CN105039522A - Detection reagent based on FoxM1 gene mutations in Wnt signal paths of peptide nucleic acid probes, PCR detection method and application of detection reagent - Google Patents
Detection reagent based on FoxM1 gene mutations in Wnt signal paths of peptide nucleic acid probes, PCR detection method and application of detection reagent Download PDFInfo
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Abstract
The invention discloses a detection reagent based on FoxM1 gene mutations in Wnt signal paths of peptide nucleic acid probes, a PCR detection method and application of the detection reagent. The detection reagent comprises primers and peptide nucleic acid fluorescent probes, wherein the primers comprises forward and reverse primers; the sequence of the forward primers is shown as SEQ ID NO.1, No.3, NO.5 and NO.7 in a sequence table; the sequence of the reverse primers is shown as SEQ ID NO.2, No.4, NO.6 and NO.8 in the sequence table; the sequence of the peptide nucleic acid fluorescent probes is shown as SEQ ID NO.9, No.10, NO.11 and NO.12 in the sequence table; the wild-type complementary peptide nucleic acid sequence is shown as SEQ ID NO.13, No.14, NO.15 and NO.16 in the sequence table. The PCR detection method is a real-time fluorescent quantitation PCR method based on the situation that peptide nucleic acid (PNA) is utilized for the fluorescent probes. Through the adoption of the detection reagent and the PCR detection method, a tool is provided for the screening of anticancer drugs, study of new targeted drugs, the basic scientific research and the like.
Description
Technical field
The invention belongs to molecular biology technical field of biological nucleic acid detection, be specifically related to a kind of detection reagent based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe, PCR detection method and application.
Background technology
Wnt signal path is extensively present in invertebrates and vertebrates, is the signal path that a class is guarded at spore process camber.Wnt signal, in the early development of animal embryo, orga-nogenesis, tissue regeneration and other physiological process, has vital effect.If the key protein in this signal paths is undergone mutation or unconventionality expression, abnormal signal is caused to activate, just the generation of possibility induced cancer.Wnt signal path comprises classical Wnt signal path and non-classical Wnt signal path, in classical path and Wnt-β-catenin signal path, the Wnt factor is by the phosphorylation of β-catenin albumen free in T suppression cell after the Frizzle/LRP5/6 cooperative expert systems on activating cells film and degraded, the core displacement of β-catenin albumen will be there is after β-catenin protein level in tenuigenin raises, β-catenin albumen in nucleus is caused to raise, in karyon, β-catenin albumen can combine Pygo2, common and the TCF/LEF-1 transcription factor family of Bcl-9 and FoxM1 albumen forms complex body and activates the transcriptional activation of Wnt signal path downstream target gene.
FoxM1 albumen is one of β-catenin downstream important member in Wnt signal path, and increasing research at present has been found that, except Pygo2 albumen, FoxM1 albumen also presents high expression level in a lot of tumour.
The core element regulatory mechanism of current research core signal path, and the expression level of some important member in cell, become a kind of key means for the treatment of tumour, although the research of Wnt signal path in cancer in recent years, and FoxM1 albumen is as the research of its expression level of Wnt signal path important member, become the major issue that research and development tumour medicine is badly in need of solving, but the sudden change of its gene in a lot of tumour cell also gets more and more simultaneously, very important effect is played to FoxM1 protein exhibits function, in lung adenocarcinoma cell, such as detect that S55C suddenlys change, R256G suddenlys change, detect that P271Q suddenlys change in dermal melanin oncocyte, the L318F sudden change detected in Urothelial Carcinoma of Bladder cell.
