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CN108949924B - Fluorescence detection kit and fluorescence detection method for gene deletion mutation - Google Patents

Fluorescence detection kit and fluorescence detection method for gene deletion mutation Download PDF

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CN108949924B
CN108949924B CN201810682300.0A CN201810682300A CN108949924B CN 108949924 B CN108949924 B CN 108949924B CN 201810682300 A CN201810682300 A CN 201810682300A CN 108949924 B CN108949924 B CN 108949924B
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CN108949924A (en
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徐小军
赵超
邢淑
付盼
徐梦佳
徐皖星
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Ningbo Institute of Material Technology and Engineering of CAS
Cixi Institute of Biomedical Engineering CIBE of CAS
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Abstract

The invention provides a fluorescent detection kit for deletion mutant genes, which comprises: a PNA capture probe; a DNA probe; a lock-type probe; a DNA polymerase; a DNA ligase; rolling circle amplification primer; a fluorescent probe. The invention also provides a method for carrying out deletion mutant gene fluorescence detection by using the kit, which comprises the following steps: 1) immobilizing the PNA capture probe on the bottom of a pore plate, and hybridizing with a target gene under the condition of a buffer solution; 2) hybridizing the target gene immobilized on the bottom of the well plate with a DNA probe; 3) hybridizing a single-stranded part on the DNA probe hybridized with the target gene with a padlock probe, performing cyclization by using ligase, and performing rolling circle amplification under the action of a rolling circle amplification primer and DNA polymerase; 4) hybridizing the fluorescent probe with the rolling circle amplification product, quenching background fluorescence, and detecting the change of fluorescence intensity.

Description

Fluorescence detection kit and fluorescence detection method for gene deletion mutation
Technical Field
The application relates to a fluorescence detection kit and a fluorescence detection method for gene deletion mutation, belonging to the technical field of chemistry and biosensing.
Background
The gene sequence deletion mutation is a kind of gene mutation due to the gene structure change caused by the deletion of base pairs in DNA molecules, and the gene deletion in human body often induces various diseases, such as the gene deletion of No. 18-21 exon of Epidermal Growth Factor (EGFR), and induces the generation of lung cancer, especially non-small cell lung cancer. Along with the occurrence and development of diseases, the deletion of mutant genes in pathological tissues or blood of a human body becomes an important physiological index for rapid and accurate diagnosis of clinical diseases. At present, qRT-PCR (quantitative reverse transcription polymerase chain reaction) and second generation gene sequencing technologies are the main detection methods for deletion mutant genes. The qRT-PCR technology can realize the amplification of a plurality of DNA molecules and has high sensitivity and specificity, but has the main defects that the reaction process needs precise temperature control, the requirement on experimental conditions is high, and the method is not suitable for field use; second generation gene sequencing technology requires specialized and expensive equipment for data acquisition and analysis, thereby limiting its widespread use.
Rolling Circle Amplification (RCA) is one of the most widely applied isothermal Amplification techniques at present, and is a method in which circular DNA is used as a template, oligonucleotides (complementary to a part of the circular template) are used as primers, and under the action of DNA polymerase (such as phi29DNA polymerase) with strand displacement, the primers are extended for one cycle to the initial extension treatment to replace the old DNA strand, and then the next cycle is continued, so that a long DNA chain containing a large number of repetitive sequences is generated. Rolling circle amplification is widely applied to various fields such as immune chip detection, cell in situ detection, whole genome DNA detection, single nucleotide polymorphism detection and the like by virtue of the properties of high specificity, high sensitivity, easiness in operation and the like.
The invention combines the rolling circle amplification technology, the graphene oxide fluorescence quenching technology and the fluorescence detection method to design a detection system aiming at the deletion mutant gene, the detection limit of the detection system reaches 1pM, and compared with other detection methods for detecting the deletion mutant gene, the detection method has the characteristics of high sensitivity, high specificity, simple and convenient operation and high speed.
Disclosure of Invention
According to one aspect of the present application, there is provided a fluorescent detection kit for deletion of a mutant gene, the kit comprising: PNA capture probe, DNA probe, padlock probe, DNA polymerase, rolling circle amplification primer and fluorescent probe.
Preferably, the PNA capture probe is a PNA sequence completely complementary to 10 to 25 base sequences near the 3 ' end of the deletion mutation site of the target gene, the carboxyl end of the PNA sequence is complementary to the 5 ' end base of the target gene, and the amino end of the PNA sequence is complementary to the 3 ' end base of the target gene.
