealkalize base probe, repeat said process, realize the amplification of signal, thereby the signal of realizing target sequence amplifies and detection; This test kit comprises the double-tagging fluorescent probe and the restriction endonuclease IV of single dealkalize base, wherein said probe is the dna single chain, one end mark fluorescent group, the other end mark quencher group, except that abasic site, its sequence and DNA target sequence mate fully, and 5 of terminal and 3 ' ends of abasic site distance probes 5 ' are more than the nucleotide residue.
Further, described probe length is traditionally arranged to be the 20-30 nucleotide residue.
It is right that fluorophor on the described probe and quencher group are selected from FRET group commonly used, as fluorophor FAM and quencher group B HQ1, and fluorophor FAM and quencher group TAMRA, fluorophor TET and quencher group B HQ2.
Than prior art, the present invention has some significant advantages in the low abundance transgenation of detection:
(1). universality, because the recognition site of restriction endonuclease IV only is double-stranded room, and the position in room can be according to target sequence flexible design to be determined: the melting temperature (Tm) that the room is cut two fragment short chains that the back forms just can reach amplification effect preferably than the melting temperature (Tm) of complete single dealkalize base probe is low more than 10 ℃.Generally speaking, null position and probe 5 ' end and 3 ' end can meet the demands more than the base at a distance of 5.So this signal amplifies and detection method goes for any target sequence.
(2). highly selective, restriction endonuclease IV thermotolerance can be worked reaction system under relatively-high temperature, thereby effectively utilized the character of mispairing melting temperature (Tm) difference, simultaneously, an object chain combines with a plurality of probes, is actually to have carried out repeatedly selecting, and selectivity has also obtained amplification, thereby realized that superelevation optionally detects, other method for amplifying signal of selectivity ratios exceed an order of magnitude.
(3). low detectability, because in testing process, an object chain combines-cuts-dissociated process with a plurality of probe circulations, send the fluorescent signal of a plurality of probes, realized the amplification of object chain signal, so detect low abundance sudden change with the inventive method, can detect and be low to moderate 1% mutant, all lower than the detectability of detection method behind other PCR.
(4). coupling, the inventive method can be easily and the PCR coupling, comprise selectivity PCR such as AS-PCR, and COLD-PCR realizes that more sensitive detects.Compare with additive method, coupling only need add involved enzyme and probe, and operation is simple.
Description of drawings
Fig. 1 uses the present invention the DNA object chain is carried out the principle schematic that signal amplifies and detects.
Fig. 2 is the fluorescent value change curve that 1 couple of lower concentration DNA of embodiment target sequence detects.
Fig. 3 uses the present invention the principle schematic that signal amplifies and detects is carried out in low abundance sudden change.
Fig. 4 is the fluorescent value change curve that 2 pairs of low abundance sudden changes of embodiment detect.
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 only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1＜lower concentration coupling dna sequence dna detects 〉
In this embodiment, target compound is the dna single chain that mates fully, and the concentration gradient of a series of dna single chains is set in the experiment, and hope can detect the least possible object chain.Detect principle referring to Fig. 1, concrete steps are as follows:
1. use the UDG enzyme to handle the double-tagging fluorescent probe that contains uridylic deoxynucleotide residue, obtain single dealkalize base probe;
2. single dealkalize base probe that will obtain mixes with the target dna sequence of different concns, adds restriction endonuclease IV then, and rapid test mixing solutions restriction endonuclease IV fluorescent value over time.
In this embodiment, the designed probe sequence is as follows:
5’-FAM-TCGTCTCCACUGAAACATACTCCATAA-TAMRA-3’(SEQ?ID?No.1)
The target sequence based composition is as follows:
5’-GTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGAGTAAG-3’(SEQ?ID?No.2)
In the 50 μ L reaction systems of different concns target sequence: probe amount: 5pmol; Target sequence amount: 5pmol, 1pmol, 0.1pmol, 10fmol, 1fmol; The restriction endonuclease IV that adds 1U in each reaction system.
Blank system: probe 5pmol, target sequence amount: 0.
Hot program: 53 ℃ of 900s, every 5s measures the first order fluorescence value.
The fluorometric assay instrument is real-time fluorescence PCR instrument rotor-gene 6000, and the sensitivity of detector during detection (gain level) is: 7.33.
Detected result: the fluorescent value change curve as shown in Figure 2,100s before reaction, fluorescent value are along with the time increases gradually, behind the 7min, in the reaction system of target sequence amount more than 1pmol, fluorescent value reaches platform.
Embodiment 2＜low abundance sudden change detects 〉
In this embodiment, system to be measured is the mixed system of wild chain, sudden change chain, and the total amount of two kinds of chains is 5pmol, but a series of different mutant proportions are set, and hope can detect alap mutant proportion.Detect principle referring to Fig. 3, concrete implementation step is as follows:
1. prepare single dealkalize base probe, method is with step 1 among the embodiment 1;
2. measure the melting temperature (Tm) of the two strands of single dealkalize base probe and wild chain, sudden change chain formation respectively, determine suitable detected temperatures;
3. single dealkalize base probe is mixed with the compound target system that contains different ratios sudden change chain, add restriction endonuclease IV, be warming up to the detected temperatures of determining in the step 2 rapidly, measure fluorescent value over time.