Peptide nucleic acid(PNA) (peptidenucleicacids, PNA) is that a class replaces the DNA analogue of sugared phosphate backbone with polypeptide backbone.It is on the basis of the first-generation, s-generation antisense agent, built by Computer Design and the third generation antisense agent of final synthetic, it is a kind of brand-new DNA analogue, namely the pentose phosphate diester linkage skeleton in DNA is instead of with the peptide chain acid amides 2-aminoethylglycine key of neutrality, remaining is identical with DNA, PNA in conjunction with DNA or RNA sequence, can form stable double-spiral structure by the form identification of Watson-Crick base pairing.Because PNA is not electronegative, and there is not electrostatic repulsion between DNA and RNA, the stability thus combined and specificity all greatly improve; Be different from the hybridization between DNA or DNA, RNA, the hybridization of PNA and DNA or RNA affects by hybridization system salt concn hardly, be much better than DNA/DNA or DNA/RNA with the hybridization ability of DNA or RNA molecule, show very high hybridization stability, excellent distinguished sequence recognition capability, not by nuclease and protease hydrolysis.
Summary of the invention
The object of the invention is for above-mentioned present situation, aim to provide a kind of signaling molecule FoxM1 transgenation situation determining core in Wnt signal path, can explain that core element FoxM1 changes in tumour cell, result repeatability, the detection reagent based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe that susceptibility is good, PCR detection method and application.
The implementation of the object of the invention is, based on the detection reagent of FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe, comprise primer and probe, described primer comprises forward primer and reverse primer, and described probe is peptide nucleic acid probe;
Described detection FoxM1 gene S55C mutant forward primer is as SEQ ID NO.1;
Described detection FoxM1 gene S55C suddenlys change reverse primer as SEQ ID NO.2;
Described detection FoxM1 gene R256G mutant forward primer is as SEQ ID NO.3;
Described detection FoxM1 gene R256G suddenlys change reverse primer as SEQ ID NO.4;
Described detection FoxM1 gene P271Q mutant forward primer is as SEQ ID NO.5;
Described detection FoxM1 gene P271Q suddenlys change reverse primer as SEQ ID NO.6;
Described detection FoxM1 gene L318F mutant forward primer is as SEQ ID NO.7;
Described detection FoxM1 gene L318F suddenlys change reverse primer as SEQ ID NO.8;
Described detection FoxM1 gene S55C suddenlys change PNA fluorescent probe as SEQ ID NO.9;
Described detection FoxM1 gene R256G suddenlys change PNA fluorescent probe as SEQ ID NO.10;
Described detection FoxM1 gene P271Q suddenlys change PNA fluorescent probe as SEQ ID NO.11.
Described detection FoxM1 gene L318F suddenlys change PNA fluorescent probe as SEQ ID NO.12;
The 5' end of described peptide nucleic acid(PNA) fluorescent probe and 3' end are modified with fluorescent reporter group and quenching group respectively, the fluorescent reporter group modifying described 5' end is: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and the quenching group modifying described 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
Detection reagent based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe carries out the method detected, and detection method is be the real time fluorescence quantifying PCR method of fluorescent probe based on peptide nucleic acid(PNA);
The reaction system of described real-time fluorescence quantitative PCR is: forward primer, reverse primer, DEPC water, have the exo-acting archaeal dna polymerase of 5' → 3', dNTPs, 10 × PCRBuffer, RNASIN, M-MLV reversed transcriptive enzyme, oligo (dT) and containing the solution of Mg ion;
The exo-acting archaeal dna polymerase of the described 5' of having → 3' is Taq enzyme.
Based on the application of the detection reagent of FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe, for the preparation of detection tumour cell and Normocellular detection reagent, described cancer cells is lung adenocarcinoma cell, dermal melanin oncocyte or bladder Urothelial cell.
The present invention adopts the PNA oligomer of distinguished sequence as probe.It is a kind of analogue of nucleic acid of synthetic due to PNA, and be achirality, uncharged molecule, avoid oligonucleotide and its target gene in conjunction with time mutually repel caused hybridization unstable because of electric charge, in conjunction with the impact not being subject to hybridization solution ionic strength, thus demonstrate extremely strong heterosis, hybrid vigor, substantially increase detection sensitivity.
