CN101423866A - PNA, probe, primer and method for detecting K-ras gene mutation - Google Patents
PNA, probe, primer and method for detecting K-ras gene mutation Download PDFInfo
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- CN101423866A CN101423866A CNA2007100476408A CN200710047640A CN101423866A CN 101423866 A CN101423866 A CN 101423866A CN A2007100476408 A CNA2007100476408 A CN A2007100476408A CN 200710047640 A CN200710047640 A CN 200710047640A CN 101423866 A CN101423866 A CN 101423866A
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Abstract
The invention discloses PNA, probe and primer for testing K-ras genetic mutation, and a method for quantitatively testing the K-ras genetic mutation in real time by the PNA, the probe and the primer. The PNA is hybridized with the 12th codon and/or the 13th codon in the first exon of wild K-ras gene; and the PNA is combined with real-time quantitative PCR to quantitatively test the mutation amount of the K-ras gene in real time. The method has the advantages of simple and convenient operation, high sensitivity and high specificity, and can be applied to early auxiliary diagnosis for pancreatic cancer and other diseases.
Description
Technical field
The present invention relates to the detection of K-ras transgenation, relate in particular to a kind of PNA, probe, primer and method of the K-ras of detection transgenation.
Background technology
K-ras gene (GenBank:NC_000012) sudden change plays an important role in the developing of carcinoma of the pancreas.Report, in carcinoma of the pancreas tissue, pancreatic juice or ight soil, blood, can detect K-ras gene the 12nd, 13 or 61 codon point mutations (Takahashi, et al.GastrointestinalEndo 2005.61:76-9; Luigi L, et al.Oncogene 2002.21:4301-6; TadaM, et al.Cancer Res 1993.53:2472-4; Lu XH, et al.Natl Med J China2001.81:1050-3; N van Heek, et al.J.Clin.Pathol.2005.58:1315-1320).
Studies show that whether the K-ras transgenation can be used as a kind of method of carcinoma of the pancreas early diagnosis in the detection bodies cell DNA.But in some experiments, find also to have the K-ras transgenation in the benign diseases such as chronic pancreatitis and Biliary Calculi, simple qualitative analysis, specificity is relatively poor, and quantitative analysis can better reflect the sudden change degree, so that good pernicious discriminating.At present the K-ras detection method of gene mutation of widespread usage is restricted fragment length polymorphism analysis method (RFLP method) and the amplification sudden change system method (ARMS method) of being obstructed.
The RFLP method is the method that PCR is combined with restriction enzyme digestion.But there is following shortcoming in this method:
(1) step is loaded down with trivial details, needs 2 day time just can finish evaluation;
(2) can only do qualitative examination, promptly can only clearly whether have the K-ras transgenation,, then need further to detect with additive method if require quantitatively;
(3) exist first round enzyme to cut the false positive that not exclusively causes.
The ARMS ratio juris is: according to 3 ' end mispairing principle, when carrying out amplified reaction, if 3 ' end base pair forms mispairing, chain extension reaction will be because of 3 ', the obstacle that 5 ' bisphosphate diester linkage forms and being obstructed, therefore, when to have only template strand be specific allelotrope, just can detect special amplified production.In the reaction system of ARMS, 2 different fluoresceins of mark on the primer, or to add method such as fluorescent probe all be to be the based gene classifying method with the real-time fluorescence PCR.
The step of ARMS method comprises:
(1) design is at the Auele Specific Primer or the probe of a certain mutation type of K-ras gene;
(2) standard substance to the known mutations amount carry out the real-time quantitative PCR detection, draw typical curve;
(3) unknown sample is carried out real-time quantitative PCR and detect,, draw not key sample sudden change amount according to above-mentioned typical curve.
But the shortcoming of this method is:
(1) every species-specific primer or probe can only be identified at a certain gene mutation type, and the K-ras gene the mutation type of the 12nd, 13 codons can have ten surplus kind, need to detect one by one;
(2) may occur because the false positive that single base mispairing causes.
Peptide nucleic acid(PNA) (Peptide Nucleic Acid, PNA) be a kind of DNA analogue, its skeleton has been replaced the phosphodiester sugar of DNA by the 2-aminoethyl glycine, it has many advantages: 1.PNA than traditional dna probe and stablized in ten minutes, the stability of correct paired DNA/PNA is higher than corresponding D NA/DNA far away, while, PNA in the PCR reaction process not can be used as the substrate of Taq enzyme, also can not degraded by other enzymes; 2. for the PNA/DNA of mispairing,, also can cause its melting temp to descend about 9-10 ℃ even have only the mispairing of a base.At present, be applied in a plurality of fields (Zhang Bingbo etc., " polymer circular " 2006,9:62-68 of peptide nucleic acid(PNA); Egholm M, et al.Nature, 1993,365:566～568).
