CN108949924A - The fluorescence detection reagent kit and fluorescence detection method of deletion mutant - Google Patents
The fluorescence detection reagent kit and fluorescence detection method of deletion mutant Download PDFInfo
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Abstract
The present invention provides a kind of fluorescence detection reagent kit of deletion mutation gene, the kit includes: PNA capture probe；DNA probe；Padlock probe；Archaeal dna polymerase；DNA ligase；Rolling circle amplification primer；Fluorescence probe.The present invention also provides the methods for using the kit to carry out the detection of deletion mutation gene by fluorescence, the described method comprises the following steps: 1) the PNA capture probe being fixed on orifice plate bottom, after hybridize under buffer conditions with target gene；2) hybridize the target gene for being fixed on orifice plate bottom with DNA probe；3) hybridize the single stranded portion on the DNA probe after hybridizing with target gene with padlock probe, and be cyclized using ligase, rolling circle amplification is carried out under the action of rolling circle amplification primer and archaeal dna polymerase；4) hybridize fluorescence probe with rolling circle amplification product, the variation of fluorescence intensity after background fluorescence is quenched.
Description
Technical field
This application involves a kind of fluorescence detection reagent kit of deletion mutant and fluorescence detection methods, belong to chemistry and life
Object field of sensing technologies.
Background technique
Gene order deletion mutation is that gene structure caused by since the missing of base-pair occurring in DNA molecular changes, and is
One of gene mutation, the gene delection in human body often induces a variety of diseases, such as the 18-21 of epidermal growth factor (EGFR)
Gene delection occurs for exon, induces the generation of lung cancer especially non-small cell lung cancer.Along with the occurrence and development of disease, people
In body pathological tissues or blood deletion mutation gene become clinical disease quickly, the important physical signs of Precise Diagnosis.Currently,
QRT-PCR (quantitative reverse transcription polymerase chain reaction) and second generation gene sequencing technology are main for deletion mutation gene
Detection method.The amplification of several DNA moleculars may be implemented in qRT-PCR technology, has very high sensitivity and specificity, however main
Defect is wanted to be that reaction process needs accurate temperature controlling, experiment condition is more demanding, is not suitable for scene and uses；Second generation gene sequencing
Technology needs profession and expensive equipment carries out data acquisition and parsing, to limit its extensive use.
Rolling circle amplification (Rolling Circle Amplification, RCA) is that most widely used constant temperature expands at this stage
One of increasing technology, it is (complementary with part cyclic template) for primer with oligonucleotide using annular DNA as template, there is strand displacement
Under archaeal dna polymerase (such as phi 29DNA polymerase) effect of effect, primer extend one is recycled to starting and extends disposition
It changes old DNA chain and continues next circulation, so repeatedly circulation, generation contain the DNA long-chain of a large amount of repetitive sequences.
It is former that rolling circle amplification by its high specific, high sensitivity and the properties such as easy to operate is widely used in immuno-chip detection, cell
In the multiple fields such as position detection, complete genome DNA detection, single nucleotide polymorphism detection.
Present invention combination rolling circle amplification, graphene oxide fluorescent quenching technology and fluorescence detection design a needle
To the detection architecture of deletion mutation gene, detection limit reaches 1pM, the detection method phase with other detection deletion mutation genes
Than having the characteristics that high sensitivity, high specific, easy to operate, quick.
Summary of the invention
According to the one aspect of the application, a kind of fluorescence detection reagent kit of deletion mutation gene is provided, which is characterized in that
The kit includes: PNA capture probe, DNA probe, padlock probe, archaeal dna polymerase, rolling circle amplification primer and fluorescence probe.
Preferably, the PNA capture probe is 10 to 25 alkali with target gene deletion mutation location proximate close to 3 ' ends
The PNA sequence of basic sequence complete complementary, the c-terminus and target gene 5 ' of the PNA sequence hold base complementrity, the PNA sequence
Aminoterminal and target gene 3 ' hold base complementrity.
In a preferred embodiment of the present invention, the PNA capture probe c-terminus (CONH2) connection 1-5
Poly glycol monomethyl ether (mPEG) monomer is to increase water solubility.
