CN1180091C - Composite gene probe structure and use - Google Patents
Composite gene probe structure and use Download PDFInfo
- Publication number
- CN1180091C CN1180091C CNB991254694A CN99125469A CN1180091C CN 1180091 C CN1180091 C CN 1180091C CN B991254694 A CNB991254694 A CN B991254694A CN 99125469 A CN99125469 A CN 99125469A CN 1180091 C CN1180091 C CN 1180091C
- Authority
- CN
- China
- Prior art keywords
- probe
- gene
- molecule
- fluorescence
- composite
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000000523 sample Substances 0.000 title claims abstract description 172
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 80
- 239000002131 composite material Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000004458 analytical method Methods 0.000 claims abstract description 11
- 230000000295 complement effect Effects 0.000 claims abstract description 7
- 238000003745 diagnosis Methods 0.000 claims abstract description 6
- 238000012360 testing method Methods 0.000 claims abstract description 4
- 206010064571 Gene mutation Diseases 0.000 claims abstract description 3
- 239000002773 nucleotide Substances 0.000 claims description 27
- 125000003729 nucleotide group Chemical group 0.000 claims description 27
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- 238000004445 quantitative analysis Methods 0.000 claims description 12
- 238000012408 PCR amplification Methods 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 7
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000000611 regression analysis Methods 0.000 claims description 3
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims description 2
- 229910052749 magnesium Inorganic materials 0.000 claims description 2
- 239000011777 magnesium Substances 0.000 claims description 2
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 abstract description 10
- 238000010791 quenching Methods 0.000 abstract description 7
- 230000000171 quenching effect Effects 0.000 abstract description 7
- 239000000203 mixture Substances 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract 1
- 238000003752 polymerase chain reaction Methods 0.000 description 29
- 239000007850 fluorescent dye Substances 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 238000013461 design Methods 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 238000013207 serial dilution Methods 0.000 description 3
- 101100433727 Caenorhabditis elegans got-1.2 gene Proteins 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010079723 Shiga Toxin Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- XUQUNBOKLNVMMK-UHFFFAOYSA-N [5-[6-[2-cyanoethoxy-[di(propan-2-yl)amino]phosphanyl]oxyhexylcarbamoyl]-6'-(2,2-dimethylpropanoyloxy)-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl] 2,2-dimethylpropanoate Chemical compound C12=CC=C(OC(=O)C(C)(C)C)C=C2OC2=CC(OC(=O)C(C)(C)C)=CC=C2C21OC(=O)C1=CC(C(=O)NCCCCCCOP(N(C(C)C)C(C)C)OCCC#N)=CC=C21 XUQUNBOKLNVMMK-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 208000015355 drug-resistant tuberculosis Diseases 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 201000009671 multidrug-resistant tuberculosis Diseases 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the structure, the using method and the application to qualitative fluorescent gene analysis, quantitative hybridization analysis, medical diagnosis and life science research of a novel composite probe. The composite probe is composed of two probes with different lengths and complementary sequences, wherein the long probe has an end 5' connected to a fluorescent molecule and an end 3' connected to an extending separation molecule, the short probe has an end 3' connected to a quenching molecule, and the long probe can be hybridized with the partial sequence of a gene to be tested. The composite probe of the present invention has the advantages of easy composition and complete fluorescent quenching, and can be widely used in the field of qualitative and quantitative gene analysis, gene mutation analysis, gene hybridization test, gene typing, etc.
Description
One, technical field
The present invention relates to a kind of biotechnology testing tool, specifically a kind of composite gene probe and using method thereof and its application in life sciences such as qualitative, the quantitative hybridization analysis of gene by fluorescence, medical diagnosis.