Summary of the invention
One of the technical problem to be solved in the present invention provides a kind of PNA of the K-ras of detection transgenation, only need a kind of PNA of design at wild-type, carry out a PCR reaction, can distinguish wild-type and mutant, it is by combining with real-time quantitative PCR, but real-time quantitative detects K-ras transgenation amount.
Two of the technical problem to be solved in the present invention provides a kind of probe of the K-ras of detection transgenation.
Three of the technical problem to be solved in the present invention provides a kind of primer of the K-ras of detection transgenation.
Four of the technical problem to be solved in the present invention provides a kind of method of the K-ras of detection transgenation.
Five of the technical problem to be solved in the present invention provides a kind of test kit of the K-ras of detection transgenation.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of PNA of the K-ras of detection transgenation, the 12nd codon and/or the 13rd codon are hybridized in this PNA and wild-type K-ras gene first exon.
Preferably, described PNA comprises at least 12 continuous bases of SEQ ID NO:1 or its complementary sequence.
More preferably, the sequence of described PNA is:
Ac-ACGCCACCAGCTC-Lysine-COOH。
In another aspect of this invention, provide a kind of probe of the K-ras of detection transgenation, described probe and the hybridization of wild-type K-ras gene target sequence, this probe sequence comprises at least 15 continuous nucleotides of SEQ ID NO:2 or its complementary sequence.
Preferably, described probe is an oligonucleotide sequence shown in the SEQ ID NO:2.
In another aspect of this invention, provide a kind of primer of the K-ras of detection transgenation, the described primer K-ras gene target sequence that is used to increase comprises that following primer is right:
(1) first primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:3, and second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:4;
(2) first primer sequences contain at least 15 continuous nucleotides of SEQ ID NO:5, and second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:6.
Preferably, described primer is to comprising: SEQ ID NO:3, SEQ ID NO:4; SEQ IDNO:5, SEQ ID NO:6.
In another aspect of this invention, provide a kind of method of the K-ras of detection transgenation, may further comprise the steps:
(1) with above-mentioned PNA, probe and primer, the plasmid standard of known mutations amount is carried out real-time quantitative PCR detect, draw typical curve;
(2) extract sample of nucleic acid;
(3) with above-mentioned PNA, probe and primer, the nucleic acid that extracts with step (2) is template, carries out real-time quantitative PCR and detects;
(4), draw the K-ras transgenation amount of sample nucleic acid according to the typical curve of step (1) gained.
In another aspect of this invention, also provide a kind of test kit of the K-ras of detection transgenation, having comprised: comprising: above-mentioned PNA, probe and primer.。
In the present invention, term " probe " is meant the one section single stranded DNA or the RNA molecule of the tape label that can discern specific nucleotide sequence, and it only combines with detected specific nucleotide sequence.Probe location is positioned as close to upstream primer.For guaranteeing binding specificity, the appropriate length of probe is generally between 15～45 Nucleotide.
In the present invention, term " primer " is meant a kind of oligonucleotide, can be natural also can be synthetic, it can be used as induce dna synthetic starting point under certain condition, can bring out synthetic and nucleic acid chains complementary primer extension product under these conditions, promptly in the presence of four kinds of different triphosphoric acid dezyribonucleosides and a kind of polymerization agent (being archaeal dna polymerase or reversed transcriptive enzyme), in a kind of suitable damping fluid and under suitable temperature, carry out above-mentioned synthetic.Preferred primer is the sub-thread oligodeoxyribonucleotide.The appropriate length of primer depends on this primer design purposes, but generally between 15～25 Nucleotide, short primer molecule needs lower temperature usually, thereby forms fully stable hybridization complex with template.Primer needn't reaction template accurate sequence, but must be fully complementary, with template hybridization and to cause DNA synthetic.
Owing to adopt technique scheme, PNA, probe, primer and the method for detection K-ras of the present invention transgenation, PNA is combined with real-time quantitative PCR, be applied to the K-ras gene test, because PNA can cover 6 bases of the 12nd, 13 codons in K-ras gene first exon simultaneously, arbitrary sudden change all can cause the mispairing of PNA/DNA, make that melting temp takes place obviously to change, therefore only need a kind of PNA of design at wild-type, carry out PCR reaction, can with the K-ras gene the 12nd, 13 codons ten surplus kind of mutant and wild-type distinguish.The inventive method is easy and simple to handle, susceptibility is high, specificity is high, but real-time quantitative detects K-ras transgenation amount, and can be applicable to the early stage auxiliary diagnosis of diseases such as carcinoma of the pancreas.