Preferably, the DNA probe is the DNA sequence with target gene deletion mutation site both ends partial sequence complete complementary
Column；It wherein, is with single-stranded shape with corresponding part at target gene deletion mutation site when the DNA probe hybridizes with target gene
Single stranded zone existing for formula.
DNA probe in the present invention hybridizes the DNA double chain to form complete complementary with normal gene, and with deletion mutation gene
When hybridization, DNA probe middle section is not combined, and is existed with single stranded form.
Preferably, the single stranded zone complete complementary at the 5 ' ends and 3 ' ends and the DNA probe of the padlock probe, the locking-type
5 ' ends of probe and the center for being located at the DNA probe single stranded zone with the tie point of the DNA probe single stranded zone at 3 ' ends.
Preferably, the archaeal dna polymerase and the rolling circle amplification primer can be visited to by the padlock probe and the DNA
The cyclic DNA of needle single stranded zone composition carries out rolling circle amplification.
The padlock probe and the DNA probe single stranded zone are capable of forming cricoid DNA under the action of DNA ligase,
To carry out subsequent rolling circle amplification.
Preferably, the fluorescence probe is the DNA signal probe with fluorescent marker, the fluorescence probe can with it is described
Repeated fragment sequence on the rolling circle amplification product of cyclic DNA combines.
Preferably, the DNA ligase is selected from least one of Ecoli DNA ligase, T4DNA ligase；More
Preferably T4DNA ligase.
The archaeal dna polymerase is selected from least one of phi29DNA polymerase, T4DNA polymerase, DNA polymerase i；More
Preferably phi 29DNA polymerase.
The rolling circle amplification primer selected from at least 15 or more the bases in cyclic DNA complementary pairing base sequence.
The side that deletion mutant fluorescence detection is carried out using the kit is provided according to another aspect of the present invention
Method the described method comprises the following steps:
1) the PNA capture probe is fixed on orifice plate bottom, after hybridize under buffer conditions with target gene；
2) hybridize the target gene for being fixed on orifice plate bottom with DNA probe；
3) hybridize the single stranded portion on the DNA probe after hybridizing with target gene with padlock probe, and using ligase into
Row cyclisation, carries out rolling circle amplification under the action of rolling circle amplification primer and archaeal dna polymerase；
4) hybridize fluorescence probe with rolling circle amplification product, the variation of fluorescence intensity after background fluorescence is quenched.
Preferably, the method that PNA capture probe is fixed in the step 1) includes that PNA probe and fixation is added to orifice plate
Liquid room temperature concussion incubation 1.5-2.5 hours, is added shielding liquid, continues concussion and be incubated for 20-40 minutes, added with after buffer solution for cleaning
Enter PBS be sealed it is spare.
The fixed orifice plate of PNA capture probe includes 96 orifice plates, 48 orifice plates, 24 orifice plates, 12 orifice plates, 6 orifice plates, orifice plate bottom belt
Active carboxyl can react to form amido bond with amino under alkaline condition.
In a preferred embodiment of the present invention, PBST is respectively adopted after incubation and PBS solution is cleaned 3 times, then plus
Enter PBS buffer solution to be saved.
Preferably, the fixer is selected from sodium bicarbonate fixer, sodium carbonate fixer, saleratus fixer, carbonic acid
At least one of Potassium fixating liquid, the shielding liquid is selected from lysine shielding liquid, arginine shields liquid, asparagine shields liquid,
Glutamine shields at least one of liquid, and the concentration of the PNA probe is 80-120 μM, the volume of PNA probe and fixer
Than for 1:(80-120).
Preferably, in the step 1), the method that PNA capture probe hybridizes with target gene includes being added after blocking liquid
Concussion is incubated for 20-40 minutes at room temperature, using the target gene that various concentration is added after buffer solution for cleaning, continues to make after concussion is incubated for
Use buffer solution for cleaning.
In the present invention, using the target gene being added after buffer solution for cleaning, concentration may include 1fM-100nM.
Preferably, the BLBs for blocking liquid to be selected from calf thymus DNA or salmon sperm dna, further preferably salmon sperm dna
BLBs block liquid.
Preferably, in the step 3), the method for cyclisation includes that DNA ligase, connection buffer and distilled water is added to exist
After being incubated for 1-2 hours under conditions of 35-40 DEG C, being incubated for 15-25 minutes under conditions of 55-65 DEG C makes the DNA ligase
Inactivation.