In a preferred embodiment of the invention, the PNA capture probe is carboxy-terminal (CONH)2) 1-5 polyethylene glycol monomethyl ether (mPEG) monomers are attached to increase water solubility.
Preferably, the DNA probe is a DNA sequence completely complementary to the sequence at both ends of the deletion mutation site of the target gene; wherein, when the DNA probe is hybridized with the target gene, the part corresponding to the deletion mutation site of the target gene is a single-stranded region existing in a single-stranded form.
The DNA probe of the present invention hybridizes to a normal gene to form a perfectly complementary DNA double strand, and when it hybridizes to a deletion mutant gene, the central portion of the DNA probe is not bound and exists in the form of a single strand.
Preferably, the 5 'and 3' ends of the padlock probe are fully complementary to the single-stranded region of the DNA probe, and the point of attachment of the 5 'and 3' ends of the padlock probe to the single-stranded region of the DNA probe is located in the center of the single-stranded region of the DNA probe.
Preferably, the DNA polymerase and the rolling circle amplification primer are capable of rolling circle amplification of a circular DNA consisting of the padlock probe and the single-stranded region of the DNA probe.
The padlock probe and the single-stranded region of the DNA probe can form circular DNA under the action of DNA ligase so as to perform subsequent rolling circle amplification.
Preferably, the fluorescent probe is a DNA signal probe with a fluorescent label, and the fluorescent probe can bind to a repetitive fragment sequence on a rolling circle amplification product of the circular DNA.
Preferably, the DNA ligase is selected from at least one of E.coli DNA ligase and T4DNA ligase; more preferably T4DNA ligase.
The DNA polymerase is at least one selected from phi29DNA polymerase, T4DNA polymerase and DNA polymerase I; more preferably phi29DNA polymerase.
The rolling circle amplification primer is selected from at least 15 bases in a base sequence which is complementary to and pairs with the circular DNA.
According to another aspect of the present invention, there is provided a method for gene deletion mutation fluorescent detection using the kit, the method comprising the steps of:
1) immobilizing the PNA capture probe on the bottom of a pore plate, and hybridizing with a target gene under the condition of a buffer solution;
2) hybridizing the target gene immobilized on the bottom of the well plate with a DNA probe;
3) hybridizing a single-stranded part on the DNA probe hybridized with the target gene with a padlock probe, performing cyclization by using ligase, and performing rolling circle amplification under the action of a rolling circle amplification primer and DNA polymerase;
4) hybridizing the fluorescent probe with the rolling circle amplification product, quenching background fluorescence, and detecting the change of fluorescence intensity.
Preferably, the method for immobilizing the PNA capture probe in step 1) comprises adding the PNA probe and the fixing solution to a well plate, performing shake incubation at room temperature for 1.5 to 2.5 hours, adding a shielding solution, performing shake incubation for 20 to 40 minutes, washing with a buffer solution, adding PBS, and performing sealed preservation for later use.
The PNA capture probe fixed pore plate comprises a 96 pore plate, a 48 pore plate, a 24 pore plate, a 12 pore plate and a 6 pore plate, wherein the bottom of the pore plate is provided with active carboxyl which can react with amino to form amido bond under alkaline condition.
In a preferred embodiment of the present invention, after incubation, the cells are washed 3 times with PBST and PBS, respectively, and then stored with PBS buffer.
Preferably, the fixing solution is at least one selected from sodium bicarbonate fixing solution, sodium carbonate fixing solution, potassium bicarbonate fixing solution and potassium carbonate fixing solution, the shielding solution is at least one selected from lysine shielding solution, arginine shielding solution, asparagine shielding solution and glutamine shielding solution, the concentration of the PNA probe is 80-120 μ M, and the volume ratio of the PNA probe to the fixing solution is 1: (80-120).
Preferably, in the step 1), the method for hybridizing the PNA capture probe and the target gene comprises adding a blocking solution, incubating at room temperature with shaking for 20-40 minutes, washing with a buffer solution, adding target genes with different concentrations, continuing to incubate with shaking, and washing with a buffer solution.
In the present invention, the target gene added after washing with a buffer may be included at a concentration of 1fM to 100 nM.
Preferably, the blocking solution is selected from the group consisting of BLBs of calf thymus DNA or salmon sperm DNA, more preferably a blocking solution of BLBs containing salmon sperm DNA.
