CN102660649A - Gene detection method with combination of COLD polymerase chain reaction (PCR) and high-resolution melting (HRM) of luna probe - Google Patents
Gene detection method with combination of COLD polymerase chain reaction (PCR) and high-resolution melting (HRM) of luna probe Download PDFInfo
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Abstract
The invention provides a gene detection method with the combination of COLD polymerase chain reaction (PCR) and high-resolution melting (HRM) of a luna probe. The method is characterized by comprising the following steps of: preparing a deoxyribonucleic acid (DNA) template, preparing a reaction system in which a luna probe with a closed 3' end is contained, performing COLD PCR amplification reaction, and performing HRM detection of the luna probe. A high-specificity luna probe HRM technology is combined on the basis of high sensitivity of a Cold-PCR technology, so that high sensitivity and specificity are ensured, and mutation detection results are accurate and reliable.
Description
Technical field
The present invention relates to the gene tester that COLD PCR combines non-marked probe HRM; Be used for detection in Gene Mutation, aspects such as genotype identification, HLA gene assembly type, gene frequency analysis, species evaluation and cultivar identification in SNP detection, methylate detection, plant and animal quality and the breeding.
Background technology
Along with the research of plant-animal molecular breeding is goed deep into, major gene mark precision is increasingly high.The molecule marker ancillary technique uses increased, and the detection essence of high precision mark is sudden change and detects.The detection of transgenation can be started with from two levels: the one, and the indirect detection of protein level; Mainly by some protein analysis technology; Analyze the variant protein matter that two mutants produces, and other changes that cause by variant protein, like colonial morphology, antibiotics resistance etc.; The 2nd, to the direct detection of genetic material (mainly being DNA), generally speaking, this type technology all is through setting up a series of electrophoresis, the analyzing DNA conformation or the characteristic of unwinding; Perhaps utilize characteristics such as DNA sex change and renaturation that dna mutation is analyzed.At present, along with the development and the application of round pcr, detection in Gene Mutation is main with the direct detection of analyzing DNA variation mainly.
The technology that detects transgenation is a lot; Common methods comprises restriction fragment length polymorphism polymerase chain reaction (Restriction Fragment Length Polymorphism polymerase chain reaction; PCR-RFLP); The detection of single stranded conformational isomery polymorphum (Single-strand conformation polymorphism, SSCP), tetra-sodium order-checking (pyrosequencing); (High-resolution melting is HRM) with Sanger order-checking (Sanger sequencing) etc. for the high resolving power melting curve.
Aforesaid method all has advantage and limitation separately.PCR-RFLP is the PCR primer amplification target material with special design, because the base mutation of specific site, insertion or disappearance number are seldom, so that no polymorphum occurs, often need carry out enzyme to corresponding pcr amplified fragment and cut processing, to detect its polymorphum; And this Technology Need uses restriction enzyme, has not only increased the detection cost, also consumes human cost, is not suitable for high-throughput, rapid detection, has limited its clinical application.Though being widely used in single base mutation, SSCP detects, its sudden change detection sensitivity only 50～80%, and be subject to sequence length to be measured, the righttest detection length of SSCP is about 100～300bp.The tetra-sodium order-checking has quite good detecting sensitivity, but needs special plant and instrument, and it is high to detect cost, is not suitable for high throughput testing.Though HRM can satisfy high-throughout detection requirement, the intrinsic bias during its PCR detects can't be avoided.The Sanger order-checking is the gold standard that generally acknowledged at present detection suddenlys change, but its detection sensitivity is very low, is merely 20%, has limited it and has found the ability of sudden change.Therefore, be badly in need of a kind of high specific, highly sensitive clinically; High-throughput; Low-cost and easy-operating sudden change detection technique satisfies the demands.
