CN111850104B - Method and kit for detecting nuclear localization of number 1 exon of human CCDC6 gene - Google Patents
Method and kit for detecting nuclear localization of number 1 exon of human CCDC6 gene Download PDFInfo
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Abstract
The invention provides a method for the nuclear localization of a No. 1 exon of a human CCDC6 gene and a kit thereof, which comprise a permeation treatment system, a blunt end treatment system, a target nucleic acid exposure treatment system, a probe locking treatment system, a signal amplification treatment system, a signal detection treatment system and a washing liquor treatment system. The method provided by the invention can be used for detecting a small amount of cells or samples without nucleic acid extraction, can observe the location and copy number of the No. 1 exon of the CCDC6 gene in cells or clinical tissue samples by amplifying target signals through the specific probe, and can be widely used for detecting the location mutation in the nucleus of the No. 1 exon of the human CCDC6 gene in solid tumors.
Description
Technical Field
The invention belongs to the field of molecular biology and oncology, relates to nucleic acid detection, and particularly relates to detection of nuclear localization of a No. 1 exon of a human CCDC6 gene.
Background
In recent years, with the rapid progress of molecular pathological diagnosis. The diagnosis and treatment of malignant tumor are obviously improved. The occurrence and development of malignant tumors are often caused by the inactivation of cancer suppressor genes and the activation of proto-oncogenes. The CCDC6 gene is a protooncogene, and the excessive activation of the expressed protein encoded by the protooncogene is considered as a driving factor of many malignant tumors. The CCDC6 gene has multiple variation modes in cancer cells, from point mutation to amplification to rearrangement. Expression of the fusion gene after the rearrangement of CCDC6 was detected in 12% of thyroid cancers, with exon1 of the CCDC6 gene and exon 12 of the RET gene on one mRNA.
Traditional detection methods require extraction of RNA from tumor tissue, reverse transcription followed by PCR product sequencing or second generation high throughput sequencing. None of these methods allows the mapping of exon1 of the CCDC6 gene in the cell nucleus.
Disclosure of Invention
The invention aims to provide a method for the nuclear localization of the No. 1 exon of the human CCDC6 gene. The method has the characteristics of less sample quantity, no need of RNA extraction, low cost, high sensitivity, good specificity, and simple and convenient operation.
The invention provides a method for detecting nuclear localization of a No. 1 Exon of a human CCDC6 gene, which comprises a permeation treatment system, a blunt end treatment system, a target nucleic acid exposure treatment system, a probe locking treatment system, a signal amplification treatment system, a signal detection treatment system and a washing liquor treatment system, wherein the probe locking treatment system comprises a CCDC6Exon1 probe and DNA ligase. The CCDC6Exon1 probe and DNA ligase are added into the reaction system at the same time, and the circular connection of the CCDC6Exon1 probe occurs while the CCDC6Exon1 probe is combined with the genome DNA.
Preferably, the CCDC6Exon1 probe sequence comprises a fluorescent probe sequence and a genome DNA binding sequence, wherein the genome DNA binding sequence is distributed at the 5 'end and the 3' end of the CCDC6Exon1 probe sequence, and the CCDC6Exon1 probe 5 'end genome DNA binding sequence and the CCDC6Exon1 probe 3' end genome DNA binding sequence respectively comprise 10-20 bases, preferably 10bp, 11112bp, 13bp,14bp,15bp, 111111bp, 17bp,18bp,19bp and 20bp; the difference between the annealing temperature of the CCDC6Exon1 probe 5 'end genome DNA binding sequence and the annealing temperature of the CCDC6Exon1 probe 3' end genome DNA binding sequence in a ligase reaction system is 3-15 ℃, preferably 3.1 ℃,10.1 ℃ and 14.3 ℃. And the Tm value of the 5' end is more than or equal to 48 ℃.
Preferably in any of the above, the method for detecting the nuclear localization of the number 1 exon of human CCDC6 gene comprises the following steps:
1) Fixing the sample, and permeating cells through a permeation treatment system;
2) Treating the cell genomic DNA with a blunt end treatment system to expose blunt ends;
3) Obtaining a genome single-stranded DNA by using a target nucleic acid exposure treatment system;
4) Using a probe locking treatment system to combine the CCDC6Exon1 probe with the single-stranded DNA in the previous step to form circular DNA (the invention is called circular probe DNA);
5) The circular probe DNA is self-replicated by using a signal amplification processing system to generate single-stranded DNA containing a repetitive sequence;
6) Binding a fluorescent probe on the single-stranded DNA containing the repetitive sequence by using a signal detection processing system;
7) Sealing;
8) And observing and recording results.