Preferably, the DNA ligase is T4DNA ligase, and the concentration of the DNA ligase is 5-9U/ μ L, the company
Connecing enzyme buffer liquid is 10 × T4DNA connection buffer, the DNA ligase, the addition volume ratio for connecting buffer and distilled water
For 2:(2-4): (24-26).
Preferably, the method for rolling circle amplification includes in the step 3), and rolling circle amplification primer, archaeal dna polymerase, polymerization is added
Buffer, bovine serum albumin(BSA), dNTP and distilled water are incubated for 0.5-1.5 hours, then at 55-65 DEG C of condition at 35-40 DEG C
Lower incubation inactivates the archaeal dna polymerase.
Preferably, the archaeal dna polymerase is phi 29DNA polymerase, and the concentration of the polymerase is 1-5U/ μ L, described
Polymerizeing buffer is 10 × phi 29DNA reaction buffer；Rolling circle amplification primer, archaeal dna polymerase, polymerization buffer, cow's serum
The addition volume ratio of albumin, dNTP and distilled water is 1:(1-5): (10-20): 1:1:(42-56).
Preferably, in the step 4), the volume ratio of fluorescence probe and the rolling circle amplification product is 1:(20-30), institute
It states fluorescence probe and mixes incubation 1-2 hours with the rolling circle amplification product.
Preferably, the quencher is the graphene oxide that concentration is 80-120 μ g/ml.
Preferably, described to be quenched including after the product for hybridizing fluorescence probe with rolling circle amplification product is cooling, addition is quenched
Agent, combination buffer and distilled water.
Preferably, the combination buffer is 10 × GO binding buffer, the quencher, combination buffer and
The addition volume ratio of distilled water is (10-20): 10:(50-60).
The beneficial effect that the application can generate includes:
1) kit provided herein is capable of providing the deletion mutant fluorescence detection based on rolling circle amplification
In involved key reagents.
2) detection method provided herein has the characteristics that high sensitivity, high specific, easy to operate, quick.
Detailed description of the invention
Fig. 1 is the schematic diagram of detection method in the present invention.
Fig. 2 is the fluorescence intensity testing result for detecting EGFR deletion mutation gene under 9 concentration gradients.
Fig. 3 is the concentration and fluorescence intensity graph of relation for detecting EGFR deletion mutation gene under 9 concentration gradients.
Fig. 4 is that the rolling circle amplification negative control experiment of detection method in the present invention runs cementing fruit.
Fig. 5 is the fluorescence intensity testing result of rolling circle amplification negative control experiment.
Fig. 6 is that the fluorescence intensity of different control groups in rolling circle amplification negative control experiment compares histogram.
Fig. 7 is the detection probe negative control fluorescence intensity testing result of detection method in the present invention.
Fig. 8 is that the fluorescence intensity of different control groups in detection probe negative control experiment compares histogram.
Fig. 9 is in the presence of 10nM EGFR normal gene, and the fluorescence intensity of different concentration of EGF R deletion mutation gene detects
As a result.
Figure 10 is the concentration and fluorescence of different concentration of EGF R deletion mutation gene in the presence of 10nM EGFR normal gene
Strength relationship curve graph.
Specific embodiment
The application is described in detail below with reference to embodiment, but the application is not limited to these embodiments.
Unless otherwise instructed, the raw material in embodiments herein is bought by commercial sources.