Description of drawings
The present invention is further detailed explanation below in conjunction with drawings and Examples.
Fig. 1 is the plasmid sequencing result figure of the embodiment of the invention 1;
Fig. 2 is the standard substance amplification curve diagram of the embodiment of the invention 1;
Fig. 3 is the canonical plotting of the embodiment of the invention 1;
Fig. 4 is the quantivative approach synoptic diagram of the embodiment of the invention 1.
Embodiment
Following examples only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The present invention adopts the real-time quantitative PCR method of associating peptide nucleic acid(PNA) (PNA) to detect the K-ras point mutation.Described K-ras gene can be the gene among the DNA that extracts from ight soil, tissue, tissue juice, cell pearl and blood.Because PNA has covered 6 bases of the 12nd, 13 codons in K-ras gene first exon simultaneously, arbitrary sudden change all can cause the mispairing of PNA/DNA, make that melting temp takes place obviously to change, so only need a kind of PNA of design at wild-type K-ras gene, carry out a PCR reaction, can distinguish wild-type and mutant.
The plasmid standard of 1 pair of known mutations amount of embodiment carries out real-time quantitative PCR and detects
1. main agents is originated:
1.1 design PNA
PNA is the PNA at the design of wild-type K-ras gene the 12nd, 13 codons, at least 12 continuous bases that comprise SEQID NO:1 or its complementary sequence, this PNA preferred sequence is Ac-ACGCCACCAGCTC-Lysine-COOH (SEQ ID NO:1), and is synthetic by Korea S panagene company.
1.2 design Tagman MGB probe
Tagman MGB probe and the hybridization of wild-type K-ras gene target sequence, this probe sequence comprises at least 15 continuous nucleotides of SEQ ID NO:2 or its complementary sequence.Described probe is preferably oligonucleotide sequence shown in the SEQID NO:2; be FAM-AGCTCCAACTACCACAAG-MGB (SEQ IDNO:2); its two ends are respectively with reporter group FAM and quenching group MGB mark, and this probe is synthetic by last sea base health engineering Services Co., Ltd.
1.3 PCR primer
The present invention's be used to increase primer of K-ras gene target sequence comprises that following primer is right:
Upstream primer F1:5 '-TACTGGTGGAGTATTTGATA-3 ' (SEQ ID NO:3);
Downstream primer R1:5 '-GAACTTTACTTTTCCTTGGGAGT-3 ' (SEQ ID NO:4);
Upstream primer F2:5 '-ATAGTGTATTAACCTTATGTGTGAC-3 ' (SEQ ID NO:5);
Downstream primer R2:5 '-TTAGCTGTATCGTCAAGGCACTCT-3 ' (SEQ ID NO:6).
Above-mentioned PCR primer is synthetic by Shanghai biotechnology Services Co., Ltd.
1.4 TA clones test kit
TA clone's test kit (Target Clone) is bought the company in TOYOBO.
1.5 plasmid extraction test kit
The plasmid extraction test kit is bought the company in TIANGEN.
1.6 Taqman realtime PCR Master Mix buys the company in TOYOBO.
2. cell strain DNA extracting
Choose pancreas cancer cell strain Panc-1, BxPC-3, CFPAC and SW1990 (cell strain is from this laboratory),, and use DNA purification kit (Tiangen company) to carry out purifying with the conventional extracting cell strain of phenol extraction method DNA.
Use the Gene NanoDrop ND1000 of company type nucleic acid trace measurement instrument to measure the extracting DNA of institute concentration (0D260/0D280=1.0～2.0).After testing, DNA concentration is respectively: Panc-1,517ng/ μ l; BxPC-3,337ng/ μ l; CFPAC, 425ng/ μ l; SW1990,425ng/ μ l.
3.PCR reaction
3.1PCR reaction solution preparation
The PCR reaction system comprises 10 * Ex Taq damping fluid, 2 μ l, dNTP mixed solution 200 μ M, and primers F 1 (SEQ ID NO:3), each 0.5 μ M of R1 (SEQ ID NO:4), dna profiling 100ng, Taq enzyme 1U is settled to 20 μ l with aseptic double-distilled water.
3.2 PCR reaction
Used instrument is German Eppendorf company 5331 type Thermal Cycler PCR instrument.The PCR reaction conditions: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 52 ℃ of annealing 40s, 72 ℃ are extended 40s, totally 35 circulations, 72 ℃ are extended 10min.