More specific preferred steps consist of:
the method comprises the following steps:
(1) After the sample to be detected is fixed, a permeable treatment system is used for permeating cells and cleaning.
(2) The cellular genomic DNA was treated using a blunt end treatment system, blunt ends were exposed, and washed.
(3) Using the target nucleic acid exposure treatment system, degrading the single-stranded DNA in the 5 '-3' direction from the blunt end, retaining the other genome single-stranded DNA, and washing.
(4) Using a probe locking treatment system, combining a CCDC6Exon1 probe on the single-stranded DNA of the genome in the previous step and forming a circular DNA (the invention is called as the circular probe DNA), cleaning, dehydrating by gradient ethanol, and airing.
(5) The circular probe DNA is self-replicated by polymerase using the genomic single-stranded DNA binding site as the origin by using a signal amplification system to generate a single-stranded DNA containing a repeat sequence of about 70 to 100kb, and the resulting DNA is washed.
(6) And (3) binding a fluorescent probe on the single-stranded DNA containing the repetitive sequence by using a signal detection processing system, and washing.
(7) Adding a sealing agent containing DAPI for sealing.
(8) The recorded results were finally observed under a fluorescence microscope.
In any of the above, the genomic DNA binding sequence of the CCDC6Exon1 probe is preferably 15 nucleotides at the 5 'end and 15 nucleotides at the 3' end of the nucleotide sequence of Seq ID NO:1, or 13 nucleotides at the 5 'end and 16 nucleotides at the 3' end of the nucleotide sequence of Seq ID NO:2, or 15 nucleotides at the 5 'end and 16 nucleotides at the 3' end of the nucleotide sequence of Seq ID NO:3, or 15 nucleotides at the 5 'end and 17 nucleotides at the 3' end of the nucleotide sequence of Seq ID NO:4, or 19 nucleotides at the 5 'end and 11 nucleotides at the 3' end of the nucleotide sequence of Seq ID NO:5, or 14 nucleotides at the 5 'end and 17 nucleotides at the 3' end of the nucleotide sequence of Seq ID NO: 6.
Preferably, in any of the above, the number of fluorescent probe sequences of the CCDC6Exon1 probe is at least 2. It is further preferable that two adjacent fluorescent probe sequences on the CCDC6Exon1 probe are connected by a DNA linker, and it is further preferable that the DNA linker is TCTT four bases. The method has the advantages that on the premise of ensuring that the CCDC6Exon1 probe can be effectively cyclized, the combination of the fluorescent probe and the obtained repetitive sequence is enhanced, the intensity of a fluorescent signal is improved, an enough physical space is reserved for a fluorophore carried and connected at the 5' end of the fluorescent probe, and the efficiency of fluorescence detection is ensured. The CCDC6Exon1 probe comprises two or more fluorescent probe sequences, so that steric hindrance caused by combination of fluorescent groups can be generated in detection, and reaction signals are reduced. The probes provided by the present application overcome the above-mentioned deficiencies, overcome steric hindrance of fluorescent signal binding, and enhance the fluorescent signal.
In any of the above, preferably, the CCDC6Exon1 probe is a nucleotide sequence as defined in any of Seq ID NO 1, seq ID NO 2, seq ID NO 3, seq ID NO 4, seq ID NO 5, seq ID NO 6 or Seq ID NO 7.
Preferably, in any of the above, the fluorescent probe is the nucleotide sequence of Seq ID NO 8.
Preferably in any of the above, the blunt end processing system comprises a restriction enzyme. Preferably, the blunt end treatment system consists of a solution containing CutSmart buffer, a restriction enzyme and nuclease-free ultrapure water, and the preferred restriction enzyme comprises at least one of FspI enzyme, cac8I enzyme or CdiI enzyme.
Preferably in any of the above, the nucleic acid exposure treatment system of interest comprises an exonuclease. The target nucleic acid exposure treatment system specifically comprises Exinuclease buffer, exonuclease and ultrapure water without the Exonuclease, wherein the Exonuclease is preferably Lambda Exonuclease; the probe locking treatment system specifically comprises a DNA Ligase buffer, ATP, a CCDC6Exon1 probe, DNA Ligase and nuclease-free ultrapure water; the signal amplification processing system specifically comprises a DNA polymerase buffer, DTT, dNTPs, DNA polymerase and nuclease-free ultrapure water; the signal detection processing system specifically comprises formamide, sodium chloride, sodium citrate, salmon sperm DNA, a fluorescent probe and nuclease-free ultrapure water; the cleaning treatment system specifically comprises: tris-HCl, naCl, tween20 and nuclease-free ultrapure water. The permeation treatment system specifically comprises: tris-HCl, EDTA, SDS and proteinase K.