Two, background technology
Polymerase chain reaction (PCR) the special genes segment that can increase continuously produces scale effect, extensively is successfully used to obtain a certain goal gene at present.PCR has broad application prospects aspect gene diagnosis equally, but some problems have limited its actual use, and subject matter has 2 points, and the one, can not be accurately quantitative; The 2nd, because too sensitive, crossed contamination easily takes place, produce false positive.For overcoming above-mentioned deficiency, people have taked many methods, as hybrid method, competition law, enzyme-linked method and uridine enzyme liberating method etc., but it is not all very successful, fluorescent energy transmits technology (fluorescence resonance energy transfer up to date, be called for short FRET, down with) be used for PCR quantitatively back the problems referred to above just solved preferably.
At present, the PCR in real time Monitoring techniques mainly contains four kinds.The TaqMan technology is by the exploitation of PE company, and this technology has mainly been utilized 5 ' 5 prime excision enzyme activity of Taq enzyme.The probe of an at first synthetic energy and the hybridization of PCR product, a kind of fluorescence molecule of 5 ' end mark of probe, the another kind of fluorescence molecule of 3 ' end mark, 3 ' end fluorescence molecule can absorb the fluorescence that 5 ' end fluorescence molecule sends.This probe does not have fluorescence under the normal circumstances, but when the PCR product was arranged in the solution, probe combined with the PCR product, activates 5 ' 5 prime excision enzyme activity of Taq enzyme, and after the fluorescence molecule that 5 ' end-grain cutting cuts off and 3 ' held fluorescence molecule to be connected, probe sent fluorescence.The fluorescence molecule number of cutting is directly proportional with the quantity of PCR product, therefore can calculate concentration [the 1.Livak KJ of original template according to the fluorescence intensity of PCR reaction solution, Flood SJ, Marmaro J.Oligonucleotideswith fluorescent dyes at opposite ends provide a quenched probesystem useful for detecting PCR product and nucleic acidhybridization.PCR Methods Appl 1995,4 (6): 357-62.2.Martell M, Gomez J, Ecteban JI, et al.High-throughput real-time reversetranscription-PCR quantitation of hepatitis C virus RNA.J ClinMicrobiol, 1999,37 (2): 327-332.3.Livak KJ.Allelicdiscrimination using fluorogenic probes and the 5 ' nucleaseassay.Genet Anal 1999 Feb; 14 (5-6): 143-9].Molecular beacons technology also is two terminal mark fluorescent molecule and quencher molecule respectively at same probe, and different with the TaqMan probe is that probe 5 ' and 3 ' end can form one 8 hairpin structure about base respectively.When fluorescence molecule and quencher molecule are contiguous, can not produce fluorescence; And when in the solution special template being arranged, probe and template hybridization, the hairpin structure of probe destroys, and produces fluorescence.Intensity of fluorescence is directly proportional with the amount of solution middle probe, therefore this technology also can be used for PCR quantitative analysis [1.TyagiS, Bratu DP, Kramer FR.Multicolor molecular beacons for allelediscrimination, Nat Biotechnol, 1998, (16): 49-53.2.Kostrikis LG, Tyagi S, Mhlanga MM, et al.Molecular beacons:spectralgenotyping of human alleles.Science, 1998,279:1228-1229.3.Bialy H.Detecting drug-resistant tuberculosis:Beacons in thedark.Nat Biotechnol, 1998,16:331.4.Piatck, AS, Tyagi S, PolAC, et al.Molecular beacon sequence analysis for detecting drugresistance in Mycobacterium tuberculosis.Nat Biotechnol, 1998,16:359-363].Amplisensor is a kind of combined probe technology.It comprises the length difference, but has two probes of complementary sequence, connects quencher on the short probe, connects fluorescence on the long probe, 7 bases G CGTCCC that long probe 5 ends have more can with the complementation of PCR primer.Before the pcr amplification, two probe hybridizations do not have fluorescence together in the solution; During pcr amplification, long probe is connected with the