3.3 electrophoresis
2.0% agarose gel electrophoresis is determined the product exactness.
4.PCR product is expressed
4.1 expression strain makes up
The PCR product is connected to E.coli DH5 α (these intestinal bacteria are by this laboratory culture gained) by the T carrier makes up plasmid.
4.1.1 try to achieve PCR product more than the X μ l: X=50Y ÷ Z (size of Ykb:PCR product, Zng/ μ l:PCR production concentration) by following formula.Connect liquid by following composition preparation T carrier: comprise sterile purified water 2 μ l, 2 * connection damping fluid 5 μ l, pTA2 carrier (50ng/ μ l) 1 μ l, T4DNA ligase enzyme 1 μ l, PCR product 1 μ l, totally 10 μ l.Reaction solution is carried out reaction in 30 minutes in room temperature (15～25 ℃).
4.1.2 preparation competent cell, and add above-mentioned connection liquid with calcium chloride method transformed into escherichia coli, and carries out X-gal and IPTG screening (concrete grammar see " molecular cloning experiment guide " third edition first volume 96-99,103 pages).
4.2 plasmid extracts
The white colony of picking screening gained adds 5ml and adds in advance in the LB substratum of penbritin (100mg/ml), 37 ℃ of joltings 12～16 hours.Extract plasmid with the little extraction reagent kit of the common plasmid of Tiangen (centrifugal column type).
4.3 plasmid order-checking
The plasmid order-checking is accepted by invitrogen company.
Sequencing result: BxPC-3 is wild-type K-ras gene (the 12nd, 13 codons is GGTGGC), and Panc-1, CFPAC and SW1990 are mutant strain, and the 12nd, 13 codons sport G respectively
ATGGC, G
TTGGC, GGTG
AThe C (see figure 1).
5. real-time quantitative PCR
5.1 standard substance preparation
According to sequencing result, choosing mutant cell strain Panc-1 is the standard substance template.
The template copy number converts: copy number=quality/molecular weight * 6.02 * 10
23
The plasmid molecule amount is calculated: MW=(A base number * 312)+(C base number * 288)+(G base number * 328)+(T base number * 303)-61 ≈ base number * 324.5; Or use software DNAMAN to calculate.
The synthetic plasmid is 3457bp in this experiment, WM ≈ 2131.19Kda, so the 1ng plasmid copy number is about 3 * 10
8
With the template doubling dilution is 10
7, 10
6, 10
5, 10
4, 10
3Copy is as the standard substance template.
5.2 TaqMan MGB probe real-time quantitative experiment
Select the specificity T aqman MGB probe (SEQ ID NO:2) at K-ras gene target sequence for use, two ends are respectively with reporter group FAM and quenching group MGB mark.Similarly, PNA can suppress wild type gene fully in the experiment, so the fluorescence volume that instrument detected can react the copy amount of mutator gene.The present invention is not owing to influenced by primer dimer, and the specificity height is quantitatively also more accurate.
5.2.1 reaction system and reaction conditions
7500 type real-time quantitative PCR instrument of instrument selection ABI company.
Reaction system comprises: 2 * Taqman realtime PCR Master Mix, primers F 2 (SEQID NO:5), each 0.4 μ M of R2 (SEQ ID NO:6), PNA (SEQ ID NO:1) 3.75 μ M, MGB probe (SEQ ID NO:2) 0.3 μ M, DNA standard substance template is 10
7, 10
6, 10
5, 10
4, 10
3Copy is settled to 25 μ l with aseptic double-distilled water.Set the negative contrast of pure water (NTC).
The PCR reaction conditions: 95 ℃ of pre-sex change 60s, 95 ℃ of sex change 15s afterwards, 70 ℃ of PNA are in conjunction with 10s, 60 ℃ of annealing 60s, totally 40 circulations.
Detected result as shown in Figure 2, Fig. 2 is the standard substance amplification curve, in the figure, five curves of rising from left to right successively respectively representative dilution multiple proportions be 10
7, 10
6, 10
5, 10
4, 10
3The standard substance template of copy.Among Fig. 2, transverse axis refers to cycle number, and the longitudinal axis refers to the fluoroscopic examination value.
5.2.2 typical curve is drawn
According to fluorescence intensity that detects in the step 5.2 and the detected result of Fig. 2, drawing standard curve (see figure 3).In Fig. 3, transverse axis refers to the logarithmic value of copy number, and the longitudinal axis refers to the Ct value, and the slope of typical curve is-6.4, and intercept (Intercept) is 56.02, and R2 is 0.998.