The invention also provides a detection kit for detecting the number 1 exon of the human CCDC6 gene by the method for detecting the nuclear localization of the number 1 exon of the human CCDC6 gene.
The invention also provides a method for identifying tumor cells, which detects the number 1 exon of the CCDC6 gene of cells by any one of the methods for detecting the nuclear localization of the number 1 exon of the human CCDC6 gene.
The working principle of the method for detecting the nuclear localization of the No. 1 exon of the human CCDC6 gene is as follows: CCDC6 is a coding gene located on human chromosome 10, q 11.21. Expression of the post-rearrangement fusion gene of the CCDC6 gene was detected in tissue samples of about 12% of thyroid cancer patients and 0.4% of lung cancer patients, and exon1 of the CCDC6 gene was detected on the mRNA thereof. According to the method, firstly, a nuclear membrane is perforated by using proteinase K, and then, the genome DNA is cut near a genome DNA target point combined with a CCDC6Exon1 probe sequence through two types of restriction endonucleases, so that a blunt end is exposed. Immediately after the digestion with exonuclease, one strand at the 5 '-3' end of the double-stranded DNA is degraded from the blunt end. To this end, the target genomic single-stranded DNA to which the CCDC6Exon1 probe was bound was exposed. Finally, the CCDC6Exon1 probe is cyclized at a binding site under the action of ligase to form closed circular single-stranded DNA (the circular single-stranded DNA of the invention is the circular probe DNA of the invention), the DNA is linearly self-replicated under the action of polymerase, a large number of DNA repetitive sequences which are not contained in human genes exist in the generated sequence, and a specific fluorescent probe is combined with the repetitive sequences to show the positioning of the CCDC6Exon1 probe in a cell nucleus. Thus, the location of exon1 of the CCDC6 gene in the nucleus is detected.
In the specific embodiment of the invention, the operation steps are as follows:
(1) After the sample to be detected is fixed, a permeable treatment system is used for permeating cells and cleaning.
(2) The blunt end treatment system was used to treat cell genomic DNA, expose blunt ends, and wash.
(3) Using the target nucleic acid exposure system, degrading the single-stranded DNA in the 5 '-3' direction of the double-stranded DNA from the blunt end, retaining the other single-stranded genomic DNA, and washing.
(4) Using a probe locking treatment system, a CCDC6Exon1 probe is combined with a target nucleic acid, simultaneously ligase is used for cyclizing the target nucleic acid, the target nucleic acid is washed and dehydrated and dried by using 70%,85% and 100% ethanol-water solution in a gradient manner.
(5) Using a signal amplification system, the circularized probe self-replicates with the single-stranded genomic DNA binding site as the origin under the action of polymerase to produce a large amount of single-stranded DNA containing a repetitive sequence, and washing.
(6) Using a signal detection processing system, combining a fluorescent probe on a single-stranded DNA containing a repetitive sequence, cleaning, and dehydrating and airing by using a 70%,85% and 100% ethanol-water solution gradient.
(7) A tableting agent is used.
(8) The recorded results were finally observed under a fluorescence microscope.
The blunt end processing system comprises 10 x CutSmart buffer, a type II endonuclease selected from FspI enzyme, cac8I enzyme or CdII enzyme and ultrapure water without an endonuclease. The method for detecting the nuclear localization of the human CCDC6 gene Exon1 can be used for gene fusion detection related to the Exon1 of the tumor cell CCDC6 gene, and for the CCDC6Exon1 gene, the preferred endonuclease FspI enzyme, cac8I enzyme or Cdii enzyme can form a flat end close to the 3' end of the CCDC6Exon1 gene, degrade single-stranded DNA in the direction of the double-stranded DNA 5' -3 ' from the flat end, and reserve another single-stranded genome DNA, and can reserve the sequence of the gene fused with the Exon1 of the CCDC6 gene, thereby providing possibility for further detection. The further detection can be the detection of other exons of the CCDC6 gene, and can also be the fusion of other genes caused by gene rearrangement. The method can be the same as or similar to the method for detecting the exon1 of the CCDC6 gene in situ, namely, the specific probe is combined with the target single-stranded genome DNA, signals are amplified through the connection and cyclization of the probe and polymerase reaction, and finally the signals are detected by the fluorescent probe; other in situ or ex situ detection methods are also possible as is known in the art. However, whether the type of the gene or the detection method for subsequent detection or whether the subsequent detection is performed, the detection of the exon1 of the human CCDC6 gene by using the method for detecting the nuclear localization of the exon1 of the human CCDC6 gene and the kit thereof provided by the application falls into the protection scope of the present invention. The target nucleic acid exposure treatment system specifically comprises 10 XExonuclease buffer and Exonuclease, wherein the Exonuclease is preferably Lambda Exonuclease; the probe lock treatment system specifically comprises 10 XDNA Ligase buffer, 100mM ATP, 50% PEG, 100uM CCDC6Exon1 probe, DNA Ligase and nuclease-free ultrapure water; the signal amplification processing system specifically comprises 10 multiplied by DNA polymerase buffer, 100mM DTT, 10mM dNTPs, DNA polymerase and nuclease-free ultrapure water; the signal detection processing system specifically comprises 20% formamide, 300mM sodium chloride, 30mM sodium citrate, 0.5ug/uL salmon sperm DNA, 100uM fluorescent probe and nuclease-free ultrapure water; the cleaning treatment system specifically comprises: tris-HCl, naCl, tween20 and nuclease-free ultrapure water. The permeation treatment system specifically comprises: tris-HCl, EDTA, SDS and proteinase K.