Embodiment 1
The design and synthesis of PNA capture probe, DNA probe, padlock probe, fluorescence probe and rolling circle amplification primer
Selecting EGFR gene deletion segment is nearby object close to the 10-25 base sequence at 3 ' ends, and design one is therewith
The PNA sequence of complete complementary is as PNA capture probe, c-terminus (CONH2) and EGFR deletion mutation gene 5 ' end base complementrity
Pairing, aminoterminal (NH2) and the end of EGFR deletion mutation gene 3 ' base pair complementarity, 1-5 mPEG is to increase for c-terminus introducing
Water solubility is condensed, one by one through cracking, purifying and structural characterization according to base sequence under the conditions of HBTU and DIEA with MBHA resin
For subsequent detection；DNA probe, padlock probe and rolling circle amplification primer are synthesized by Shanghai Jie Li Bioisystech Co., Ltd, in detail
Thin base sequence is as shown in table 1:
1 EGFR deletion mutation gene of table and normal gene, probe and primer sequence
Embodiment 2
The fluorescence detection of EGFR deletion mutation gene
The fixation of PNA capture probe on (1) 96 orifice plate
1 μ L100 μM PNA capture probe and 100 μ L NaHCO are added in every hole3The end of fixer, PNA capture probe is dense
Degree is 1.0 μM, shakes (600-700rpm) at room temperature and is incubated for 2 hours, and the rear 50 μ L lysines that are added shield liquid, continues room temperature concussion
(600-700rpm) 0.5 hour is cleaned four times with 300 1 × PBST of μ L, and 250 1 × PBS of μ L are cleaned four times, 250 1 × PBS of μ L
It stores for future use, the used time removes 250 1 × PBS of μ L, and 200 μ L BLBs are added and block liquid, room temperature shakes (600-700rpm) 0.5
Hour, rear remove blocks liquid, is directly used in and detects in next step；
(2) capture of EGFR deletion mutation gene
Be added in 100 μ L systems EGFR deletion mutation gene and 1 × PBS buffer solution be configured to 9 concentration gradients (0fM,
1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM), 5 repetitions of each concentration, room temperature shakes (600-700rpm)
15 minutes, EGFR deletion mutation gene is hybridized with PNA capture probe and is fixed on orifice plate, cleans four with 200 1 × PBS of μ L
It is secondary；
(3) DNA probe and EGFR deletion mutation gene hybridize
1 μ L10 μM DNA probe is added in 99 μ 1 × PBS buffer solution of L, is hybridized with target gene, room temperature concussion
(600-700rpm) 15 minutes is cleaned four times with 200 1 × PBS of μ L；
(4) padlock probe and DNA probe hybridize
1 μ L10 μM padlock probe is added in 99 μ 1 × PBS buffer solution of L, with the single stranded zone being not associated on DNA probe
Hybridization, room temperature shake (600-700rpm) 15 minutes, are cleaned four times with 200 1 × PBS of μ L；
(5) T4DNA ligase connects
T4DNA ligase, 10 μ 10 × T4DNA of L ligase buffer solutions and the 36 μ L that 4 μ L 7.0U/ μ L are added in every group are bis-
Water is steamed, stationary incubation 1 hour at 16 DEG C, being placed on inactivates T4DNA ligase；
(6) rolling circle amplification
1 μ l, 100 μM of primers, 2 μ L 1.0U/ μ L phi 29DNA polymerases, 10 10 × phi of μ L are added in every group
29DNA polymerization reaction buffer, 1 μ l 10mg/ml BSA, 1 μ l 10mM dNTP, 35 μ L distilled waters, stationary incubation 3 at 37 DEG C
Hour, being placed on inactivates phi 29DNA polymerase；
(7) fluorescence signal detects
10 μM of fluorescence probes of 20 μ l rolling circle amplification products and 1 μ l are taken to be incubated for 1 minute at 85 DEG C, room temperature shakes (600-
It is 700rpm) 15 minutes cooling, add 20 μ l, 100 μ g/ml graphene oxide, 10 μ l 10 × graphene oxide combination buffers
With 49 μ l distilled waters, room temperature shakes (600-700rpm) 10 minutes, operation is strictly protected from light, finally with Fluorescence Spectrometer in 648nm
The variation of measurement system emission spectrum under excitation wavelength, and record the fluorescence intensity in the presence of various concentration target gene.As a result
As shown in Figures 2 and 3, with the increase of detection EGFR deletion mutation mrna concentration, the fluorescence intensity detected also enhances therewith, most
Low detection sensitivity reaches 1pM.