Compared with prior art, advantage of the present invention and positively effect are: FoxM1 gene be in newfound Wnt signal path β-catenin proteins downstream play the gene of critical function, the invention provides the reagent of FoxM1 detection in Gene Mutation in direct-detection Wnt signal path, the sudden change of FoxM1 gene can be gone out in transcriptional level rapid detection by quantitative real-time PCR by means of described detection reagent, and with common real-time fluorescence quantitative PCR unlike, the peptide nucleic acid(PNA) PNA fluorescent probe that we use is sensitiveer, the present invention is the screening of cancer therapy drug, the Mechanism Study of new targeted drug both provides very strong instrument.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of peptide nucleic acid(PNA) described in the embodiment of the present invention;
Fig. 2 is the saltant type of S55C described in the embodiment of the present invention and reference wild-type product pcr amplification figure;
Fig. 3 is the saltant type of R256G described in the embodiment of the present invention and reference wild-type product pcr amplification figure;
Fig. 4 is the saltant type of P271Q described in the embodiment of the present invention and reference wild-type product pcr amplification figure;
Fig. 5 is the saltant type of L318F described in the embodiment of the present invention and reference wild-type product pcr amplification figure.
Embodiment
Based on the detection reagent of Pygo2 transgenation in the Wnt signal path of peptide nucleic acid probe, described primer comprises forward primer and reverse primer, and described probe is peptide nucleic acid probe, it is characterized in that:
Described detection Pygo2 gene R46S mutant forward primer is as SEQ ID NO.1;
Described detection Pygo2 gene R46S suddenlys change reverse primer as SEQ ID NO.2;
Described detection Pygo2 gene P98H mutant forward primer is as SEQ ID NO.3;
Described detection Pygo2 gene P98H suddenlys change reverse primer as SEQ ID NO.4;
Described detection Pygo2 gene R334Q mutant forward primer is as SEQ ID NO.5;
Described detection Pygo2 gene R334Q suddenlys change reverse primer as SEQ ID NO.6;
Described detection Pygo2 gene R356P mutant forward primer is as SEQ ID NO.7;
Described detection Pygo2 gene R356P suddenlys change reverse primer as SEQ ID NO.8;
Described detection Pygo2 gene R46S suddenlys change PNA fluorescent probe as SEQ ID NO.9;
Described detection Pygo2 gene P98H suddenlys change PNA fluorescent probe as SEQ ID NO.10;
Described detection Pygo2 gene R334Q suddenlys change PNA fluorescent probe as SEQ ID NO.11;
Described detection Pygo2 gene R356P suddenlys change PNA fluorescent probe as SEQ ID NO.12.
The 5' end of described peptide nucleic acid(PNA) fluorescent probe and 3' end are modified with fluorescent reporter group and quenching group respectively, the fluorescent reporter group modifying described 5' end is: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and the quenching group modifying described 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
Detection reagent is also containing contrast agent, and contrast agent is the peptide nucleic acid sequence with the complementation of FoxM1 gene wild-type,
Described specific binding comprises FoxM1 gene 55 codon wild-type PNA sequence as SEQ ID NO.13;
Described specific binding comprises FoxM1 gene 256 codon wild-type PNA sequence as SEQ ID NO.14;
Described specific binding comprises FoxM1 gene 271 codon wild-type PNA sequence as SEQ ID NO.15;
Described specific binding comprises FoxM1 gene 318 codon wild-type PNA sequence as SEQ ID NO.16.
The mass spectrum of the present invention's peptide nucleic acid(PNA) used is shown in Fig. 1.
Carry out the method detected by the detection reagent based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe, detection method is PCR detection method, and described PCR detection method is be the real time fluorescence quantifying PCR method of fluorescent probe based on peptide nucleic acid(PNA);
The reaction system of described real-time fluorescence quantitative PCR is: forward primer, reverse primer, DEPC water, have the exo-acting archaeal dna polymerase of 5' → 3', dNTPs, 10 × PCRBuffer, RNASIN, M-MLV reversed transcriptive enzyme, oligo (dT) and containing the solution of Mg ion.
The exo-acting archaeal dna polymerase of the described 5' of having → 3' is Taq enzyme.