The invention has the advantages and positive effects that:
1. the method provided by the invention does not need nucleic acid extraction. Thus, detection can be performed with a small amount of cells or clinical tissue samples.
2. The method provided by the invention amplifies a target signal through a specific probe. Thus, the location and copy number of exon1 of the CCDC6 gene in cells or clinical tissue samples can be observed.
3. The method provided by the invention can be used for detecting the nuclear localization mutation of the No. 1 exon of the human CCDC6 gene in all solid tumors. Therefore, the method has the advantage of wide applicability.
Drawings
FIG. 1 is a schematic diagram of the method for detecting the nuclear localization of the exon1 of human CCDC6 gene of the present invention.
FIG. 2 is a preferred embodiment of the method for detecting nuclear localization of exon1 of human CCDC6 gene of the present invention, and 2 is the fluorescence detection result of KTC cells
FIG. 3 shows the fluorescence detection result of TPC-1 cells in the preferred embodiment of the method for detecting the nuclear localization of exon1 of human CCDC6 gene of the present invention
FIG. 4 shows the result of detecting the nuclear localization of exon1 of human CCDC6 gene in KTC tumorigenic paraffin tissue section sample in the preferred embodiment of the method of the present invention.
FIG. 5 shows the result of the preferred embodiment 3 of the method for detecting the nuclear localization of the exon1 of human CCDC6 gene in the TPC-1 paraffin tissue section sample.
FIG. 6 shows the method for detecting the nuclear localization of exon1 of human CCDC6 gene in the preferred embodiment of the present invention 5 Probe 1 detects KTC cells
FIG. 7 shows results of KTC cell detection by Probe 2 in accordance with preferred embodiment of the present invention for detecting the nuclear localization of exon1 of human CCDC6 gene, FIG. 8 shows results of KTC cell detection by Probe 3 in accordance with preferred embodiment of the present invention for detecting the nuclear localization of exon1 of human CCDC6 gene, FIG. 5
FIG. 9 shows the method for detecting the nuclear localization of exon1 of human CCDC6 gene in accordance with the present invention, preferred embodiment 5 is the result of detecting KTC cells by probe 4
FIG. 10 shows the result of detecting the nuclear localization of exon1 of human CCDC6 gene in the preferred embodiment of the present invention 5 Probe 5 for KTC cells
FIG. 11 shows the result of detecting nuclear localization of exon1 of human CCDC6 gene in the preferred embodiment of the present invention 5 probe 6 for detecting KTC cells
FIG. 12 shows the result of detecting the nuclear localization of exon1 of human CCDC6 gene in the preferred embodiment of the present invention, probe 7 in the preferred embodiment of the present invention detects KTC cells
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
Example 1
Embodiment 1 provides a method for in-nucleus localization of exon1 of human CCDC6 gene, which comprises a transparent processing system, a blunt end processing system, a target nucleic acid exposure processing system, a probe locking processing system, a signal amplification processing system and a signal detection processing system.