Preferably, in the step 3), the circularization method comprises adding DNA ligase, ligation buffer and double distilled water, incubating for 1-2 hours at 35-40 ℃, and then incubating for 15-25 minutes at 55-65 ℃ to inactivate the DNA ligase.
Preferably, the DNA ligase is T4DNA ligase, the concentration of the DNA ligase is 5-9U/mu L, the ligase buffer is 10 XT 4DNA ligation buffer, and the adding volume ratio of the DNA ligase, the ligation buffer and double distilled water is 2: (2-4): (24-26).
Preferably, the rolling circle amplification method in step 3) comprises adding rolling circle amplification primers, DNA polymerase, polymerization buffer, bovine serum albumin, dNTP and double distilled water, incubating at 35-40 ℃ for 0.5-1.5 hours, and incubating at 55-65 ℃ for 15-25 minutes to inactivate the DNA polymerase.
Preferably, the DNA polymerase is phi29DNA polymerase, the concentration of the polymerase is 1-5U/mu L, and the polymerization buffer is 10 x phi29DNA reaction buffer; the volume ratio of the rolling circle amplification primer, the DNA polymerase, the polymerization buffer solution, the bovine serum albumin, the dNTP and the double distilled water is 1: (1-5): (10-20): 1: 1: (42-56).
Preferably, in the step 4), the volume ratio of the fluorescent probe to the rolling circle amplification product is 1: (20-30), and incubating the fluorescent probe and the rolling circle amplification product for 1-2 hours in a mixing way.
Preferably, the quencher is graphene oxide at a concentration of 80-120 μ g/ml.
Preferably, the quenching comprises adding a quenching agent, a binding buffer and double distilled water after cooling a product of hybridization of the fluorescent probe and the rolling circle amplification product.
Preferably, the binding buffer is 10 × GO binding buffer, and the addition volume ratio of the quencher, the binding buffer and double distilled water is (10-20): 10: (50-60).
The beneficial effects that this application can produce include:
1) the kit provided by the application can provide key reagents involved in gene deletion mutation fluorescence detection based on rolling circle amplification technology.
2) The detection method provided by the application has the characteristics of high sensitivity, high specificity, simplicity and convenience in operation and rapidness.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2 shows the result of detecting the fluorescence intensity of EGFR deletion mutant gene under 9 concentration gradients.
FIG. 3 is a graph showing the relationship between the concentration of EGFR deletion mutant gene and the fluorescence intensity when 9 concentration gradients are detected.
FIG. 4 shows the gel running result of the rolling circle amplification negative control experiment according to the detection method of the present invention.
FIG. 5 shows the result of fluorescence intensity detection in rolling circle amplification negative control experiment.
FIG. 6 is a comparison histogram of fluorescence intensity of different control groups in the rolling circle amplification negative control experiment.
FIG. 7 shows the result of detecting the fluorescence intensity of the negative control of the detection probe in the detection method of the present invention.
FIG. 8 is a comparison histogram of fluorescence intensity of different control groups in the test probe negative control experiment.
FIG. 9 shows the results of fluorescence intensity measurements of different concentrations of EGFR deletion mutant gene in the presence of 10nM of EGFR normal gene.
FIG. 10 is a graph showing the relationship between the concentration of EGFR deletion mutant gene at various concentrations and the fluorescence intensity in the presence of 10nM EGFR normal gene.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.