COLD PCR is a kind of method that improves the sudden change detection sensitivity that Li in 2008 etc. report on Nature Medicine, and its ultimate principle is to exist its solvent temperature of double-stranded DNA of base mismatch to change.Li in 2009 etc. propose COLD PCR is combined the unknown mutation of HRM technology for detection lower concentration on Clinical Chemistry.Though advantages such as that this method has concurrently is quick, simple and easy, sensitivity are subject to the limitation of its PCR quality, instrument and dyestuff to a great extent.
Summary of the invention
The objective of the invention is to set up a kind of highly sensitive and high specific; Low cost and high-throughput; Quick and easy, and not limited to by the mutational site, can detect the gene tester that known mutations also can detect unknown mutation; Be applied to the diagnosis of non-disease and the detection in Gene Mutation of therapeutic purpose, aspects such as genotype identification, HLA gene assembly type, gene frequency analysis, species evaluation and cultivar identification in SNP detection, methylate detection, plant and animal quality and the breeding.
In order to achieve the above object; The invention provides the gene tester that a kind of COLD PCR combines non-marked probe HRM, it is characterized in that concrete steps are: the preparation dna profiling; The preparation reaction system; The non-marked probe that contains 3 ' end sealing in the described reaction system, carry out the COLD pcr amplification reaction after, carry out non-marked probe HRM again and detect.
Principle of the present invention is following:
COLD PCR combines the gene tester of non-marked probe HRM, and the composite amplification bonding probes technology under the promptly low denaturation temperature combines the HRM detection method of non-marked probe.(1) COLD-PCR (composite amplification under the low denaturation temperature, Li Dengren.2008) solved some limitation that low-content gene mutation detects, in the pcr amplification process, utilized the abundant amplicon that contains sudden change of crucial denaturation temperature, no matter where sudden change is positioned at sequence.The main attribute of COLD-PCR is that the enrichment two mutants is so that the direct order-checking behind PCR.Because the base order-checking costs an arm and a leg, complicated operation, is effective solution through the examination of high-throughput scanning sequence therefore.(2) to melt (HRM) be a kind of technology that can be used for unknown mutation before the preparatory examination order-checking to high resolving power, but be not sure of the identity of concrete detection transgenation.The mispairing of single oligonucleotide that sudden change causes will cause the Tm value of dna double chain to change.The intensity of variation of Tm value depends on the sequence and the position thereof in mutational site.Usually in the dna sequence dna of the about 200bp of length, 0.2～1.5 ℃ change takes place in single oligonucleotide sudden change can causing Tm.High resolving power melting curve (HRM) analysis is on the basis of melting curve, through using saturated fluorescence dyestuff and high resolution instrument, improves its detection sensitivity.Can distinguish the difference that single oligonucleotide sudden change causes 0.2～1.5 ℃ of Tm value according to the melting curve shape variation though HRM analyzes, HRM is subject to PCR quality, instrument and dyestuff to a great extent.(3) the present invention adds the non-marked probe of 3 ' end sealing on the basis of HRM, can hybridize with the ssDNA that asymmetric PCR produces, and in the HRM analytic process, forms the probe segment melting curve.The Tm value difference of the corresponding generation of its single stranded oligonucleotide mispairing is different from 2～8 ℃, and it is promptly distinguishable to need not specific instrument and dyestuff.The melting curve of non-marked probe-PCR-HRM can be divided into two portions: probe segment and amplicon section; Wherein probe segment can detect the known mutations site; Guaranteed the highly sensitive that detects; The amplicon section can be brought into play the effect of checking in the unknown mutation in detecting sequence, guaranteed the high accuracy that detects.Therefore,, combine non-marked probe HRM sudden change scanning and COLD-PCR, carry out the direct order-checking of known mutations subsequently and identify for the unknown mutation of enrichment, rapid screening and evaluation lower concentration.The COLD-PCR detection technique has increased the detectivity of non-marked probe HRM, can differentiate the site and the type of low levels sudden change through the order-checking of positive sudden change sample subsequently, has improved the evaluating ability to the lower concentration sudden change.Result's specificity is also very important to clinical application in sudden change detects, and the present invention at first adds specific probe in original technology, to improve the specificity of detected result.Nucleic probe (nucleic acid probe) know-why is base pairing.Two nucleic acid strands of complementary form double-stranded through annealing, this process is called nucleic acid hybridization.Nucleic probe is meant the nucleic acid fragment of the known array that has affinity tag, its can and with its complementary nucleic acid array hybridizing, form double-stranded, so can be used for the detection of specific gene sequence in the determined nucleic acid sample.