PCR primer under the effect of ligase enzyme, mix template for primer part as half nested primer in long probe-primer complex, the cancellation probe discharges, destroyed FRET, thereby generation fluorescence, intensity of fluorescence [the 1.Chen S that is directly proportional with the template amount that when amplification adds, Yee A, Griffiths M, Wu KY, et al.A rapid, sensitive and automatedmethod for detection of Salmonella species in foods using AG-9600Ampl iSensor Analyzer.J Appl Microbiol, 1997,83 (3): 314-321.2.Chiang PW, Song WJ, Wu KY, et al.Use of a fluorescent-PCR reactionto detect genomic sequence copy number and transcriptionalabundance.Genome Res, 1996,6 (10): 1013-1026.3.Chen S, Xu R, Yee A, et al.An automated fluorescent PCR method for detectionof shiga toxin-producing Escherichia coli in foods.Appl EnvironMicrobiol, 1998,64 (11): 4210-4216] '.LightCycler is a kind of PCR quantitative technique that Roche company develops recently, the characteristics of this technology also are that fluorescence molecule and quencher molecule are marked at respectively on two different probes, produce luminescence probe and cancellation probe, 5 ' end of luminescence probe connects fluorescence molecule, and 3 ' end of cancellation probe connects quencher molecule.Since two probes of design can with same adjacent sequence hybridization of chain of template, the fluorescence molecule of two probes and quencher molecule are closely adjacent during hybridization, thereby FRET takes place and make fluorescent quenching.The degree of fluorescent quenching and the amount of starting template are inversely proportional to, can carry out PCR quantitative analysis [1.Sagner G with this, Goldstein C, Miltenburg RV.Detection of multiple reporter dyesin real-time, on line PCR analysis with the LightCycler system.Roche Molecular Biochemicals BIOCHEMICA, 1999,2:7-11.2.ReiserA, Geyer M, Miltenburg RV, et al.Mutation detection usingmulti-color detection on the LightCycler system.Roche MolecularBiochemicals BIOCHEMICA, 1999,2:12-15].All there are some defectives in above-mentioned FRET technology, and is not thorough as cancellation, synthetic and mark complexity, and the cost height, non-specific amplification is not easily distinguishable or the like.The inventor has invented a kind of new FRET probe technique on the basis of comprehensive prior art advantage, this technology has overcome the deficiencies in the prior art to a great extent.
Three, summary of the invention
The object of the present invention is to provide a kind of new composite gene probe.
Another object of the present invention is to provide the using method of described combined probe.
A further object of the present invention is to provide described combined probe in life science Application for Field such as qualitative, the quantitative hybridization analysis of gene by fluorescence, medical diagnosiss.
Ultimate principle of the present invention is as follows: as shown in Figure 1, at first synthetic two probes, fluorescent probe 5 ' is held and is connected with reporter group R, and 3 ' end is connected with and extends blocker molecule B, and cancellation probe 3 ' end is connected with quenching group Q, and the cancellation probe can be hybridized with fluorescent probe 5 ' end.Two probes in conjunction with the time fluorescent probe fluorescence that sends absorbed by the cancellation probe, do not have fluorescence to produce in the solution; The fluorescence that fluorescent probe sends during two probe separates is not absorbed by the cancellation probe, has fluorescence to produce in the solution.Based on this design, the inventor has synthesized combined probe, when not having template in the pcr amplification reaction liquid, and the composite gene probe specific combination, solution does not have fluorescence to produce; When in the reaction solution template being arranged, fluorescent probe preferentially combines with template under comparatively high temps, thereby two probe separates generation fluorescence, and fluorescence intensity is directly proportional with template number in the solution, can carry out the PCR quantitative assay in view of the above.