Example 1
Design and synthesis of PNA capture probe, DNA probe, padlock probe, fluorescent probe and rolling circle amplification primer
Selecting 10-25 base sequences near the 3' end near the EGFR gene deletion site as objects, designing a PNA sequence completely complementary with the EGFR gene deletion site as PNA capture probe, carboxyl end (CONH)2) Complementary pairing with EGFR deletion mutant gene 5' end base, amino end (NH)2) Complementary pairing with the base of the 3' end of the EGFR deletion mutant gene, introducing 1-5 mPEG at the carboxyl end to increase water solubility, condensing MBHA resin one by one according to the base sequence under the conditions of HBTU and DIEA, and performing cracking, purification and structural characterization for subsequent detection; DNA probes, padlock probes and rolling circle amplification primers were synthesized by Shanghai Jili Biotechnology Ltd, and the detailed base sequences are shown in Table 1:
TABLE 1 EGFR deletion mutant and Normal genes, probes and primer sequences
Figure BDA0001710855050000061
Example 2
Fluorescent detection of EGFR deletion mutant genes
(1) Immobilization of PNA capture probes on 96-well plates
mu.L of 100. mu.M PNA capture probe and 100. mu.L NaHCO were added to each well3The final concentration of the PNA capture probe was 1.0. mu.M in the fixative, incubated at room temperature with shaking (600-Then 250 μ L of 1 XPBS is removed, 200 μ L of BLBs blocking solution is added, the mixture is shaken at room temperature (600-;
(2) capture of EGFR deletion mutant Gene
EGFR deletion mutant gene and 1 XPBS buffer solution are added into a 100 mu L system to be configured into 9 concentration gradients (0fM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM), each concentration is 5 times repeated, the mixture is shaken at room temperature (600-;
(3) hybridization of DNA probes to EGFR deletion mutant genes
1 u L10 u M DNA probe is added into 99 u L1 x PBS buffer solution, and hybridization with the target gene, room temperature shaking (600-;
(4) hybridization of padlock probes with DNA probes
mu.L of 10. mu.M padlock probe was added to 99. mu.L of 1 XPBS buffer, hybridized with unbound single-stranded region on DNA probe, shaken (600-;
(5) t4DNA ligase ligation
Adding 4 μ L of 7.0U/. mu.L T4DNA ligase, 10 μ L of 10 XT 4DNA ligase buffer solution and 36 μ L double distilled water to each group, standing and incubating at 16 ℃ for 1 hour, and then placing at 65 ℃ for 20 minutes to inactivate T4DNA ligase;
(6) rolling circle amplification
To each group were added 1. mu.l of 100. mu.M primer, 2. mu.l of 1.0U/. mu.L phi29DNA polymerase, 10. mu.L of 10 XPhi 29DNA polymerization buffer, 1. mu.l of 10mg/ml BSA, 1. mu.l of 10mM dNTP, and 35. mu.L of double distilled water, and the mixture was incubated at 37 ℃ for 3 hours and then left at 65 ℃ for 20 minutes to inactivate the phi29DNA polymerase;
(7) fluorescence signal detection
20 mu l of the rolling circle amplification product and 1 mu l of 10 mu M fluorescent probe are incubated for 1 minute at 85 ℃, vibrated at room temperature (600 plus 700rpm) for cooling for 15 minutes, then 20 mu l of 100 mu g/ml graphene oxide, 10 mu l of 10 multiplied graphene oxide binding buffer and 49 mu l of double distilled water are added, vibrated at room temperature (600 plus 700rpm) for 10 minutes, strictly protected from light, finally a fluorescence spectrometer is used for measuring the change of the emission spectrum of the system under the excitation light wavelength of 648nm, and the fluorescence intensity when target genes with different concentrations exist is recorded. As shown in FIGS. 2 and 3, the fluorescence intensity was increased with the increase of the concentration of the EGFR deletion mutant gene, and the lowest detection sensitivity was 1 pM.
Example 3
Negative control for fluorescent detection of EGFR deletion mutant gene
(1) Immobilization of PNA capture probes on 96-well plates
mu.L 100. mu.M PNA capture probe and 99. mu.L NaHCO were added to each well3The final concentration of the PNA capture probe is 1.0 mu M, the PNA capture probe is incubated for 2 hours at room temperature by shaking (600-700rpm), then 50 mu L of lysine shielding solution is added, the PNA capture probe is further shaken for 0.5 hours at room temperature (600-700rpm), washed with 300 mu L of 1 XPBST for four times, washed with 250 mu L of 1 XPBS for four times, and stored with 250 mu L of 1 XPBS for standby, when the PNA capture probe is used, the 250 mu L of 1 XPBS is removed, 200 mu L of BLBs blocking solution is added, the PNA capture probe is shaken for 0.5 hours at room temperature, the blocking solution is removed, and the PNA capture probe is directly used for next detection;