Compared with prior art, advantage of the present invention is:
(1) the present invention is based on the highly sensitive of COLD round pcr, the non-marked probe HRM technology in conjunction with high specific has guaranteed height sensitivity and specificity, makes the sudden change detected result accurately and reliably.
(2) the present invention combines at traditional C OLD PCR to add the non-marked probe on the basis of HRM method, has avoided the limitation that traditional method is subject to PCR quality, instrument and dyestuff.
Description of drawings
Fig. 1 combines the sensitivity map of the gene tester of non-marked probe HRM for COLD PCR; Among the figure, on behalf of 0% mutant (wild-type), 2,1 represent 0.1% mutant, 3 to represent 0.5% mutant, 4 to represent 1% mutant, 5 to represent 3% mutant, 6 to represent 8% mutant, 7 to represent 10% mutant, 8 to represent 20% mutant, 9 to represent 50% mutant, 10 to represent 100% mutant, 11 to represent water.
Fig. 2 is the amplified production figure of Sanger PCR sequencing PCR detection COLD PCR sensitivity experiment, and among the figure, on behalf of C base, 2,1 represent the T base.
Embodiment
Specify the present invention below in conjunction with embodiment.
1. serial dilution KRAS wild-type cell strain (SW480colon carcinoma cells) and the strain of KRAS mutant cell (AsPC-1 metastatic pancreatic carcinoma cells) genomic dna is with the sensitivity of assessment institute construction method.
1.1.DNA the preparation of template:
1.1.1. (Qiagen, Germany) extraction human colon adenocarcinoma cell SW480 (K-ras wild-type), people shift the DNA of pancreatic cancer cell AsPC-1 (K-ras mutant) to use Blood Mini Kit.
1.1.2. with ultraviolet spectrophotometer (U-3010; HITACHI) record the concentration of SW480 and AsPC-1; The result is respectively 48.6ng/ μ l and 49.2ng/ μ l, and the DNA of KRAS mutant (AsPC-1) and wild-type (SW480) mixed by the volume ratio of 1: 2,1: 5,1: 10,1: 12.5,1: 33.3,1: 100,1: 200 and 1: 1000 obtains dna profiling.
2.COLD PCR-HRM reaction:
2.1. dispose 20 μ l reaction systems: comprise that 2 * Premix Taq [contains 1.25U/25 μ l TaKaRa Taq HS; 0.4mM dNTP mixture and 3mM Mg2+; (TaKaRa; China)] 10 μ l, 0.67 μ M forward primer K-ras-F0.5 μ l, 10 μ M reverse primer K-ras-R, 0.5 μ l, 10 μ M non-marked probe K-ras-P (sangon, China) 0.5 μ l, optical dye SYTO-9 (Invitrogen, CA) 0.6 μ l, dna profiling 2 μ l, H
2O 5.9 μ l.
K-ras-F：5’-gcctgctgaaaatgactgaa-3’
K-ras-R：5’-gaatggtcctgcaccagtaa-3’
K-ras-P:5 '-aacttgtggtagttggagctggtggcgtaggcaagag-3 ' (3 ' end sealing)
2.2. carry out the COLD pcr amplification reaction, program is following: 95 ℃ of preparatory sex change 10min; Get into 80.6 ℃ of sex change 30s of circulation, 56 ℃ of annealing 30s, 72 ℃ are extended 30 ℃, totally 20 circulations; Get into 95 ℃ of sex change 30s of circulation, 56 ℃ of annealing 30s, 72 ℃ are extended 30 ℃, totally 40 circulations; 72 ℃ are extended 5min, and 40 ℃ of cooling 30s (Mastercycler Gradient, Eppendorf).