5.2.3 quantivative approach
Select the plasmid standard (Pancl) with a series of doubling dilutions for use, be divided into two groups, do not add PNA in one group of standard substance reaction system, another group adds PNA (final concentration is 3.75 μ M).Simultaneously, template to be checked is divided into two groups too.After reaction finishes, utilize two standard sets product production standard curve respectively, carry out the quantitative of sample to be checked respectively.Wherein, for the reaction that does not add PNA, wild-type and mutant all increase, and the template amount that the result calculates is total copy number of wild type gene and mutated genes; And the reaction of adding PNA does not have amplification because wild type gene is suppressed, and the template amount of gained is the copy number (see figure 4) of mutated genes contained in the sample as a result.
The detection of K-ras transgenation in embodiment 2 clinical samples (is example with blood and faecal samples)
1. collect clinical carcinoma of the pancreas, chronic pancreatitis patient and normal people's ight soil and blood, extracting DNA.
2. blood DNA method for extracting: 1. anticoagulated whole blood 0.5～12ml; 2. add 500～700ulTT damping fluid, behind the thermal agitation, refrigerated centrifuge left the heart 5 minutes with per minute 12000; 3. collecting precipitation repeats 2,3 steps, and is colourless until being precipitated as; 4. add 200ulPCR lysate A; 5. 37 ℃ of water-baths are spent the night; 6. extract PCR lysate (about 200ul), add 200ul phenol chloroform, fully behind the mixing, left the heart 20 minutes with per minute 12000; 7. get supernatant, add the abundant mixing of 200ul chloroform, 12000 rev/mins centrifugal 10 minutes; 8. get supernatant, add the abundant mixing of 100ul ammonium acetate and 2 times of volume dehydrated alcohols (about 700ul), leave standstill 1 minute after, 12000 rev/mins are centrifugal 10 minutes; 9. abandon supernatant and add 1ml 70% washing with alcohol, 12000 rev/mins centrifugal 5 minutes; 10. abandon supernatant, natural air drying with 100ulTE liquid dissolution precipitation, is sub-packed in-80 ℃ of preservations.
3. faeces DNA method for extracting: adopt the QIAamp DNA Stool MiniKit test kit of Qiagen company to carry out.
4.DNA method for measurement of concentration is with the step 2 of embodiment 1.
5. according to the 5.2.1 of embodiment 1, the PNA that sample is carried out the K-ras gene clamps down on the quantitative PCR method detection.
6. interpretation of result:, draw the ratio of carcinoma of the pancreas K-ras transgenation copy number and mutant and wild-type according to typical curve.
Sequence table
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Claims (9)
1. a PNA who detects the K-ras transgenation is characterized in that, the 12nd codon and/or the 13rd codon are hybridized in this PNA and wild-type K-ras gene first exon.
2. PNA as claimed in claim 1 is characterized in that, this PNA comprises at least 12 continuous bases of SEQ ID NO:1 or its complementary sequence.
3. PNA as claimed in claim 1 is characterized in that, the sequence of this PNA is:
Ac-ACGCCACCAGCTC-Lysine-COOH。
4. a probe that detects the K-ras transgenation is characterized in that, described probe and the hybridization of wild-type K-ras gene target sequence, and this probe sequence comprises at least 15 continuous nucleotides of SEQ ID NO:2 or its complementary sequence.
5. probe as claimed in claim 4 is characterized in that, described probe is an oligonucleotide sequence shown in the SEQ ID NO:2.
6. a primer that detects the K-ras transgenation is characterized in that, the described primer K-ras gene target sequence that is used to increase comprises that following primer is right:
(1) first primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:3, and second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:4;
(2) first primer sequences contain at least 15 continuous nucleotides of SEQ ID NO:5, and second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:6.
7. primer as claimed in claim 6 is characterized in that, described primer is to comprising: SEQ IDNO:3, SEQ ID NO:4; SEQ ID NO:5, SEQ ID NO:6.
8. a method that detects the K-ras transgenation is characterized in that, may further comprise the steps:
(1) with claim 1 described PNA, the described probe of claim 4 and the described primer of claim 6, the plasmid standard of known mutations amount is carried out real-time quantitative PCR detect, draw typical curve;
(2) extract sample of nucleic acid;
(3) with the described PNA of claim 1, the described probe of claim 4 and the described primer of claim 6, the nucleic acid that extracts with step (2) is template, carries out real-time quantitative PCR and detects;
(4), draw the K-ras transgenation amount of sample nucleic acid according to the typical curve of step (1) gained.
9. a test kit that detects the K-ras transgenation is characterized in that, comprising: the described PNA of claim 1, the described probe of claim 4 and the described primer of claim 6.
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