Embodiment 3
The negative control of EGFR deletion mutation gene by fluorescence detection
The fixation of PNA capture probe on (1) 96 orifice plate
1 μ L100 μM PNA capture probe and 99 μ L NaHCO are added in every hole3Fixer, the final concentration of PNA capture probe
It is 1.0 μM, shakes (600-700rpm) at room temperature and be incubated for 2 hours, the rear 50 μ L lysines that are added shields liquid, continue room temperature concussion
(600-700rpm) 0.5 hour is cleaned four times with 300 1 × PBST of μ L, and 250 1 × PBS of μ L are cleaned four times, 250 1 × PBS of μ L
It stores for future use, the used time removes 250 1 × PBS of μ L, and 200 μ L BLBs are added and block liquid, room temperature shakes (600-700rpm) 0.5
Hour, rear remove blocks liquid, is directly used in and detects in next step；
(2) capture of EGFR gene
The experiment is divided into 5 groups: a, b, c, d, e in 96 orifice plates, 1 μ L, 1 μM of EGFR is wherein added in a, c, d group and lacks
Mutated gene is lost, 1 μ L, 1 μM of EGFR normal gene is added to make negative control in b group, target gene is not added in e group to make sky
White control, then be separately added into 1 × PBS buffer solution and be configured to 100 μ L systems, every group of five repetitions, room temperature shakes (600-
700rpm) 15 minutes, EGFR gene is hybridized with PNA capture probe and is fixed on orifice plate, cleans four with 200 μ L1 × PBS
It is secondary；
(3) DNA probe and EGFR gene hybridize
It is separately added into 1 μ L10 μM DNA probe in this four groups of a, b, d and e, DNA probe is not added to make blank control in c group,
Every group is added 1 × PBS buffer solution and is configured to 100 μ L systems, is hybridized with target gene, room temperature shakes (600-700rpm) 15
Minute, it is cleaned four times with 200 1 × PBS of μ L；
(4) padlock probe and DNA probe hybridize
It is separately added into 1 μ L10 μM padlock probe in this four groups of a, b, c and e, padlock probe is not added to make blank pair in d group
According to every group is added 1 × PBS buffer solution and is configured to 100 μ L systems, hybridizes with the single stranded zone being not associated on DNA probe, room temperature shake
It swings (600-700rpm) 15 minutes, is cleaned four times with 200 1 × PBS of μ L；
(5) T4DNA ligase connects
T4DNA ligase, 10 μ 10 × T4DNA of L ligase buffer solutions and the 36 μ L that 4 μ L 7.0U/ μ L are added in every group are bis-
Water is steamed, stationary incubation 1 hour at 16 DEG C, being placed on inactivates T4DNA ligase；
(6) rolling circle amplification
1 μ l, 100 μM of primers, 2 μ L 1.0U/ μ L phi 29DNA polymerases, 10 10 × phi of μ L are added in every group
29DNA polymerization reaction buffer, 1 μ l 10mg/ml BSA, 1 μ l 10mM dNTP, 35 μ L distilled waters, stationary incubation 3 at 37 DEG C
Hour, being placed on inactivates phi 29DNA polymerase；
(7) polyacrylamide gel is imaged
Prepare 10% polyacrylamide gel (40% acrylamide of 1.5ml/methylene diacrylamide, 0.6ml 10 ×
TAE-Mg buffer, 60 μ L 10%APS, 8 μ L TEMED and 3.9ml distilled waters), with gel electrophoresis system under 110V voltage
It runs glue 1.5 hours, 500bp Marker is compared, and dyes 30 minutes through GelRed, gel is placed in Labworks image acquisition and analysis software
In, rolling circle amplification product is detected in the UV lamp, as a result as shown in figure 4, showing that the positive control of EGFR deletion mutation gene has
Rolling circle amplification product；EGFR normal gene is as negative control and without target gene as equal nothing under blank control both of these case
Rolling circle amplification product；Blank control is had also been made to DNA probe and padlock probe, the results showed that without rolling circle amplification product.Pass through
This experiment shows the detection architecture to the accuracy of EGFR deletion mutation genetic test.
(8) fluorescence signal detects
10 μM of fluorescence probes of 20 μ l rolling circle amplification products and 1 μ l are taken to be incubated for 1 minute at 85 DEG C, room temperature shakes (600-
It is 700rpm) 15 minutes cooling, add 20 μ l, 100 μ g/ml graphene oxide, 10 μ l 10 × graphene oxide combination buffers
With 49 μ l distilled waters, room temperature shakes (600-700rpm) 10 minutes, operation is strictly protected from light, finally with Fluorescence Spectrometer in 648nm
Measurement system emission spectrum under excitation wavelength, each autofluorescence under the conditions of record positive control, negative control and blank control
Intensity.As a result (as illustrated in Figures 5 and 6) shows that the fluorescence intensity of positive controls is most strong, and average value reaches 112.7, and negative right
According to group and blank control group without obvious fluorescence intensity, DNA probe and padlock probe blank control group, should also without obvious fluorescence intensity
Fluorescence signal testing result matches with cementing fruit is run, and demonstrates again that the detection architecture is applied to EGFR deletion mutation genetic test
Accuracy and practicability.