2.3. carry out the HRM detection reaction, program is following: 95 ℃ of sex change 2min, 40 ℃ of hybridization 2min, 65-85 ℃ of high resolving power melting curve analyzed, 40 ℃ of cooling 30s (Rotor-Gene Q, Qiagen).
3. the judgement of detected result: observe the melting curve shape, sample to be measured and negative control are compared (referring to Fig. 1).It is unimodal from figure, can to see that 0% mutant (wild-type) sample has formed wild-type probe about 76.5 ℃, about 73.6 ℃, fuses flat-satin; 100% mutant sample has formed the mutant probe about 73.6 ℃ unimodal, about 76.5 ℃, fuses flat-satin; The tendency no significant difference of the melting curve of the melting curve of 0.1% mutant sample and 0% mutant (wild-type) sample, detected result is a wild-type; 0.5%～50% mutant sample has all formed the probe peak at 73.6 ℃ and 76.5 ℃, and the result is the heterozygous mutant type.COLD PCR combines non-marked probe HRM method can identify the sudden change of 0.5-100%, and its sensitivity is 0.5%.
Embodiment 2
1. use DNAMini Kit (Qiagen, Germany) DNA of extraction 36 routine carcinoma of the pancreas adenoma (PA) tissues (disease group), 24 routine digestive tube benign disease group pathological tissues (disease control group), 25 routine surperficial healthy human peripheral bloods (normal healthy controls group).
2.COLD PCR-HRM reaction:
2.1. dispose 20 μ l reaction systems: comprise that 2 * Premix Taq [contains 1.25U/25 μ l TaKaRa Taq HS; 0.4mM dNTP mixture and 3mM Mg2+; (TaKaRa; China)] 10 μ l, 0.67 μ M forward primer K-ras-F0.5 μ l, 10 μ M reverse primer K-ras-R0.5 μ l, 10 μ M non-marked probe K-ras-P (sangon, China) 0.5 μ l, optical dye SYTO-9 (Invitrogen, CA) 0.6 μ l, dna profiling 2 μ l, H
2O 5.9 μ l.
K-ras-F：5’-gcctgctgaaaatgactgaa-3’
K-ras-R：5’-gaatggtcctgcaccagtaa-3’
K-ras-P:5 '-aacttgtggtagttggagctggtggcgtaggcaagag-3 ' (3 ' end sealing)
2.2. carry out the COLD pcr amplification reaction, program is following: 95 ℃ of preparatory sex change 10min; Get into 80.6 ℃ of sex change 30s of circulation, 56 ℃ of annealing 30s, 72 ℃ are extended 30 ℃, totally 20 circulations; Get into 95 ℃ of sex change 30s of circulation, 56 ℃ of annealing 30s, 72 ℃ are extended 30 ℃, totally 40 circulations; 72 ℃ are extended 5min, and 40 ℃ of cooling 30s (Mastercycler Gradient, Eppendorf).
2.3. carry out the HRM detection reaction, program is following: 95 ℃ of sex change 2min, 40 ℃ of hybridization 2min, 65-85 ℃ of high resolving power melting curve analyzed, 40 ℃ of cooling 30s (Rotor-Gene Q, Qiagen).
3.TA-DNA clone and order-checking:
3.1.PCR amplification:
3.1.2. dispose 100 μ l reaction systems: comprise that 2 * Premix Taq [contains 1.25U/25 μ l TaKaRa Taq HS; 0.4mM dNTP mixture and 3mM Mg2+; (TaKaRa; China)] 50 μ l, 10 μ M forward primer K-ras-F, 2.5 μ l, 10 μ M reverse primer K-ras-R2.5 μ l, optical dye SYTO-9 (Invitrogen, CA) 3 μ l, dna profiling 4 μ l, H
2O 38 μ l.