Based on the detection method of FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe, detecting step is as follows:
1) primer and the PNA probe in these sites is detected for the design of FoxM1 gene 55,256,271,318 codon mutation type;
2) for the 4 section PNA sequences of FoxM1 gene 55,256,271,318 Codon sequences design with these site wild-type complementations;
3) build the plasmid containing FoxM1 genic mutation type and wild-type, and calculate copy number, make reference material;
4) mRNA in cell to be measured is extracted;
5) with above-mentioned detection FoxM1 transgenation reagent, fluorescence quantitative PCR detection is carried out to reference material and sample, and judge whether FoxM1 gene 55,256,271,318 codon undergos mutation.
The final concentration of Taq enzyme described in described PCR reaction solution is 0.01U/ μ L ~ 0.05U/ μ L, the final concentration of described dNTPs is 0.2 ~ 0.6mM, the final concentration of described 10 × PCRBuffer is 1 ×, the final concentration of described RNASIN is 40U/ μ L ~ 60U/ μ L, the final concentration of described M-MLV reversed transcriptive enzyme is 200U/ μ L ~ 320U/ μ L, described MgCl
2final concentration be 1.5 ~ 5.0mM, solvent is DEPC water, and the final concentration of described forward primer is 0.05 ~ 0.9 μM, and the final concentration of described reverse primer is 0.05 ~ 0.9 μM, and the final concentration of described fluorescent probe is 0.05 ~ 0.9 μM.
The response procedures of described real-time fluorescence quantitative PCR is: 42 DEG C of reverse transcription 20min; 94 DEG C of denaturations, 2min; 95 DEG C of sex change, 30s; 58 DEG C, 45s; Carry out 40 circulations.
Experimental system of the present invention can carry out on any real-time fluorescence quantitative PCR instrument, and its PCR reaction kit is AppliedBiosystems company 7500 type quantitative real time PCR Instrument.Above-mentioned primer is placed in eight unions, carries out real-time fluorescence quantitative PCR detection, and experimental implementation is simple, low cost, result repeatability, and susceptibility is good, is a kind of important means of the research tumour related drugs mechanism of action and basic scientific research.
Based on the detection reagent of Pygo2 transgenation in the Wnt signal path of peptide nucleic acid probe for the preparation of detection tumour cell and Normocellular detection reagent, described cancer cells is lung adenocarcinoma cell, dermal melanin oncocyte or bladder Urothelial cell.
The present invention is illustrated by pancreatic cancer cell.
PNAC-1 (human pancreas cancer PNAC-1 clone) clone purchased from American ATCC in the present invention, the RPMI-1640 substratum that culturing cell uses and 10% foetal calf serum are all purchased from handsome company, and other reagent are mainly purchased from precious biotechnology (Dalian) company limited.
Utilize reagent of the present invention to detect Wnt signal path Pygo2 transgenation in cell and comprise following concrete steps:
According to American National Biotechnology Information center (NationalCenterforBiotechnologyInformation, NCBI) (http://www.ncbi.nlm.nih.gov) hFoxM1mRNA sequence (NCBIReferenceSequence:NM_202002.2) of reporting, the FoxM1 primer using the PrimerExpressSoftwareforReal-TimePCR software design of AppliedBiosystems company exploitation special and probe.