The object of the present invention is achieved like this: (1) preparation composite gene probe, its two probe different by length but that have a complementary sequence constitutes, long probe 5 ' end connects fluorescence molecule, 3 ' end connects the extension blocker molecule, short probe 3 ' end connects quencher molecule, and described long probe can be hybridized with the partial sequence of gene to be checked.Long probe in the composite gene probe is made up of 20-40 Nucleotide, and short probe is made up of 5-25 Nucleotide, and long probe is made up of 25-28 Nucleotide in the preferably combination, and short probe is made up of 15-20 Nucleotide.The Tm value of long probe should be higher than short probe more than 2 ℃ in the composite gene probe.Fluorescence molecule that connects on the probe and quencher molecule number can be 1-5, consider cost and synthetic accessibility, preferably 1, fluorescence molecule is fluorescein, rhodamine etc., quencher molecule is that non-fluorescence molecule such as paramethyl red, long wave excite rhodamine or fluorescence molecule such as rhodamine, in order to reduce the fluorescence background, preferably select non-fluorescence molecule, particularly long wave excites rhodamine, it is than paramethyl red cancellation efficient height, and the wavelength of fluorescence that fluorescence molecule sends on the long probe must be by cancellation molecular absorption on the short probe.Extension blocker molecule on the long probe prevents that primer dimer from forming and the generation of non-specific fluorescence, and the extension blocker molecule is phosphoric acid, glycerine etc., preferably phosphoric acid.
According to a further aspect in the invention, the invention discloses the using method of composite gene probe in gene test, it comprises the steps:
(1) preparation composite gene probe, its two probe different by length but that have a complementary sequence constitutes, and long probe 5 ' end connects fluorescence molecule, and 3 ' end connects and extends blocker molecule, short probe 3 ' end connects quencher molecule, and long probe can be hybridized with the partial sequence of gene to be detected.
(2) according to the sequence of gene to be checked, design and synthesize a pair of upstream and downstream primer, primer Tm value should be lower than long probe, and primer does not overlap with probe and the two ends of contiguous probe, 1-100 the Nucleotide in distance probes two ends.
(3) template is added in the reaction mixture contain probe, primer, PCR damping fluid, magnesium or mn ion, dNTP carry out conventional PCR, 25-60 the circulation of increasing is advisable with 60-500 base as the length nucleic acid of template, preferably 70-120 base.When each cycle annealing or extension, read fluorescent value.
(4) with the logarithm of template initial concentration the cycle number of threshold fluorescence is done regression analysis, the production standard curve carries out quantitative analysis to the concentration of gene to be detected.Threshold fluorescence is meant the fluorescence intensity of the tested gene that doubles the background fluorescence variation coefficient.
According to above-mentioned detection step, composite gene probe of the present invention can be extensively, be advantageously used in fields such as qualitative, the quantitative hybridization analysis of gene by fluorescence, medical diagnosis, especially has more advantage in polygene detection simultaneously and somatotype, multidigit point gene mutation analysis.
Compared with prior art, described technological method has following characteristics: (1) adopts non-fluorescent quenching agent, and background is low; (2) little to the amplification efficiency influence; (3) probe design, synthetic, mark and purifying are convenient.In a word,, fluorescent quenching synthetic easy owing to having thoroughly and to amplification efficiency influences advantages such as little, and this technology has bigger application value.
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Four, description of drawings
The schematic diagram of Fig. 1 composite gene probe fluorescence qualitative and quantitative analysis
The synoptic diagram of Fig. 2 probe and design of primers
Fig. 3 composite gene probe quantitative pcr amplification curve
The specific detection curve of Fig. 4 composite gene probe
The detection curve of the susceptibility that Fig. 5 composite gene probe detects
The influence curve that Fig. 6 amplified production length detects composite gene probe PCR
The influence curve that Fig. 7 cancellation probe detects PCR
The typical curve of Fig. 8 composite gene probe quantitative PCR detection
Five, embodiment
For further specifying composite gene probe technology and application, describe with reference to the following example, these embodiment do not limit the present invention in any way for explanation.
The design of embodiment one primer and probe and synthetic
1. the design of primer and probe
According to composite gene probe fluorescence qualitative and quantitative analysis principle,, designed primer P according to the dna sequence dna of target molecule HBV to be checked
1, P
1', P
1", P
2, P
2', fluorescent probe F and cancellation probe q, q
1, q
2, the position of primer and probe is referring to Fig. 2, and its sequence sees Table 1.