(2) capture of EGFR Gene
The experiment was divided into 5 groups in 96-well plates: a. b, c, d and e, wherein 1 mu L of 1 mu M EGFR deletion mutant gene is added into the a, c and d groups, 1 mu L of 1 mu M EGFR normal gene is added into the b group to be used as negative control, the target gene is not added into the e group to be used as blank control, 1 XPBS buffer solution is respectively added to prepare a 100 mu L system, each group is five-repeated, the system is oscillated at room temperature (600 + 700rpm) for 15 minutes, the EGFR gene and the PNA capture probe are hybridized and fixed on a pore plate, and the hybridization is carried out by 200 mu L1 XPBS for four times;
(3) hybridization of DNA probes to EGFR Gene
Adding 1 uL 10 uM DNA probe into the four groups of a, b, d and e respectively, adding no DNA probe into the group c to serve as blank control, adding 1 XPBS buffer solution into each group to prepare a 100 uL system, hybridizing with the target gene, shaking at room temperature (600- & gt 700rpm) for 15 minutes, and washing with 200 uL 1 XPBS four times;
(4) hybridization of padlock probes with DNA probes
Adding 1 uL 10 uM padlock probes to the four groups of a, b, c and e respectively, adding no padlock probe to the group d to serve as blank control, adding 1 XPBS buffer solution to each group to prepare a 100 uL system, hybridizing with the unbound single-stranded region on the DNA probe, shaking at room temperature (600-700rpm) for 15 minutes, and washing with 200 uL 1 XPBS four times;
(5) t4DNA ligase ligation
Adding 4 μ L of 7.0U/. mu.L T4DNA ligase, 10 μ L of 10 XT 4DNA ligase buffer solution and 36 μ L double distilled water to each group, standing and incubating at 16 ℃ for 1 hour, and then placing at 65 ℃ for 20 minutes to inactivate T4DNA ligase;
(6) rolling circle amplification
To each group were added 1. mu.l of 100. mu.M primer, 2. mu.l of 1.0U/. mu.L phi29DNA polymerase, 10. mu.L of 10 XPhi 29DNA polymerization buffer, 1. mu.l of 10mg/ml BSA, 1. mu.l of 10mM dNTP, and 35. mu.L of double distilled water, and the mixture was incubated at 37 ℃ for 3 hours and then left at 65 ℃ for 20 minutes to inactivate the phi29DNA polymerase;
(7) polyacrylamide gel imaging
Preparing 10% polyacrylamide gel (1.5ml of 40% acrylamide/methylene bisacrylamide, 0.6ml of 10 XTAE-Mg buffer solution, 60 mu L of 10% APS, 8 mu L of TEMED and 3.9ml of double distilled water), running the gel for 1.5 hours at the voltage of 110V by using a gel electrophoresis system, taking 500bp Marker as a control, staining the gel for 30 minutes by GelRed, placing the gel in a gel imaging analysis system, detecting a rolling ring amplification product under an ultraviolet lamp, and showing that the positive control of the EGFR deletion mutant gene has the rolling ring amplification product as shown in figure 4; the EGFR normal gene is used as a negative control and the non-target gene is used as a blank control, and rolling circle amplification products do not exist in the two conditions; blank controls were also made for the DNA probe and padlock probe, and the results showed no rolling circle amplification product. The experiment shows that the detection system has the accuracy of detecting the EGFR deletion mutant gene.
(8) Fluorescence signal detection
Incubating 20 μ l of the rolling circle amplification product with 1 μ l of 10 μ M fluorescent probe at 85 ℃ for 1 minute, oscillating at room temperature (600 + 700rpm) for cooling for 15 minutes, adding 20 μ l of 100 μ g/ml graphene oxide, 10 μ l of 10 × graphene oxide binding buffer solution and 49 μ l of double distilled water, oscillating at room temperature (600 + 700rpm) for 10 minutes, strictly avoiding light, finally measuring the emission spectrum of the system by using a fluorescence spectrometer at the excitation wavelength of 648nm, and recording the respective fluorescence intensity under the conditions of positive control, negative control and blank control. The results (as shown in fig. 5 and 6) show that the fluorescence intensity of the positive control group is strongest, the average value reaches 112.7, the negative control group and the blank control group have no obvious fluorescence intensity, the DNA probe and the padlock probe blank control group also have no obvious fluorescence intensity, the detection result of the fluorescence signal is identical with the running gel result, and the accuracy and the practicability of the detection system applied to the detection of the EGFR deletion mutant gene are proved again.