As shown in FIG. 1, the principle of the method for the nuclear localization of the number 1 exon of human CCDC6 gene provided by the invention is shown as follows: the nuclear membrane of the cell is firstly perforated by proteinase K, and then the genome DNA is cut near the genome DNA target point combined with the CCDC6Exon1 probe sequence by two types of restriction endonucleases, so that the blunt end is exposed. Immediately after the degradation of one strand at the 5 '-3' end in the double-stranded DNA from the blunt end by the action of exonuclease. Thus, the target genomic single-stranded DNA to which the CCDC6Exon1 probe was bound was exposed. And finally, cyclizing the CCDC6Exon1 probe at the binding site under the action of ligase to form closed circular single-stranded DNA. The closed circular single-stranded DNA formed by the CCDC6Exon1 probe is linearly self-replicated under the action of polymerase, a large number of DNA repetitive sequences which are not existed on human genes are generated in the sequence, wherein the fluorescent probe sequence included in the CCDC6Exon1 probe forms a repetitive sequence which is complementary with the nucleotide shown in Seq ID NO:8 in the replication process, and the specific fluorescent probe is combined with the sequence which is complementary with the nucleotide shown in the large number of repetitive Seq ID NO:8 to show the positioning of the CCDC6Exon1 probe in cell nucleus. Thus, the location of exon1 of the CCDC6 gene in the nucleus was detected.
The permeation treatment system specifically comprises: tris-HCl, EDTA, SDS and proteinase K, wherein the concentration of the proteinase is 5 ug/mL-30 ug/mL. These reagents are all biochemical reagents commonly used in the art.
Moreover, the blunt end processing system specifically includes: 1 XCutSmart buffer, fspI enzyme and nuclease-free ultrapure water, wherein the formulation of the 1 XCutSmart buffer is 50mM/L potassium acetate, 20mM/L Tri-acetate, 10mM/L magnesium acetate, 0.1mg/mL BSA, and 0.5U/uL FspI enzyme. These reagents are all biochemical reagents commonly used in the art.
Moreover, the target nucleic acid exposure treatment system specifically includes: 1 XExonuclease buffer, lambda Exonuclease and ultrapure water without nuclease, wherein the formula of the 1 XExonuclease buffer is 50mM/L potassium acetate, 20mM/L Tri-acetate, 10mM/L magnesium acetate, 0.1mg/mL BSA and 0.4U/. Mu.L Lambda Exonuclease. These reagents are all biochemical reagents commonly used in the art.
Moreover, the probe lock processing system specifically includes: 1 XDNA Ligase buffer, 500nM/L ATP, T 4 DNA Ligase, CCDC6Exon1 probe and nuclease-free ultrapure water, wherein the 1 XDNA Ligase buffer formula is 40mM/L Tris-HCl, 10mM/L magnesium chloride, 10mM/L DTT, 0.5mM/L ATP, T 4 DNA ligase 0.05Weiss U/. Mu.L, CCDC6Exon1 probe sequence as shown in Seq ID NO:2, final concentration of 100. Mu.M/L. These reagents are all biochemical reagents commonly used in the art.
Moreover, the signal amplification processing system specifically includes: 1 XDNA polymerase buffer, 1mM/L DTT, 2.5mM/L dNTPs, DNA polymerase and nuclease-free ultrapure water, wherein the formula of the 1 XDNA polymerase buffer is 33mM/L Tris-acetate, 10mM/L magnesium acetate, 66mM/L potassium acetate, 0.1% (v/v) Tween20, phi29 DNA polymerase 1U/L. These reagents are all biochemical reagents commonly used in the art.
Moreover, the signal detection processing system specifically includes: 20% (v/v) formamide, 0.3M/L NaCl, 0.03M/L sodium citrate, 0.5ug/uL salmon sperm DNA, a fluorescent probe sequence and nuclease-free ultrapure water, wherein the fluorescent probe sequence is 5 'Cy3-X-3', and X is a nucleotide sequence shown in Seq ID NO: 8. The final concentration was 100. Mu.M/L. These reagents are all biochemical reagents commonly used in the art.
Moreover, the cleaning treatment system specifically includes: tris-HCl, naCl and Tween20, and nuclease-free ultrapure water, wherein the concentration of the Tris-HCl is 0.1M/L, the concentration of the NaCl is 0.15M/L, and the concentration of the Tween20 is 0.05% (v/v). These reagents are all biochemical reagents commonly used in the art.
The method for nuclear localization of the exon1 of the human CCDC6 gene described in example 1 comprises the following steps:
(1) After the sample to be detected is well fixed, a permeation treatment system is used for permeating cells. After the in vitro cells are fixed, the cells are treated for 3 to 4 minutes at the temperature of 37 ℃; clinical tissue samples were processed at 37 ℃ for 15 to 20 minutes. After the completion, the liquid is discarded, and the mixture is put into ultrapure water, and finally dehydrated and dried by 70 percent, 85 percent and 100 percent of ethanol aqueous solution.
(2) Blunt-end treatment system was used to expose blunt ends of genomic DNA. A reaction system was prepared by diluting 10 XCutSmart buffer with nuclease-free ultrapure water to 1X, the final concentration of FspI enzyme was 0.5U/. Mu.L, and treating at 37 ℃ for 1 hour. And after the completion, discarding the liquid, cleaning with a cleaning solution, and then discarding the cleaning solution.