Embodiment 4
The detection probe negative control of EGFR deletion mutation gene by fluorescence detection
The experiment is divided into 5 groups: 1,2,3,4,5,1 μ l, 10 μM of fluorescence probes are added in every group, is not added and appoints in 1 and 2 group
What detects gene, is separately added into 20 μ l 10nM EGFR deletion mutation gene rolling circle amplification products, 20 μ l 10nM in 3,4 and 5 groups
10 μM of padlock probes of EGFR normal gene rolling circle amplification product and 1 μ l, every group of 5 repetitions are incubated for 1 minute, room temperature at 85 DEG C
Shake (600-700rpm) cooling 15 minutes, every group of 10 μ l 10 × graphene oxide combination buffer of addition, in addition to 1 group other
Four groups are added 20 μ l, 100 μ g/ml graphene oxide, are eventually adding distilled water and are configured to 100 μ l detection architectures.Room temperature concussion
(600-700rpm) 10 minutes, is strictly protected from light operation, and with Fluorescence Spectrometer, measurement system emits light under 648nm excitation wavelength
Spectrum, the respective fluorescence intensity under the conditions of record positive control, negative control and blank control.As a result as shown in FIG. 7 and 8, this table
The mean value of fluorescence intensity of bright 100nM fluorescence probe in the presence of without gene and graphene oxide is detected is 295.22,
The mean value of fluorescence intensity of positive controls is 112.7, and negative control group (4 and 5 groups) and blank control group (2 groups) are without bright
Aobvious fluorescence intensity, this not only illustrates that fluorescence probe detects rolling circle amplification product enough under 100nM concentration, but also also turns out that this is glimmering
Light probe can be used for the fluorescence detection of EGFR deletion mutation gene rolling ring product.
Embodiment 5
The fluorescence detection of mixed base EGFR deletion mutation gene because in
The fixation of PNA capture probe on (1) 96 orifice plate
1 μ L100 μM PNA capture probe and 100 μ L NaHCO3 fixers are added in every hole, the end of PNA capture probe is dense
Degree is 1.0 μM, shakes (600-700rpm) at room temperature and is incubated for 2 hours, and the rear 50 μ L lysines that are added shield liquid, continues room temperature concussion
(600-700rpm) 0.5 hour is cleaned four times with 300 1 × PBST of μ L, and 250 1 × PBS of μ L are cleaned four times, 250 1 × PBS of μ L
It stores for future use, the used time removes 250 1 × PBS of μ L, and 200 μ L BLBs are added and block liquid, room temperature shakes (600-700rpm) 0.5
Hour, rear remove blocks liquid, is directly used in and detects in next step；
(2) capture of EGFR gene
The EGFR deletion mutation gene of 9 concentration gradients mixes 1 μ L1 μM EGFR normal gene, with 1 × PBS buffer solution
It is configured to the 100 μ L systems of 0fM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 5 repetitions of each concentration,
Room temperature shakes (600-700rpm) 15 minutes, and EGFR mixed base is fixed on orifice plate due to hybridizing with PNA capture probe, uses
200 1 × PBS of μ L are cleaned four times；
(3) DNA probe and EGFR mixed base because hybridize
1 μ L10 μM DNA probe is added in 99 μ 1 × PBS buffer solution of L, with EGFR mixed base because being hybridized, room
Temperature concussion (600-700rpm) 15 minutes, is cleaned four times with 200 1 × PBS of μ L；
(4) padlock probe and DNA probe hybridize
1 μ L10 μM padlock probe is added in 99 μ 1 × PBS buffer solution of L, with the single stranded zone being not associated on DNA probe
Hybridization, room temperature shake (600-700rpm) 15 minutes, are cleaned four times with 200 1 × PBS of μ L；
(5) T4DNA ligase connects
T4DNA ligase, 10 μ 10 × T4DNA of L ligase buffer solutions and the 36 μ L that 4 μ L 7.0U/ μ L are added in every group are bis-
Water is steamed, stationary incubation 1 hour at 16 DEG C, being placed on inactivates T4DNA ligase；
(6) rolling circle amplification
1 μ l, 100 μM of primers, 2 μ L 1.0U/ μ L phi 29DNA polymerases, 10 10 × phi of μ L are added in every group