Example 4
Detection probe negative control for EGFR deletion mutant gene fluorescence detection
The experiment was divided into 5 groups: 1. 2, 3, 4 and 5, adding 1 mul of 10 MuM fluorescent probe into each group, adding no detection gene into the groups 1 and 2, respectively adding 20 mul of 10nM EGFR deletion mutant gene rolling circle amplification product, 20 mul of 10nM EGFR normal gene rolling circle amplification product and 1 mul of 10 MuM padlock probe into the groups 3, 4 and 5, respectively, incubating 5 repeats in each group at 85 ℃ for 1 minute, shaking at room temperature (600 and 700rpm) for 15 minutes, adding 10 mul of 10 Xoxidized graphene binding buffer solution into each group, adding 20 mul of 100 Mug/ml oxidized graphene into the four groups except the group 1, and finally adding double distilled water to prepare a detection system of 100 mul. Shaking at room temperature (600-700rpm) for 10 minutes, strictly avoiding light, measuring the emission spectrum of the system by using a fluorescence spectrometer at the excitation wavelength of 648nm, and recording the respective fluorescence intensity under the conditions of positive control, negative control and blank control. The results are shown in fig. 7 and 8, which indicate that the mean fluorescence intensity of the 100nM fluorescent probe in the absence of the detection gene and graphene oxide is 295.22, the mean fluorescence intensity of the positive control group is 112.7, and the negative control groups (groups 4 and 5) and the blank control group (group 2) have no significant fluorescence intensity, which not only indicates that the fluorescent probe is sufficient for detecting the rolling circle amplification product at a concentration of 100nM, but also proves that the fluorescent probe can be used for the fluorescent detection of the rolling circle product of the EGFR deletion mutant gene.
Example 5
Fluorescence detection of EGFR deletion mutant genes in mixed genes
(1) Immobilization of PNA capture probes on 96-well plates
Adding 1 mu L of 100 mu M PNA capture probe and 100 mu L NaHCO3 stationary solution into each well, wherein the final concentration of the PNA capture probe is 1.0 mu M, oscillating (600-700rpm) at room temperature for 2 hours, then adding 50 mu L lysine shielding solution, continuing oscillating (600-700rpm) at room temperature for 0.5 hour, washing with 300 mu L1 XPBST for four times, washing with 250 mu L1 XPBS for four times, storing with 250 mu L1 XPBS for later use, removing 250 mu L1 XPBS when in use, adding 200 mu L BLBs blocking solution, oscillating (600-700rpm) at room temperature for 0.5 hour, then removing the blocking solution, and directly using for next detection;
(2) capture of EGFR Gene
Mixing 1 uL 1 uM EGFR normal gene with 9 concentration gradient EGFR deletion mutant genes, configuring 100 uL systems of 0fM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM and 10nM with 1 XPBS buffer solution, each concentration is 5 times repeated, shaking at room temperature (600-;
(3) hybridization of DNA probes to EGFR Mixed Gene
Adding 1 μ L of 10 μ M DNA probe into 99 μ L of 1 XPBS buffer solution, hybridizing with EGFR mixed gene, shaking at room temperature (600-700rpm) for 15 minutes, and washing with 200 μ L of 1 XPBS four times;
(4) hybridization of padlock probes with DNA probes
mu.L of 10. mu.M padlock probe was added to 99. mu.L of 1 XPBS buffer, hybridized with unbound single-stranded region on DNA probe, shaken (600-;
(5) t4DNA ligase ligation
Adding 4 μ L of 7.0U/. mu.L T4DNA ligase, 10 μ L of 10 XT 4DNA ligase buffer solution and 36 μ L double distilled water to each group, standing and incubating at 16 ℃ for 1 hour, and then placing at 65 ℃ for 20 minutes to inactivate T4DNA ligase;
(6) rolling circle amplification
To each group were added 1. mu.l of 100. mu.M primer, 2. mu.l of 1.0U/. mu.L phi29DNA polymerase, 10. mu.L of 10 XPhi 29DNA polymerization buffer, 1. mu.l of 10mg/ml BSA, 1. mu.l of 10mM dNTP, and 35. mu.L of double distilled water, and the mixture was incubated at 37 ℃ for 3 hours and then left at 65 ℃ for 20 minutes to inactivate the phi29DNA polymerase;
(7) fluorescence signal detection