(3) Using the target nucleic acid exposure system, the single-stranded DNA is degraded from the blunt end in the 5 '-3' direction, and the other genomic single-stranded DNA is retained. An Exonuclease reaction system was prepared, 10 XExonuclease buffer was diluted to 1X with nuclease-free ultrapure water, the final concentration of Lambda Exonuclease was 0.4U/. Mu.L, and the mixture was treated at 37 ℃ for 0.5 hour. After completion, the liquid is discarded, the cleaning liquid is used for cleaning, and then the cleaning liquid is discarded.
(4) Using a probe lock treatment system, a CCDC6Exon1 probe was used to form circular DNA. A probe lock treatment system was prepared by diluting 10 XDNA Ligase buffer with nuclease-free ultrapure water to 1X, 10mM/L ATP to 0.5mM/L, T 4 The final concentration of DNA ligase was 0.05Weiss U/. Mu.L and the treatment was carried out at 37 ℃ for 0.5 hour. And (3) after the completion, removing the liquid, cleaning with a cleaning solution, removing the cleaning solution, and finally dehydrating and airing 70%,85% and 100% ethanol aqueous solution.
(5) Using a signal amplification system, the circular probe DNA is self-replicated by polymerase from the site of single-stranded genomic DNA binding to generate a single-stranded DNA having a large number of repetitive sequences. A signal amplification system was prepared by diluting 10 XDNA polymerase buffer with nuclease-free ultrapure water to 1X, 100mM/L DTT to 1mM/L,10mM/L dNTPs to 0.25mM/L, and DNA polymerase to a final concentration of 1U/. Mu.L, and treating at 44 ℃ for 1 hour. After completion, the liquid is discarded, the cleaning liquid is used for cleaning, and then the cleaning liquid is discarded.
(6) Using a signal detection processing system, fluorescent probes are bound to a single-stranded DNA containing a repetitive sequence. Preparing a probe combination treatment system, wherein the final concentration of formamide is 20% (v/v), the final concentration of NaCl is 0.3M/L, the final concentration of sodium citrate is 0.03M/L, 10 mu g/uL salmon sperm DNA is diluted to 0.5 mu g/mu L, the final concentration of a fluorescent probe is 100 mu M/L, and the treatment is carried out at 37 ℃ for 10 minutes. And (3) after the completion, removing the liquid, cleaning with a cleaning solution, removing the cleaning solution, and finally dehydrating and airing 70%,85% and 100% ethanol aqueous solution.
(7) Adding a sealing agent containing DAPI, and sealing.
(8) The recorded results were finally observed under a fluorescence microscope.
Example 2
To better understand the usage and effect of the kit of the present invention, example 2 provides a kit for detecting the nuclear localization of exon1 of human CCDC6 gene, and the detection is performed by using the method for detecting the nuclear localization of exon1 of human CCDC6 gene provided in example 1. The CCDC6Exon1 probe in example 2 was the nucleotide sequence described in Seq ID NO:2 (Probe 2).
Example 2 KTC and TPC-1 in vitro cell lines were tested.
Thyroid cancer cell lines, KTC and TPC-1 cell lines, were obtained from ATCC in the USA. Respectively inoculating two kinds of cells on a diagnosis glass slide, fixing the cells for 10 minutes by using 4 percent PFA after the cells adhere to the wall, then treating the cells in ultrapure water for 3 minutes, taking out the sample, drying the sample in the air, dropwise adding 20 mu L of permeation treatment reaction liquid, treating the sample in a wet box for 3 minutes at 37 ℃, and placing the sample in a washing liquid cylinder for washing once; adding 15 mu L of blunt end treatment reaction liquid, treating for 1 hour at 37 ℃ in a wet box, and putting into a washing liquid cylinder for washing once; adding 15 mu L of target nucleic acid exposure treatment reaction liquid, treating for 0.5 hour at 37 ℃ in a wet box, and putting into a washing liquid cylinder for washing once; adding 15 mu L of probe locking treatment reaction liquid, treating for 0.5 hour at 37 ℃ in a wet box, putting into a washing liquid cylinder for washing once, and performing gradient treatment and dehydration by using ethanol; after air drying, adding 15 mu L of signal amplification treatment reaction liquid, treating for 1 hour at 44 ℃ in a wet box, and putting into a washing liquid cylinder for washing once; adding 15 μ L of reaction solution for signal detection, processing in a wet box at 37 deg.C in dark for 15 min, washing in a washing liquid cylinder, and dehydrating by ethanol gradient; after drying, adding a proper amount of tabletting agent, and sealing; and (5) observing the result under a fluorescence microscope. Blue nuclei under the microscope were used as localization, and green fluorescence spots were used as positive results, as shown in FIGS. 2 and 3.