The sequence of table 1 probe and primer and position
Title sequence length position
(Nucleotide) (base pair)
F 5’-ACT?TCC?GGA AAC?TAC?TGT?TGT?TAG?ACG?A-B-3’ 28 2328-2357
q 5’-GTA?GTT?TCC?GGA?AGT-Dabcyl-3’ 15 2328-2342
q
1 5’-ACA?ACA?GTA?GTT?TCC?GGA?AGT-Dabcyl-3’ 21 2328-2348
q
2 5’-TCG?TCTAAC?AAC?AGT?AGT?TTC-Dabcyl-3’ 21 2337-2357
P
1 5’-GGA?GTG?TGG?ATT?CGC?ACT?CCT?C-3’ 22 2269-2290
P
1’ 5’-AGA?CCA?CCA?AAT?GCC?CCT?ATC?TT-3’ 23 2298-2321
P
1” 5’-GTC?CTA?CTG?TTC?AAG?CCT?CCA?AGC?TG-3’ 26 1856-1881
P
2 5’-CG?AGA?TTG?AGA?TCT?TCT?GCG?ACG?CGG-3’ 26 2418-2443
P
2’ 5’-TTC?TTC?TTC?TAG?GGG?ACC?TGC?CT-3’ 23 2364-2386
1.1 primer:
Upstream primer has three, P
122 Nucleotide are arranged, with composite gene probe at a distance of 37 Nucleotide; P
1' 23 Nucleotide are arranged, with composite gene probe at a distance of 6 Nucleotide; P
2' 26 Nucleotide are arranged, with composite gene probe at a distance of 446 Nucleotide.
Totally two of downstream primers, P
226 Nucleotide are arranged, with composite gene probe at a distance of 6 Nucleotide; P
2' 26 Nucleotide are arranged, with composite gene probe at a distance of 61 Nucleotide.
1.2 fluorescent probe
Fluorescent probe and the complementation of target sequence minus strand are made up of 28 Nucleotide, and its 5 ' end is with a fluorescein molecule, and 3 ' end is with one to extend blocker molecule phosphoric acid.
1.3 cancellation probe
Cancellation probe and fluorescent probe 5 ' end are complementary, the contiguous quencher molecule paramethyl red of its 3 ' end.Designed three cancellation probes altogether, q contains 15 Nucleotide, q
1And q
2All contain 21 Nucleotide, q
1With q
2On the position at a distance of 6 Nucleotide, and q
1Than long 6 Nucleotide of q.
2. primer and probe is synthetic
Synthesis material: four kinds of bases, methyl red CPG, phosphoric acid CPG, fluorescein phosphoramidite are available from U.S. Glen Research company, and it is synthetic automatically that primer and probe all adopt PE company to produce 391A type dna synthesizer.Being modified in the building-up process of probe finished automatically.Synthetic 55 ℃ of cuttings of strong aqua and the deprotection 15 hours of finishing is after the anti-phase purification column of Micro Pure II (SolidPhase Sciences) purifying.Probe is further used high performance thin layer chromatography plate (Sillica gel-60GF 254 10 * 20cm E.MERK company) purifying.At last, quantitative with uv-spectrophotometric instrument (BeckmanDu640).
The general step that embodiment two composite gene probe PCR detect.
The PCR reaction mixture contains 10 * PCR damping fluid, 2 microlitres, the dNTPs0.2 mmole/liter, MgCl
23 mmoles/liter.Reaction system 20 microlitres, contain PCR reaction mixture 15 microlitres, each 0.1 microgram of upstream and downstream primer, Taq DNA polymerase (promega company) 0.5U, fluorescent probe (fluorescence intensity is the 4000-5000 flat light emission) 0.04 microgram, cancellation probe 0.04 microgram, the mol ratio of fluorescent probe and cancellation probe is 1: 2, standard substance are with 10 times of serial dilutions, and final concentration is 10
2Nanogram/microlitre-10
-3Between nanogram/microlitre, get 1 microlitre as template.With 96 special orifice plates (Techne H1 TEMP 96), on the Britain Genuis of Techne company pcr amplification instrument, increase, reaction conditions is 94 ℃, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 40 seconds.After per 5 circulation extensions finish, take out Sptting plate,, measure fluorescence intensity with Finland Wallac VICTOM 1420 multiple labeling detectors.With cycle number the composite gene probe property analysis is carried out in fluorescence intensity mapping.Logarithm with starting template concentration carries out regression analysis formulation typical curve to the cycle index that produces threshold fluorescence, promptly can be used for the nucleic acid concentration of unknown sample is carried out quantitative analysis.