K-ras-F：5’-gcctgctgaaaatgactgaa-3’
K-ras-R：5’-gaatggtcctgcaccagtaa-3’
3.1.3. carry out pcr amplification reaction, program is following: 95 ℃ of preparatory sex change 10min; Get into 95 ℃ of sex change 30s of circulation, 56 ℃ of annealing 30s, 72 ℃ are extended 30 ℃, totally 40 circulations; 72 ℃ are extended 5min, and 40 ℃ of cooling 30s (Mastercycler Gradient, Eppendorf).
Carry out cloning and sequencing 3.2. send the PCR product outside China big gene.
4. the judgement of detected result: use COLD PCR to combine non-marked probe HRM method in the PA disease group, to detect 26 (72.2%) example sudden changes, all do not detect sudden change in other 2 groups.The detected result of cloning and sequencing method is in full accord with it.(table 1)
KRAS sudden change detected result in the table 1 carcinoma of the pancreas glandular tissue
To sum up, the inventive method can detect the KRAS sudden change in the PA tissue accurately, and its sensitivity is merely 0.5%, and specificity and Sanger order-checking are quite.
Claims (1)
1. the gene tester of a COLD PCR combination non-marked probe HRM is characterized in that the preparation dna profiling; The preparation reaction system; The non-marked probe that contains 3 ' end sealing in the described reaction system, carry out the COLD pcr amplification reaction after, carry out non-marked probe HRM again and detect.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106957902A (en) * | 2016-11-01 | 2017-07-18 | 复旦大学 | A kind of non-marked probe for being used to detect drug-induced deafness related locus |
| WO2018096349A1 (en) * | 2016-11-25 | 2018-05-31 | Imperial Innovations Limited | Primers, methods and kits for diagnosing and predicting therapy response of cancers by cold-pcr based amplification of mutation-rich regions of kras, egfr and p53 |
-
2012
- 2012-05-17 CN CN2012101545694A patent/CN102660649A/en active Pending
Non-Patent Citations (7)
| Title |
|---|
| 《Clinical Chemistry》 20091231 Coren A. Milbury 等 COLD-PCR-Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations 2130-2143 1 第55卷, 第12期 * |
| COREN A. MILBURY 等: "COLD-PCR–Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations", 《CLINICAL CHEMISTRY》 * |
| GUO W 等: "Unlabeled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in pancreatic adenocarcinoma tissues", 《CLIN CHEM LAB MED》 * |
| JIN LI 等: "Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing", 《NATURE MEDICINE》 * |
| TU NGUYEN-DUMONT 等: "Description and Validation of High-Throughput Simultaneous Genotyping and Mutation Scanning by High-Resolution Melting Curve Analysis", 《HUMAN MUTATION》 * |
| 张春燕: "外周血游离DNAKRAS基因突变检测方法的建立和临床应用", 《中国优秀硕士学位论文全文数据库》 * |
| 沈薇 等: "高分辨率熔解曲线及其在分子诊断中的应用", 《检验医学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106957902A (en) * | 2016-11-01 | 2017-07-18 | 复旦大学 | A kind of non-marked probe for being used to detect drug-induced deafness related locus |
| CN106957902B (en) * | 2016-11-01 | 2020-12-04 | 复旦大学 | Non-labeled probe for detecting drug-induced deafness related sites |
| WO2018096349A1 (en) * | 2016-11-25 | 2018-05-31 | Imperial Innovations Limited | Primers, methods and kits for diagnosing and predicting therapy response of cancers by cold-pcr based amplification of mutation-rich regions of kras, egfr and p53 |
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Application publication date: 20120912 |