29DNA polymerization reaction buffer, 1 μ l 10mg/ml BSA, 1 μ l 10mM dNTP, 35 μ L distilled waters, stationary incubation 3 at 37 DEG C
Hour, being placed on inactivates phi 29DNA polymerase；
(7) fluorescence signal detects
10 μM of fluorescence probes of 20 μ l rolling circle amplification products and 1 μ l are taken to be incubated for 1 minute at 85 DEG C, room temperature shakes (600-
It is 700rpm) 15 minutes cooling, add 20 μ l, 100 μ g/ml graphene oxide, 10 μ l 10 × graphene oxide combination buffers
With 49 μ l distilled waters, room temperature shakes (600-700rpm) 10 minutes, operation is strictly protected from light, finally with Fluorescence Spectrometer in 648nm
The variation of measurement system emission spectrum under excitation wavelength, and record the fluorescence intensity in the presence of various concentration target gene.As a result
As shown in Figures 9 and 10, with the increase of EGFR deletion mutation mrna concentration, the rolling circle amplification product fluorescence intensity detected also with
Enhancing, lowest detection sensitivity reaches 1pM.This shows that the detection method is still quasi- in the presence of 10nM EGFR normal gene
Really, effectively and reliably.The true detection sample situation of the design simulation of the experiment is in patient's Non-Small Cell Lung Carcinoma
The detection of EGFR deletion mutation gene lays the foundation.
The above is only several embodiments of the application, not does any type of limitation to the application, although this Shen
Please disclosed as above with preferred embodiment, however not to limit the application, any person skilled in the art is not taking off
In the range of technical scheme, a little variation or modification are made using the technology contents of the disclosure above and is equal to
Case study on implementation is imitated, is belonged in technical proposal scope.
Sequence table
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Chinese Academy of Sciences Ningbo Material Technology and Engineering Institute
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Claims (10)
1. a kind of fluorescence detection reagent kit of deletion mutant, which is characterized in that the kit include: PNA capture probe,
DNA probe, padlock probe, DNA ligase, archaeal dna polymerase, rolling circle amplification primer and fluorescence probe.
2. kit according to claim 1, which is characterized in that the PNA capture probe is and target gene deletion mutation
PNA sequence of the location proximate close to 10 to 25 base sequence complete complementaries at 3 ' ends, the c-terminus and target base of the PNA sequence
Because 5 ' hold base complementrity, the aminoterminal and target gene 3 ' of the PNA sequence hold base complementrity.
3. kit according to claim 1, which is characterized in that the DNA probe is and target gene deletion mutation site
The DNA sequence dna of both ends partial sequence complete complementary；Wherein, when the DNA probe hybridizes with target gene, with target gene deletion mutation
Corresponding part is with single stranded zone existing for single stranded form at site；
Preferably, the single stranded zone complete complementary at the 5 ' ends and 3 ' ends and the DNA probe of the padlock probe, the padlock probe
5 ' end and 3 ' hold the center for being located at the DNA probe single stranded zone with the tie point of the DNA probe single stranded zone；
Preferably, the archaeal dna polymerase and the rolling circle amplification primer can to the cyclic DNA formed by the padlock probe into
Row rolling circle amplification；
Preferably, the fluorescence probe is the DNA signal probe with fluorescent marker, and the fluorescence probe can be with the ring-type
The repeated fragment sequence of DNA rolling circle amplification product combines.
4. kit according to claim 3, which is characterized in that the DNA ligase is selected from Ecoli DNA connection
At least one of enzyme, T4 DNA ligase；
The archaeal dna polymerase is selected from least one of phi29 archaeal dna polymerase, T4 archaeal dna polymerase, DNA polymerase i；
The rolling circle amplification primer selected from at least 15 or more the bases in cyclic DNA complementary pairing base sequence.