20 mu l of the rolling circle amplification product and 1 mu l of 10 mu M fluorescent probe are incubated for 1 minute at 85 ℃, vibrated at room temperature (600 plus 700rpm) for cooling for 15 minutes, then 20 mu l of 100 mu g/ml graphene oxide, 10 mu l of 10 multiplied graphene oxide binding buffer and 49 mu l of double distilled water are added, vibrated at room temperature (600 plus 700rpm) for 10 minutes, strictly protected from light, finally a fluorescence spectrometer is used for measuring the change of the emission spectrum of the system under the excitation light wavelength of 648nm, and the fluorescence intensity when target genes with different concentrations exist is recorded. As shown in FIGS. 9 and 10, the fluorescence intensity of the detected rolling circle amplification product is increased with the increase of the concentration of the EGFR deletion mutant gene, and the lowest detection sensitivity reaches 1 pM. This indicates that the detection method is still accurate, effective and reliable in the presence of 10nM EGFR normal gene. The design of the experiment simulates the real condition of a detection sample, and lays a foundation for the detection of EGFR deletion mutant genes in non-small cell lung cancer tissues of patients.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Sequence listing
<110> Cixi biomedical engineering institute of Ningbo Industrial technology institute of Chinese academy of sciences
Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences
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Claims (17)

1. A fluorescence detection kit for gene deletion mutation is characterized by comprising: PNA capture probe, DNA probe, padlock probe, DNA ligase, DNA polymerase, rolling circle amplification primer and fluorescent probe;
the DNA probe is a DNA sequence which is completely complementary with the sequence of the two end parts of the target gene deletion mutation site; wherein, when the DNA probe is hybridized with a target gene, the part corresponding to the deletion mutation site of the target gene is a single-stranded region in a single-stranded form;
the sequence of the PNA capture probe is as follows: h2NOC-CTACACTCAAA- (mPEG)3-NH 2;
the sequence of the DNA probe is as follows:
5’-TCCTTGTTGGCTTTCGGAGATGTTGCTTCTCTTAATTCCTTGATAGCGACGGGAA-3’；
the sequence of the padlock probe is as follows:
5’-P-AAGCAACATCTCAACTATCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGTTCTGGAAAGCGGAATTAAGAG-3’；
the sequence of the rolling circle amplification primer is as follows:
5’-GCTGAGACATGACGAGTC-3’；
the sequence of the fluorescent probe is as follows:
Cy5-CTAACGGTCACTAATACG-3’。
2. the kit of claim 1, wherein the PNA capture probe is a PNA sequence completely complementary to 10 to 25 bases near the 3 ' end of the deletion mutation site of the target gene, wherein the carboxyl end of the PNA sequence is complementary to the 5 ' base of the target gene and the amino end of the PNA sequence is complementary to the 3 ' base of the target gene.
3. The kit of claim 1, wherein the 5 'and 3' ends of the padlock probe are fully complementary to the single-stranded region of the DNA probe, and the point of attachment of the 5 'and 3' ends of the padlock probe to the single-stranded region of the DNA probe is located in the center of the single-stranded region of the DNA probe.
4. The kit of claim 1, wherein the DNA polymerase and the rolling circle amplification primer are capable of rolling circle amplification of circular DNA formed by the padlock probe.
5. The kit of claim 1, wherein the fluorescent probe is a DNA signaling probe with a fluorescent label capable of binding to a repetitive fragment sequence of a circular DNA rolling circle amplification product.
6. The kit according to claim 3, wherein the DNA ligase is selected from at least one of E-coli DNA ligase, T4DNA ligase;
the DNA polymerase is at least one selected from phi29DNA polymerase, T4DNA polymerase and DNA polymerase I.
7. Method for the fluorescent detection of gene deletion mutations for non-diagnostic therapeutic purposes using a kit according to any of claims 1 to 6, characterized in that it comprises the following steps:
1) immobilizing the PNA capture probe on the bottom of a pore plate, and hybridizing with a target gene under the condition of a buffer solution;
2) hybridizing the target gene immobilized on the bottom of the well plate with a DNA probe;
3) hybridizing a single-stranded part on the DNA probe hybridized with the target gene with a padlock probe, performing cyclization by using ligase, and performing rolling circle amplification under the action of a rolling circle amplification primer and DNA polymerase;
4) hybridizing the fluorescent probe with the rolling circle amplification product, quenching background fluorescence, and detecting the change of fluorescence intensity.
8. The detection method according to claim 7, wherein the method for immobilizing the PNA capture probe in step 1) comprises adding the PNA probe and the immobilizing solution to a well plate, performing shake incubation at room temperature for 1.5-2.5 hours, adding a shielding solution, performing further shake incubation for 20-40 minutes, washing with a buffer solution, adding PBS, and performing sealed preservation for later use.