Example 3
Example 3 similar to example 2, except that the detection kit for nuclear localization of exon1 of human CCDC6 gene was used to detect paraffin tissue section samples. The CCDC6Exon1 probe in example 3 is the nucleotide sequence described in Seq ID NO:2 (Probe 2).
After TKC and TPC-1 cells are inoculated on the back of a nude mouse to form a tumor, the tumor is taken out and embedded into a paraffin sample, a paraffin tissue slice is cut into 5 mu m thick, 50 mu L of transparent treatment reaction liquid is dripped after pretreatment, the solution is treated for 15 minutes in a wet box at 37 ℃, and the solution is placed in a washing liquid cylinder to be washed once; adding 50 mu L of blunt end treatment reaction liquid, treating for 1 hour at 37 ℃ in a wet box, and putting into a washing liquid cylinder for washing once; adding 50 mu L of target nucleic acid exposure treatment reaction liquid, treating for 0.5 hour at 37 ℃ in a wet box, and putting into a washing liquid cylinder for washing once; adding 50 mu L of probe locking treatment reaction liquid, treating for 0.5 hour at 37 ℃ in a wet box, putting into a washing liquid cylinder for washing once, and performing gradient treatment on ethanol for dehydration; after air drying, adding 50 mu L of signal amplification treatment reaction liquid, treating for 2 hours at 44 ℃ in a wet box, and putting into a washing liquid cylinder for washing once; adding 50 μ L of reaction solution for signal detection, processing in a wet box at 37 deg.C in dark for 10 min, washing in a washing liquid cylinder, and dehydrating by ethanol gradient; after drying, adding a proper amount of tabletting agent, and sealing; and (5) observing the result under a fluorescence microscope. Blue nuclei under the microscope were used as localization and green fluorescence spots as positive results, as shown in FIGS. 4 and 5.
Example 4
Example 4 similar to examples 1 to 3 except that the CCDC6Exon1 probe sequence was the nucleotide sequence described in Seq ID No. 7 (probe 7) and the two types of restriction endonucleases used were the Cac8I enzymes. The number of fluorescent probe sequences in the CCDC6Exon1 probe sequence described in Seq ID No. 7 was 1, and the results are shown in FIG. 12.
Example 5
The method of example 5 is similar to examples 1 to 4 except that the CCDC6Exon1 probe sequence is selected from the nucleotide sequences described in Seq ID NO:1 (Probe 1), seq ID NO:3 (Probe 3), seq ID NO:4 (Probe 4), seq ID NO:5 (Probe 5), and Seq ID NO:6 (Probe 6). Two types of restriction endonucleases employed are the FspI enzyme, the Cac8I enzyme or the CdII enzyme. The results are shown in FIGS. 6, 8, 9, 10 and 11.
As shown in the table I, the basic information of each CCDC6Exon1 probe is shown, and the Tm value is calculated under the condition of the ligase reaction system.
FIGS. 6 to 12 are comparisons of the results of detection of 7 probes according to the preferred embodiment of the present invention, and it can be seen that although the probes 1 to 7 are all capable of detecting fluorescence signals, the change of the genomic DNA binding sequence (base number, recognition site, GC content) on the CCDC6Exon1 probe causes a difference in the detection efficiency and fluorescence intensity of the CCDC6Exon1 probe. The number of fluorescent probe sequences contained in the CCDC6Exon1 probe also affects the detection efficiency of the probe. The 7 probes provided by the invention can effectively detect fluorescent signals through screening among a plurality of probes, and further preferably the difference between the annealing temperature of the CCDC6Exon1 probe 5' end genome DNA binding sequence and the annealing temperature of the CCDC6Exon1 probe 3' end genome DNA binding sequence in a ligase reaction system is 3-15 ℃, and the Tm value of the 5' end is more than or equal to 48 ℃, so that the invention has good detection results.