Embodiment three
Getting the HBV plasmid DNA is template, becomes 10 with 10 times of serial dilutions
2Nanogram/microlitre-10
-3The concentration gradient of nanogram/microlitre is got 1 microlitre as template, with F and q
1Be composite gene probe, press embodiment two operations, carry out pcr amplification.As shown in Figure 3, the starting template amount that adds in visible fluorescence response and the reaction system is relevant, and each template amount standard reaction hole fluorescence intensity meets the pcr amplification rule with the change that cycle number increases, promptly fluorescence intensity presents the change of index sample between certain cycle number, then is tending towards the platform sample and changes when high cycle number.
The specificity of embodiment four composite gene probes
With F and q
1For meeting probe, setting gradually template concentrations is 10
2, 10
0, 10
-2The standard orifice of nanogram is provided with no pattern hole again, and no fluorescent probe hole, no cancellation probe aperture and no composite gene probe hole are carried out composite gene probe according to the operation steps of embodiment two and detected in contrast.Experimental result as shown in Figure 4, increase along with the PCR cycle number, each standard orifice fluorescence intensity presents the fluorescence response relevant with the template initial concentration, and in no pattern hole, no fluorescent probe hole, no cancellation probe aperture and do not have in the composite gene probe hole, fluorescence intensity does not change because of the increase of cycle number, proves that thus composite gene probe has good specificity.
The quantitative scope and the sensitivity of embodiment five composite gene probes
With F and q
1Be composite gene probe, with template by concentration 10
2Nanogram/microlitre, 10 times of serial dilutions to 10
-7Nanogram/microlitre is got 1 microlitre and is carried out pcr amplification by embodiment two operations, detects the quantitative scope and the sensitivity of composite gene probe.As shown in Figure 5, the template amount is 10
2Nanogram and 10
-4Reacting hole between the nanogram changes visible corresponding fluorescence response with cycle number, shows that the application composite gene probe can be to content 10
2-10
-4Sample in the nanogram scope carries out accurate quantitative analysis, and this composite gene probe can detect content and be low to moderate 10
-4The target molecule of nanogram/microlitre.
Embodiment six influences the quantitative factor of composite gene probe
1. the influence of expanding fragment length
With F and q
1Be composite gene probe,, carry out pcr amplification by embodiment two with different primer pairings.With the P1-P2 pairing, as shown in Figure 6A, amplified production length is 119bp; P1 "-P2 ' pairing, shown in Fig. 6 B, amplified production length is 89bp; P1 '-P2 pairing, shown in Fig. 6 C, amplified production length is 588bp.When amplified production length was 119bp, each normal content reacting hole changed with cycle number and presents corresponding fluorescence response, and amplified production length is when being 89bp and 587bp, and fluorescence response appears in the reacting hole of only high template amount, and fluorescence intensity is low.The above results shows that PCR product length has tangible influence to the quantitative analysis of composite gene probe, and amplified production is oversize or too short, all is unfavorable for the quantitative analysis of composite gene probe.
2. the influence of cancellation probe length and position
With primer P
1-P
2Cancellation probe q and fluorescent probe F (Fig. 7 A), q are adopted in pairing respectively
1With F (Fig. 7 B), q
2Constitute composite gene probe with F (Fig. 7 C), press embodiment two operations, carry out pcr amplification.The result shows, the cancellation probe length between 15-21 Nucleotide, with the fluorescence molecule position when 6 Nucleotide, the quantitative analysis of composite gene probe is not had tangible influence.