FoxM1S55C：
FoxM1-F:5'-ACATCAGAGGAGGAACCTAAGAGAT-3'；
FoxM1-R:5'-GGGTGGTTAATAATCTTGATCCCA-3'；
FoxM1(PNA)-P:5'FAM-TGGCAGAGTCCAACTGTTGCAAGT-BHQ3'；
FoxM1-PNA：TGGCAGAGTCCAACTCTTGCAAGT
Target sequence:
CAAGTGAAACATCAGAGGAGGAACCTAAGAGATCCCCTGCCCAACAGGAGTCTAATCAAGCAGAGGCCTCCAAGGAAGTGGCAGAGTCCAACTCTTGCAAGTTTCCAGCTGGGATCAAGATTATTAACCACCCCACCATGCCC
FoxM1R256G:
FoxM1-F:5'-AATTCGCCATCAACAGCACTG-3'；
FoxM1-R:5'-GGTCCTCAATCCACGTATAGATGTC-3'；
FoxM1(PNA)-P:5'FAM-AGGAAGGGCATGACTTTGAA-BHQ3'；
FoxM1-PNA：AGGAAGCGCATGACTTTGAA
Target sequence:
ATGGCCATGATACAATTCGCCATCAACAGCACTGAGAGGAAGCGCATGACTTTGAAAGACATCTATACGTGGATTGAGGACCACTTT
FoxM1P271Q：
FoxM1-F:5'-GACATCTATACGTGGATTGAGGACC-3'；
FoxM1-R:5'-GAGACCTTGCCATTGGCAGA-3'；
FoxM1(PNA)-P:5'FAM-TTTCAATACTTTAAGCACATTGCC-BHQ3'；
FoxM1-PNA：TTTCCCTACTTTAAGCACATTGCC
Target sequence:
GAAAGACATCTATACGTGGATTGAGGACCACTTTCCCTACTTTAAGCACATTGCCAAGCCAGGCTGGAAGAACTCCATCCGCCACAACCTTTCCCTGCACGACATGTTTGTCCGGGAGACGTCTGCCAATGGCAAGGTCTCCTT
FoxM1L318F:
FoxM1-F:5'-TCTGCCAATGGCAAGGTCTC-3'；
FoxM1-R:5'-TCGGTCGTTTCTGCTGTGATT-3'；
FoxM1(PNA)-P:5'FAM-CCAACCGCTACTTCACATTGGA-BHQ3'；
FoxM1-PNA：CCAACCGCTACTTGACATTGGA
Target sequence:
GGAGACGTCTGCCAATGGCAAGGTCTCCTTCTGGACCATTCACCCCAGTGCCAACCGCTACTTGACATTGGACCAGGTGTTTAAGCCACTGGACCCAGGGTCTCCACAATTGCCCGAGCACTTGGAATCACAGCAGAAACGACCGAATCCAG
Above: F:forward, forward; HW-F represents the forward primer for detecting hookworm nucleic acid.
R:reverse, oppositely; HW-R represents the reverse primer for detecting hookworm nucleic acid.
P:probe, fluorescent probe; HW-P represents the fluorescent probe for detecting hookworm nucleic acid, and this fluorescent probe is TaqMan fluorescent probe.
In embodiments of the present invention, the fluorescent reporter group modifying the 5' end of fluorescent probe can be: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5; The quenching group modifying the 3' end of fluorescent probe can be: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL, this fluorescent reporter group and quenching group do not affect the amplification of quantitative fluorescent PCR, only the model of instrument used need be selected to arrange detectable fluorescent signal scope according to the fluorescent reporter group of probe and quenching group.The fluorescent probe fluorescent reporter group that the embodiment of the present invention provides: the excitation wavelength of FAM, HEX, TET and FAM is 470-650nm, and reception wavelength is 500-700nm; Quenching group: Eclipse, TAMRA, BHQ1.Way of purification after primer synthesis can be: HAP, PAGE and HPLC way of purification.
Two, human lung adenocarcinoma Calu-3 clone is cultivated and is gone down to posterity
1) cell cultures
All cells system uses RPMI-1640 substratum (Invitrogen, Carlsbad, CA), 10% foetal calf serum (Invitrogen, Carlsbad, CA), in 37 DEG C, cultivate under 5%CO2 environment.
2) passage
First use sterilizing suction pipe by the nutrient solution sucking-off in Tissue Culture Dish, add PBS buffer solution for cleaning 2 times, appropriate trypsinase is slowly dripped in cell, treat cell rounding, the DMEM substratum containing 10% foetal calf serum of 3ml is added after adjustment angle cell can move, after repeatedly blowing and beating gently, basis of microscopic observation cellular form also counts, according to the content of cell in culture dish by appropriate passage in the culture dish of other sterilizings, put into 5%CO2 incubator after adding the DMEM substratum of 5ml.