9. The detection method according to claim 8, wherein the fixing solution is at least one selected from sodium bicarbonate fixing solution, sodium carbonate fixing solution, potassium bicarbonate fixing solution and potassium carbonate fixing solution, and the shielding solution is at least one selected from lysine shielding solution, arginine shielding solution, asparagine shielding solution and glutamine shielding solution.
10. The detection method according to claim 7, wherein in step 1), the method for hybridizing the PNA capture probe and the target gene comprises adding blocking solution, incubating at room temperature with shaking for 20-40 minutes, washing with buffer solution, adding target gene with different concentrations, continuing to incubate with shaking, and washing with buffer solution.
11. The detection method as set forth in claim 10, wherein the blocking solution is selected from the group consisting of BLBs of calf thymus DNA or salmon sperm DNA.
12. The detection method as claimed in claim 7, wherein in the step 3), the circularization method comprises adding DNA ligase, ligation buffer and double distilled water, incubating for 1-2 hours at 35-40 ℃, and then incubating for 15-25 minutes at 55-65 ℃ to inactivate the DNA ligase.
13. The method of claim 7, wherein the DNA ligase is T4DNA ligase and the ligase buffer is 10 XT 4DNA ligase buffer.
14. The detection method according to claim 7, wherein the rolling circle amplification method in step 3) comprises adding rolling circle amplification primers, DNA polymerase, polymerization buffer, bovine serum albumin, dNTP and double distilled water, incubating for 0.5-1.5 hours at 35-40 ℃, and further incubating for 15-25 minutes at 55-65 ℃ to inactivate the DNA polymerase.
15. The method of claim 7, wherein the DNA polymerase is phi29DNA polymerase and the polymerization buffer is 10 XPhi 29DNA reaction buffer.
16. The detection method according to claim 7, wherein the quenching comprises adding a quencher, a binding buffer and double distilled water after cooling the product of the hybridization of the fluorescent probe with the rolling circle amplification product.
17. The detection method according to claim 7, wherein the quencher is graphene oxide at a concentration of 80 to 120 μ g/ml.
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US12129516B2 (en) 2020-02-07 2024-10-29 10X Genomics, Inc. Quantitative and automated permeabilization performance evaluation for spatial transcriptomics

Families Citing this family (36)

* Cited by examiner, † Cited by third party
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US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
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US11981960B1 (en) 2020-07-06 2024-05-14 10X Genomics, Inc. Spatial analysis utilizing degradable hydrogels
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
US11981958B1 (en) 2020-08-20 2024-05-14 10X Genomics, Inc. Methods for spatial analysis using DNA capture
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
EP4214330A1 (en) * 2020-10-22 2023-07-26 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
AU2021409136A1 (en) 2020-12-21 2023-06-29 10X Genomics, Inc. Methods, compositions, and systems for capturing probes and/or barcodes
WO2022177367A1 (en) * 2021-02-18 2022-08-25 한국과학기술원 Composition for multiplex detection of target nucleic acid, based on isothermal amplification and fluorescent ring pattern, and detection method using same
EP4347879A1 (en) 2021-06-03 2024-04-10 10X Genomics, Inc. Methods, compositions, kits, and systems for enhancing analyte capture for spatial analysis
EP4196605A1 (en) 2021-09-01 2023-06-21 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
CN114456802B (en) * 2022-02-18 2023-04-07 南京农业大学 Ratio type silver nano-cluster fluorescent probe and preparation method and application thereof
CN117089605B (en) * 2023-05-08 2024-05-03 中国科学院苏州生物医学工程技术研究所 FQ-RCA-based RNA isothermal real-time genotyping method

Citations (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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 (3)

* Cited by examiner, † Cited by third party
Title
Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non–Small-Cell Lung Cancer to Gefitinib;Thomas J. Lynch, M.D.,等;《The NEW ENGLAND JOURNAL of MEDICINE》;20040520;第350卷(第21期);第2129-2139页 *
DNA的滚环扩增技术研究进展;王晓亮等;《食品工业科技》;20120511;第33卷(第16期);第358页摘要、第358页右栏最后一段至第360页右栏第2段 *
Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide;Chaesun Hong等;《Analytical chemistry》;20160223;第88卷(第6期);第2999-3004页 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12129516B2 (en) 2020-02-07 2024-10-29 10X Genomics, Inc. Quantitative and automated permeabilization performance evaluation for spatial transcriptomics

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