Embodiment seven HBV DNA quantitative analyses
2 routine HBV DNA positive serums and 2 routine HBV DNA negative serum samples are got 40uL respectively and are added HBV lysate 10uL, and mixing is put 98 ℃ and boiled 15 minutes, and centrifugal 10 minutes of 14000rpm gets 2uL as template.With F and q
1Be composite gene probe.Press embodiment two operations, carry out pcr amplification.Fig. 8 is a known content (10
2-10
-4Nanogram) the threshold fluorescence cycle number C that standard reaction hole produces
TTo the typical curve of starting template amount logarithm mapping, by the C of serum to be checked
TValue obtains the HBV DNA serum content of serum to be checked according to typical curve, and 2 parts of HBV negative serums are not seen fluorescence response, the C of 2 parts of HBV DNA positive serums
TValue is respectively 9 and 14, and its serum HBV dna content is respectively 8.75 mcg/ml and 0.85 mcg/ml.
Claims (12)
1. composite gene probe, its two probe different by length but that have a complementary sequence constitutes, and long probe 5 ' end connects fluorescence molecule, and 3 ' end connects and extends blocker molecule, short probe 3 ' end connects quencher molecule, and described long probe can be hybridized with the partial sequence of gene to be detected.
2. composite gene probe according to claim 1, wherein said long probe is made up of 20-40 Nucleotide, and described short probe is made up of 5-25 Nucleotide.
3. composite gene probe according to claim 2, wherein said long probe is made up of 25-28 Nucleotide, and described short probe is made up of 15-20 Nucleotide.
4. composite gene probe according to claim 2, the extension blocker molecule that connects on the wherein said long probe is phosphoric acid or glycerine.
5. composite gene probe according to claim 2, the quencher molecule that connects on the wherein said short probe are that methyl red, long wave excite rhodamine or rhodamine.
6. composite gene probe according to claim 4, the extension blocker molecule that connects on the wherein said long probe is a phosphoric acid.
7. composite gene probe according to claim 5, the quencher molecule that connects on the wherein said short probe is that long wave excites rhodamine.
8. according to each described composite gene probe of claim 1-7, the fluorescence molecule that connects on its middle probe and the quantity of quencher molecule are 1-5.
9. composite gene probe according to claim 8, wherein the quantity of fluorescence molecule that connects on the composite gene probe and quencher molecule is 1.
10. composite gene probe according to claim 1, it can be used for, and gene by fluorescence is qualitative, in quantitative analysis and the medical diagnosis.
11. composite gene probe according to claim 10, it can be used for polygene detection and somatotype, multidigit point gene mutation analysis.
12. the using method of a composite gene probe, its concrete operations step is as follows:
(1) preparation composite gene probe, its two probe different by length but that have a complementary sequence constitutes, and long probe 5 ' end connects fluorescence molecule, and 3 ' end connects and extends blocker molecule, short probe 3 ' end connects quencher molecule, and long probe can be hybridized with the partial sequence of gene to be detected.
(2) the upstream and downstream primer of synthetic testing gene;
(3) carry out pcr amplification: comprise template, probe, primer, PCR damping fluid, magnesium or mn ion, dNTP in the reaction solution of amplification, the cycle number of amplification is 25-60; Read fluorescent value when each cycle annealing or extension;
(4) with the logarithm of template initial concentration the cycle number of threshold fluorescence is done regression analysis, the production standard curve carries out quantitative analysis to the concentration of gene to be detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991254694A CN1180091C (en) | 1999-12-08 | 1999-12-08 | Composite gene probe structure and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991254694A CN1180091C (en) | 1999-12-08 | 1999-12-08 | Composite gene probe structure and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1261665A CN1261665A (en) | 2000-08-02 |
| CN1180091C true CN1180091C (en) | 2004-12-15 |
Family
ID=5283949