Cell counting formula (individual/ml): (4 large lattice total cellular score) × 10
4× extension rate/4
Three, the extraction of total serum IgE
1) outwell substratum, after PBS cleaning, directly 1mlTrizol is injected culturing bottle (wherein cell 5 × 10
6individual/ml), suction is evenly repeatedly;
2) in the centrifuge tube that lysate is housed, add the chloroform (for 1/5 of Trizol cumulative volume) of 0.2ml, vibration is mixed 30 seconds, left at room temperature 5 minutes;
3) 12000rpm4 DEG C centrifugal 15 minutes, phase-splitting is three layers.Upper strata: RNA (being about 60% of Trizol); Middle: DNA; Lower floor: protein (phenol-chloroform);
4) careful Aspirate supernatant, transfers in another EP pipe.The supernatant volume that 1ml lysate produces is about 0.4 ~ 0.6ml.DNA and protein are contained in organic phase and middle layer, avoid touching;
5) supernatant liquor adds the Virahol of about 0.5ml, and vibration is mixed 30 seconds.Left at room temperature 10 minutes;
6) 12000rpm4 DEG C centrifugal 10 minutes;
7) side at the centrifugal end is formed by RNA precipitation.Supernatant liquor is abandoned in careful suction, notes avoiding suction to abandon RNA precipitation;
8) centrifuge tube adds 75% ethanol (1mlTrizol is 1ml ethanol purge DNA at least) of 1ml precooling, and vibration is mixed 30 seconds, precipitation is vibrated, centrifugal 1 ~ 2 minute of room temperature 12000rpm.Inhale as far as possible and abandon supernatant liquor, prevent RNA from precipitating and lose.Repeat above cleaning step once.In 75% ethanol, RNA at least preserves 1 week at 4 DEG C, at least preserves 1 year for-20 DEG C;
9) room temperature selecting liquidity is little, is inverted centrifuge tube on filter paper, dry RNA, but can not complete drying (5 ~ 10 minutes).With DEPC water 15 μ L dissolution precipitation, hatch 10 ~ 15 minutes for 55-60 DEG C.
10) RNA purity detecting
Drip 2 μ LRNA solution in ultramicrospectrophotometer (model: P330-311), and read OD260/OD280 ratio in instrument.
Four, construction recombination plasmid
1) DNA fragmentation containing FoxM1 genic mutation type and wild-type is carried out pcr amplification;
2) PCR primer is carried out double digestion;
Carrier and PCR primer carry out double digestion (reaction system is 30ul, 37 DEG C, and enzyme cuts 2 hours) by once condition respectively;
3) (operating according to test kit specification sheets) is reclaimed in rubber tapping after double digestion product electrophoresis;
4) digestion products is connected with plasmid vector;
Above-mentioned double digestion product is through purifying (wherein the rubber tapping of carrier digestion products is reclaimed, and it is identical with above-mentioned PCR primer purification step that PCR fragment enzyme cuts rear purification step), and under the effect of T4DNA ligase enzyme, 16 DEG C of connections are spent the night.Linked system is as follows: carrier, 2ul; PCR fragment, 6ul; 10xT4buffer, 1ul; T4DNAligase, 1ul.
5) transformation of E. coli competence;
Getting above-mentioned connecting fluid 5 μ l is transformed in previously prepared DH5 α Competent cell, ice bath 30 minutes, 42 DEG C of heat shock 2min, put 5min on ice, add 1mlLB nutrient solution 37 DEG C of shaking table 45min, centrifugal 5000rpm, 1-5min are (centrifugal too not of a specified duration, in order to avoid too real), be finally uniformly coated on containing on the antibiotic LB flat board of 100ng/ml (100-150ul).Flat board is inverted overnight incubation at 37 DEG C.Picking positive colony bacterium colony turns to draw and contains on the antibiotic LB flat board of 100ng/ml to another block, and to be numbered it, to be inverted overnight incubation for 37 DEG C.
6) QIAGEN test kit extracting plasmid (carrying out to specifications), makes reference material.