5. the method for carrying out deletion mutant fluorescence detection using kit described in any one of claim 1-4,
It is characterized in that, the described method comprises the following steps:
1) the PNA capture probe is fixed on orifice plate bottom, after hybridize under buffer conditions with target gene；
2) hybridize the target gene for being fixed on orifice plate bottom with DNA probe；
3) hybridize the single stranded portion on the DNA probe after hybridizing with target gene with padlock probe, and carry out ring using ligase
Change, rolling circle amplification is carried out under the action of rolling circle amplification primer and archaeal dna polymerase；
4) hybridize fluorescence probe with rolling circle amplification product, the variation of fluorescence intensity after background fluorescence is quenched.
6. detection method according to claim 5, which is characterized in that fix the side of PNA capture probe in the step 1)
Method includes that PNA probe and fixer is added to orifice plate, and room temperature concussion is incubated for 1.5-2.5 hours, and shielding liquid is added, continues to shake
It is incubated for 20-40 minutes, is sealed with addition PBS after buffer solution for cleaning spare；
Preferably, it is solid to be selected from sodium bicarbonate fixer, sodium carbonate fixer, saleratus fixer, potassium carbonate for the fixer
Determine at least one of liquid, the shielding liquid is selected from lysine shielding liquid, arginine shielding liquid, asparagine and shields liquid, paddy ammonia
Amide shields at least one of liquid.
7. detection method according to claim 5, which is characterized in that in the step 1), PNA capture probe and target gene
The method of hybridization includes being added after blocking liquid concussion incubation 20-40 minutes at room temperature, different using being added after buffer solution for cleaning
The target gene of concentration continues to use buffer solution for cleaning after concussion is incubated for；
Preferably, the BLBs for blocking liquid to be selected from calf thymus DNA or salmon sperm dna, preferably comprises the BLBs of salmon sperm dna
Block liquid.
8. detection method according to claim 5, which is characterized in that in the step 3), the method for cyclisation includes being added
After DNA ligase, connection buffer and distilled water are incubated for 1-2 hours under conditions of 35-40 DEG C, then at 55-65 DEG C of condition
Lower incubation inactivates the DNA ligase；
Preferably, the DNA ligase is T4 DNA ligase, and the ligase buffer solution is 10 × T4 DNA connection buffering
Liquid.
9. detection method according to claim 5, which is characterized in that the method for rolling circle amplification includes in the step 3),
Rolling circle amplification primer, archaeal dna polymerase, polymerization buffer, bovine serum albumin(BSA), dNTP and distilled water is added, is incubated at 35-40 DEG C
It educates 0.5-1.5 hours, being incubated under conditions of 55-65 DEG C inactivates the archaeal dna polymerase；
Preferably, the archaeal dna polymerase is 29 archaeal dna polymerase of phi, and the polymerization buffer is 10 × phi, 29 DNA reaction
Buffer.
10. detection method according to claim 5, which is characterized in that described to be quenched including expanding fluorescence probe and rolling ring
After the product cooling for increasing products thereof, quencher, combination buffer and distilled water is added；
Preferably, the quencher is the graphene oxide that concentration is 80-120 μ g/ml.
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Families Citing this family (1)
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017861A (en) * | 2014-04-23 | 2014-09-03 | 常州方圆制药有限公司 | Nucleic acid detection method |
| CN104955963A (en) * | 2012-11-14 | 2015-09-30 | 欧凌科公司 | Rca reporter probes and their use in detecting nucleic acid molecules |
-
2018
- 2018-06-27 CN CN201810682300.0A patent/CN108949924B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104955963A (en) * | 2012-11-14 | 2015-09-30 | 欧凌科公司 | Rca reporter probes and their use in detecting nucleic acid molecules |
| CN104017861A (en) * | 2014-04-23 | 2014-09-03 | 常州方圆制药有限公司 | Nucleic acid detection method |
Non-Patent Citations (4)
| Title |
|---|
| CHAESUN HONG等: "Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide", 《ANALYTICAL CHEMISTRY》 * |
| THOMAS J. LYNCH, M.D.,等: "Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non–Small-Cell Lung Cancer to Gefitinib", 《THE NEW ENGLAND JOURNAL OF MEDICINE》 * |
| 周国华主编: "《SNP检测技术与个体化药物治疗》", 28 February 2015 * |
| 王晓亮等: "DNA的滚环扩增技术研究进展", 《食品工业科技》 * |
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