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB991254694A Expired - Lifetime CN1180091C (en) | 1999-12-08 | 1999-12-08 | Composite gene probe structure and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1180091C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088144A (en) * | 2013-01-30 | 2013-05-08 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Detection kit of nucleic acid of klebsiella pneumoniae KPC (Klebsiella Pneumoniae Carbapenmase) type carbapenemases gene |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ597400A (en) * | 2004-02-18 | 2013-06-28 | Chromocell Corp | Methods and Materials Using Signaling Probes |
| EP2591432A4 (en) * | 2010-07-08 | 2017-05-10 | Prime Genomics, Inc. | System for the quantification of system-wide dynamics in complex networks |
| CN102985825B (en) * | 2011-05-24 | 2015-08-19 | 戴立军 | A kind of bioanalytical reagent and using method thereof |
| CN103866013A (en) * | 2014-03-06 | 2014-06-18 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Orientia tsutsugamushi disease nucleic acid detection kit |
| CN105441554A (en) * | 2015-12-29 | 2016-03-30 | 上海赛安生物医药科技有限公司 | Primer probe system for SEPT9 (septin-9) gene methylation detection and kit adopting primer probe system |
| CN105441557B (en) * | 2015-12-29 | 2020-04-17 | 上海达澈生物科技有限公司 | MLH1 gene methylation detection primer-probe system and its kit |
| CN105441558B (en) * | 2015-12-29 | 2020-02-07 | 厦门市同普生物科技有限公司 | MGMT gene methylation detection primer probe system and kit thereof |
| CN109652516A (en) * | 2018-12-29 | 2019-04-19 | 中国人民解放军军事科学院军事医学研究院 | A kind of structure and purposes of double chain oligonucleotide nucleic acid probe |
-
1999
- 1999-12-08 CN CNB991254694A patent/CN1180091C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088144A (en) * | 2013-01-30 | 2013-05-08 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Detection kit of nucleic acid of klebsiella pneumoniae KPC (Klebsiella Pneumoniae Carbapenmase) type carbapenemases gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1261665A (en) | 2000-08-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4540844B2 (en) | Fluorescence quantitative detection system for nucleic acids | |
| CN102344960B (en) | Quantification of gene expression | |
| JP4457001B2 (en) | MET / FRET based method for target nucleic acid detection in which donor / acceptor moieties are on complementary strands | |
| JP6099684B2 (en) | Detection of target nucleic acid sequences by repetitive exonucleolytic cleavage reaction | |
| JP5385973B2 (en) | Simultaneous detection of multiple nucleic acid sequences in a reaction | |
| EP2630262B1 (en) | Detection of target nucleic acid sequences using dual-labeled immobilized probes on solid phase | |
| US9222124B2 (en) | Method for the simultaneous detection of multiple nucleic acid sequences in a sample | |
| CN1236395A (en) | Nucleotide homogeneity real-time assay | |
| EP1838880A2 (en) | Methods and compositions for increased dynamic range detection of nucleic acid molecules | |
| US20220090195A1 (en) | Structure and application of double-stranded oligonucleotide nucleic acid probe | |
| CN1297488A (en) | Method for detecting target nucleic acid by PCR | |
| JP2012529266A (en) | Detection of multiple nucleic acid sequences in reaction cartridges | |
| US20210310056A1 (en) | Real-time multiplexed hydrolysis probe assay using spectrally identifiable microspheres | |
| US6063572A (en) | Method of assay of nucleic acid sequences | |
| CN105002285A (en) | Liquid-stage chip constant-temperature detection method for tiny RNA | |
| WO2016170121A1 (en) | Method for the simultaneous detection of multiple nucleic acid sequences in a sample | |
| EP2705160B1 (en) | Nucleotides and oligonucleotides comprising a label associated through a linker | |
| CN1180091C (en) | Composite gene probe structure and use | |
| JP2004532044A5 (en) | ||
| US9512470B2 (en) | Method for the simultaneous detection of multiple nucleic acid sequences in a sample | |
| JP2006501836A (en) | Detection system | |
| CN105063190A (en) | Solid chip constant temperature detection method of MiRNA | |
| Sinicropi et al. | Gene expression profiling utilizing microarray technology and RT-PCR | |
| JP2003506068A5 (en) | ||
| CN1340711A (en) | Preparing process and application of detection probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20041215 |
|
| CX01 | Expiry of patent term |