Five, real-time fluorescence quantitative PCR
Get the total serum IgE 1-5 μ g of extraction, add PCR reaction solution, PCR reaction solution comprises: sterilized water, have the exo-acting archaeal dna polymerase of 5' → 3', dNTPs, 10 × PCRBuffer, RNASIN, M-MLV reversed transcriptive enzyme, oligo (dT) and containing the solution of Mg ion.Wherein, concentration be 5U/ μ L there is 5' → 3' exo-acting archaeal dna polymerase 0.3 μ L, concentration is the dNTPs2 μ L of 10mmol/L, 10 × PCRBuffer5 μ L, concentration is the RNASIN0.6 μ L of 40U/ μ L, concentration is the M-MLV reversed transcriptive enzyme 0.6 μ L of 200U/ μ L, and concentration is the MgCl of 25mmol/L
2solution 5 μ L, adding sterilized water to volume is 50 μ L.Wherein, having the exo-acting archaeal dna polymerase of 5' → 3' can be Taq enzyme.
Pcr amplification: the reactive tank each reaction tubes being put into quantitative fluorescent PCR instrument, mark fluorescent radical species, sample ID and type are set, (this product fluorescent reporter group is FAM, HEX, TAT to the Taqman fluorescence probe of selection, fluorescent quenching group is Eclipse), definition sample well, and the amplification program that according to the form below provides carries out pcr amplification:
Table 1 is pcr amplification reaction amplification program
Fluorescent value is read in the end of a period of the 3rd step of amplification program.
The saltant type of S55C described in the embodiment of the present invention and reference wild-type product pcr amplification figure are shown in Fig. 2, and in figure, three bars, upper, middle and lower line respectively represents the amplification curve of FoxM1 the 55th bit codon mutation type reference material, sample and reference wild-type product.Described R256G saltant type and reference wild-type product pcr amplification figure are shown in Fig. 3; In figure, three bars, upper, middle and lower line respectively represents the amplification curve of FoxM1 the 256th bit codon mutation type reference material, sample and reference wild-type product.Described P271Q saltant type and reference wild-type product pcr amplification figure are shown in Fig. 4; In figure, three bars, upper, middle and lower line respectively represents the amplification curve of FoxM1 the 271st bit codon mutation type reference material, sample and reference wild-type product.Described L318F saltant type and reference wild-type product pcr amplification figure are shown in Fig. 5, and in figure, three bars, upper, middle and lower line respectively represents the amplification curve of FoxM1 the 318th bit codon mutation type reference material, sample and reference wild-type product.As seen from the figure in adenocarcinoma of lung Calu-3 clone the 55th, 256 bit codons are saltant type, and the 271st, 318 bit codons are wild-type.
Six, data analysis judges:
This saltant type corresponding with this pattern detection site of selected institute sample and reference wild-type sample wells, contrast three hole pcr amplification curve (CT simultaneously
arepresent sample aperture CT value, CT
wrepresent wild-type CT value, CT
mrepresent saltant type CT value):
Work as CT
w< CT
a≤ CT
mtime, show that this sample exists sudden change;
Work as CT
w=CT
atime, show that this sample is wild-type.
Claims (7)
1. based on the detection reagent of FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe, comprise primer and probe, described primer comprises forward primer and reverse primer, and described probe is peptide nucleic acid probe, it is characterized in that:
Described detection FoxM1 gene S55C mutant forward primer is as SEQ ID NO.1;
Described detection FoxM1 gene S55C suddenlys change reverse primer as SEQ ID NO.2;
Described detection FoxM1 gene R256G mutant forward primer is as SEQ ID NO.3;
Described detection FoxM1 gene R256G suddenlys change reverse primer as SEQ ID NO.4;
Described detection FoxM1 gene P271Q mutant forward primer is as SEQ ID NO.5;
Described detection FoxM1 gene P271Q suddenlys change reverse primer as SEQ ID NO.6;
Described detection FoxM1 gene L318F mutant forward primer is as SEQ ID NO.7;
Described detection FoxM1 gene L318F suddenlys change reverse primer as SEQ ID NO.8;
Described detection FoxM1 gene S55C suddenlys change PNA fluorescent probe as SEQ ID NO.9;
Described detection FoxM1 gene R256G suddenlys change PNA fluorescent probe as SEQ ID NO.10;
Described detection FoxM1 gene P271Q suddenlys change PNA fluorescent probe as SEQ ID NO.11.