Sequence listing
<110> Pidelog Biotechnology Ltd
<120> method for detecting human CCDC6 gene No. 1 exon nuclear localization and kit thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cctcctgcag tgcctccctc gcatcaatac cgatcatccc ctcgcatcaa taccgatcat 60
cgcaggtcgc ggttct 76
<210> 2
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctcctgcagt gccccctcgc atcaataccg atcattcttc ccctcgcatc aataccgatc 60
atcgcaggtc gcggttctc 79
<210> 3
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctcctgcagt gccttccctc gcatcaatac cgatcattct tcccctcgca tcaataccga 60
tcatcgcagg tcgcggttct c 81
<210> 4
<211> 82
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcctgcagtg ccttgccctc gcatcaatac cgatcattct tcccctcgca tcaataccga 60
tcatcgcagg tcgcggttct cc 82
<210> 5
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttctcctcct gcagtgcctc cctcgcatca ataccgatca ttcttcccct cgcatcaata 60
ccgatcatcg caggtcgcgg 80
<210> 6
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcctgcagtg ccttccctcg catcaatacc gatcattctt cccctcgcat caataccgat 60
catcgcaggt cgcggttctc c 81
<210> 7
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctcctgcagt gccttccttt tacgaccctc gcatcaatac cgatcatctc ttcgcaggtc 60
gcggttctc 69
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccctcgcatc aataccgatc atc 23
Claims (5)
1. A kit for detecting the nuclear localization of the No. 1 Exon of a human CCDC6 gene comprises a permeation treatment system, a blunt end treatment system, a target nucleic acid exposure treatment system, a probe locking treatment system, a signal amplification treatment system, a signal detection treatment system and a washing liquor treatment system, and is characterized in that the probe locking treatment system comprises a CCDC6Exon1 probe and DNA ligase, the CCDC6Exon1 probe sequence comprises a fluorescent probe sequence and a genome DNA binding sequence, and the CCDC6Exon1 probe is a nucleotide sequence as described in any one of Seq ID NO 1, seq ID NO 2, seq ID NO 3, seq ID NO 4, seq ID NO 5, seq ID NO 6 or Seq ID NO 7; the blunt end processing system comprises a restriction enzyme which enables a blunt end to be formed near the 3' end of the CCDC6Exon1 gene, and the restriction enzyme cuts the genomic DNA near the genomic DNA target point combined by the CCDC6Exon1 probe sequence.
2. The kit for detecting nuclear localization of exon1 of human CCDC6 gene according to claim 1, comprising the steps of:
1) Fixing the sample, and permeating cells through a permeation treatment system;
2) Treating the cell genomic DNA with a blunt end treatment system to expose blunt ends;
3) Obtaining a genome single-stranded DNA by using a target nucleic acid exposure treatment system;
4) Combining a CCDC6Exon1 probe with the single-stranded DNA in the previous step to form circular DNA by using a probe locking treatment system;
5) Using a signal amplification processing system, the circular probe DNA is replicated automatically to generate a single-stranded DNA containing a repetitive sequence;
6) Using a signal detection processing system, and combining a fluorescent probe on the single-stranded DNA containing the repetitive sequence;
7) Sealing;
8) And observing and recording results.
3. The kit for detecting the nuclear localization of the exon1 of the human CCDC6 gene according to claim 2, wherein the fluorescent probe is the nucleotide sequence of Seq ID NO 8.
4. The kit for detecting nuclear localization of exon1 of human CCDC6 gene according to claim 3, wherein the target nucleic acid exposure treatment system comprises exonuclease.
5. The kit for detecting the nuclear localization of the exon1 of the human CCDC6 gene of claim 3, wherein the endonuclease is selected from at least one of FspI enzyme, cac8I enzyme or CdII enzyme.
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| CN202010828465.1A CN111850104B (en) | 2020-08-18 | 2020-08-18 | Method and kit for detecting nuclear localization of number 1 exon of human CCDC6 gene |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652506A (en) * | 2018-12-20 | 2019-04-19 | 派德洛格(天津)生物科技有限公司 | Fluorescent in situ detects the method and kit of 21 exon p.L858R of Human epidermal growth factor receptor gene mutation |
| CN109652505A (en) * | 2018-12-20 | 2019-04-19 | 派德洛格(天津)生物科技有限公司 | Fluorescent in situ detects the method and kit of 20 exon p.T790M of Human epidermal growth factor receptor gene mutation |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652506A (en) * | 2018-12-20 | 2019-04-19 | 派德洛格(天津)生物科技有限公司 | Fluorescent in situ detects the method and kit of 21 exon p.L858R of Human epidermal growth factor receptor gene mutation |
| CN109652505A (en) * | 2018-12-20 | 2019-04-19 | 派德洛格(天津)生物科技有限公司 | Fluorescent in situ detects the method and kit of 20 exon p.T790M of Human epidermal growth factor receptor gene mutation |
Non-Patent Citations (1)
| Title |
|---|
| Tereza Halkova MSc等.A novel RET/PTC variant detected in a pediatric patient with papillary thyroid cancer without ionization history.《Human Pathology》.2015,第46卷 * |
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