In this embodiment, the designed probe sequence is as follows:
5’-FAM-TCTCCAC
GA
ACATACTCCA-BHQ1-3’(SEQ?ID?No.3)
The sudden change chain-ordering is composed as follows:
5’-GTTTTAAATTATGGAGTATGT
TCTGTGGAGACGAGAGTAAG-3’(SEQ?ID?No.4)
Wild chain-ordering is composed as follows:
5’-GTTTTAAATTATGGAGTATGT
TCTGTGGAGACGAGAGTAAG-3’(SEQ?ID?No.5)
(black matrix underscore mark be the position of mutating alkali yl)
50 μ L reaction systems of different ratios mutant: probe amount: 5pmol; Mutant proportion: 100%, 10%, 5%, 2.5%, 1%, the combination chain total amount is 5pmol; The restriction endonuclease IV that adds 1U in each reaction system.
Blank group: probe amount: 5pmol; Mutant proportion: 0%.
Hot program: 52.5 ℃ of 600s, every 5s measures the first order fluorescence value.
The fluorometric assay instrument is real-time fluorescence PCR instrument rotor-gene 6000, and the sensitivity of detector during detection (gain level) is: 6.77.
Detected result: the fluorescent value change curve as shown in Figure 4, fluorescent value is along with the time increases gradually, in the system of mutant content 100%, fluorescent value reaches platform rapidly.
Claims (10)
1. the method that the DNA target sequence is carried out the signal amplification and detects comprises the steps:
1) the double-tagging fluorescent probe of the single dealkalize base of preparation, this probe is a single stranded DNA, an end mark fluorescent group, the other end mark quencher group, except that abasic site, its sequence and DNA target sequence mate fully, and 5 of terminal and 3 ' ends of abasic site distance probes 5 ' are more than the nucleotide residue;
2) described probe is mixed with system to be measured, add restriction endonuclease IV then, carrying out real-time fluorescence under 47-60 ℃ detected temperatures measures, under detected temperatures, probe combines with the target sequence specificity, formed double-stranded room is discerned and is cut by restriction endonuclease IV, probe fragment after the cutting disintegrates down from target sequence, send fluorescence, and the target sequence that discharges continues to combine with another probe specificity, repeat said process, the signal of realizing target sequence amplifies and detection, and detected fluorescence intensity is high more to show that the content of target sequence in the system to be measured is high more, wherein, described detected temperatures is lower than the melting temperature (Tm) of the two strands of probe and target sequence formation, but is higher than the melting temperature (Tm) of the two strands that other sequences form in probe and the system to be measured.
2. the method for claim 1 is characterized in that, step 1) prepares the double-tagging fluorescent probe of described single dealkalize base by chemical synthesis.
3. the method for claim 1, it is characterized in that step 1) preparation earlier contains the double-tagging fluorescent probe in single uridylic deoxynucleotide site, with uridylic-DNA-glycosylase it is handled then, excise the uridylic base, obtain the double-tagging fluorescent probe of single dealkalize base.
4. the method for claim 1 is characterized in that, described DNA target sequence is meant the dna sequence dna that contains the mutational site.
5. the method for claim 1 is characterized in that, described probe length is the 20-30 nucleotide residue.
6. the method for claim 1 is characterized in that, described fluorophor and quencher group be following groups to one of: FAM and TAMRA, FAM and BHQ1, perhaps TET and BHQ2.
7. test kit that the DNA target sequence is carried out that signal amplifies and detects, under certain detected temperatures, utilize restriction endonuclease IV that the double-tagging fluorescent probe that specificity is incorporated into the single dealkalize base on the DNA target sequence is cut, the probe fragment is disintegrated down from target sequence, produce fluorescent signal, simultaneously, the target sequence that discharges that dissociates continues to combine with another probe specificity, repeat said process, thereby the signal of realizing target sequence amplifies and detection; This test kit comprises the double-tagging fluorescent probe and the restriction endonuclease IV of single dealkalize base, wherein said probe is the dna single chain, one end mark fluorescent group, the other end mark quencher group, except that abasic site, its sequence and DNA target sequence mate fully, and 5 of terminal and 3 ' ends of abasic site distance probes 5 ' are more than the nucleotide residue.
8. test kit as claimed in claim 7 is characterized in that, described probe length is the 20-30 nucleotide residue.
9. test kit as claimed in claim 7 is characterized in that, described fluorophor and quencher group be following groups to one of: FAM and TAMRA, FAM and BHQ1, perhaps TET and BHQ2.
10. test kit as claimed in claim 7 is characterized in that, described DNA target sequence is meant the dna sequence dna that contains the mutational site.
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| CN109661580A (en) * | 2016-07-29 | 2019-04-19 | 塞尔玛生物技术有限责任公司 | The improvement of digital counting method |
| CN113897418A (en) * | 2021-06-28 | 2022-01-07 | 华中科技大学 | Probe and kit for detecting DNA point mutation and application thereof |
| US11693001B2 (en) | 2015-10-07 | 2023-07-04 | Selma Diagnostics Aps | Flow system and methods for digital counting |
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Application publication date: 20111228 |