Described detection FoxM1 gene L318F suddenlys change PNA fluorescent probe as SEQ ID NO.12;
The 5' end of described peptide nucleic acid(PNA) fluorescent probe and 3' end are modified with fluorescent reporter group and quenching group respectively, the fluorescent reporter group modifying described 5' end is: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and the quenching group modifying described 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
2. the detection reagent based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe according to claim 1, is characterized in that: detection reagent is also containing contrast agent, and contrast agent is the peptide nucleic acid sequence with the complementation of FoxM1 gene wild-type,
Described specific binding comprises FoxM1 gene 55 codon wild-type PNA sequence as SEQ ID NO.13;
Described specific binding comprises FoxM1 gene 256 codon wild-type PNA sequence as SEQ ID NO.14;
Described specific binding comprises FoxM1 gene 271 codon wild-type PNA sequence as SEQ ID NO.15;
Described specific binding comprises FoxM1 gene 318 codon wild-type PNA sequence as SEQ ID NO.16.
3. the method detected is carried out by the detection reagent based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe according to claim 1, it is characterized in that: detection method is PCR detection method, described PCR detection method is be the real time fluorescence quantifying PCR method of fluorescent probe based on peptide nucleic acid(PNA);
The reaction system of described real-time fluorescence quantitative PCR is: forward primer, reverse primer, DEPC water, have the exo-acting archaeal dna polymerase of 5' → 3', dNTPs, 10 × PCRBuffer, RNASIN, M-MLV reversed transcriptive enzyme, oligo (dT) and containing the solution of Mg ion;
The exo-acting archaeal dna polymerase of the described 5' of having → 3' is Taq enzyme.
4. the detection method based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe according to claim 3, is characterized in that: detecting step is as follows:
1) primer and the PNA probe in these sites is detected for the design of FoxM1 gene 55,256,271,318 codon mutation type;
2) for the 4 section PNA sequences of FoxM1 gene 55,256,271,318 Codon sequences design with these site wild-type complementations;
3) build the plasmid containing FoxM1 genic mutation type and wild-type, and calculate copy number, make reference material;
4) mRNA in cell to be measured is extracted;
5) with above-mentioned detection FoxM1 transgenation reagent, fluorescence quantitative PCR detection is carried out to reference material and sample, and judge whether FoxM1 gene 55,256,271,318 codon undergos mutation.
5. the detection method based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe according to claim 3, it is characterized in that: the final concentration of Taq enzyme described in described PCR reaction solution is 0.01U/ μ L ~ 0.05U/ μ L, the final concentration of described dNTPs is 0.2 ~ 0.6mM, the final concentration of described 10 × PCRBuffer is 1 ×, the final concentration of described RNASIN is 40U/ μ L ~ 60U/ μ L, the final concentration of described M-MLV reversed transcriptive enzyme is 200U/ μ L ~ 320U/ μ L, described MgCl
2final concentration be 1.5 ~ 5.0mM, solvent is DEPC water, and the final concentration of described forward primer is 0.05 ~ 0.9 μM, and the final concentration of described reverse primer is 0.05 ~ 0.9 μM, and the final concentration of described fluorescent probe is 0.05 ~ 0.9 μM.
6. the detection method based on FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe according to claim 3, is characterized in that: the response procedures of described real-time fluorescence quantitative PCR is: 42 DEG C of reverse transcription 20min; 94 DEG C of denaturations, 2min; 95 DEG C of sex change, 30s; 58 DEG C, 45s; Carry out 40 circulations.
7. the application based on the detection reagent of FoxM1 transgenation in the Wnt signal path of peptide nucleic acid probe according to claim 1, it is characterized in that: for the preparation of detection tumour cell and Normocellular detection reagent, described cancer cells is lung adenocarcinoma cell, dermal melanin oncocyte or bladder Urothelial cell.
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| CN201711188151.4A CN107893105A (en) | 2015-06-24 | 2015-06-24 | The detection reagent that FoxM1 genes R256G is mutated in Wnt signal paths based on peptide nucleic acid probe |
| CN201711188141.0A CN107828871A (en) | 2015-06-24 | 2015-06-24 | The detection reagent that FoxM1 genes P271Q is mutated in Wnt signal paths based on peptide nucleic acid probe |
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