CN107385016A - A kind of molecular beacon probe and kit for detecting KRAS gene mutation - Google Patents

A kind of molecular beacon probe and kit for detecting KRAS gene mutation Download PDF

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CN107385016A
CN107385016A CN201710449386.8A CN201710449386A CN107385016A CN 107385016 A CN107385016 A CN 107385016A CN 201710449386 A CN201710449386 A CN 201710449386A CN 107385016 A CN107385016 A CN 107385016A
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probe
molecular beacon
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kras gene
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孙喜元
王季武
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Cheng Mei Bio Tech Ltd Suzhou
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Abstract

A kind of kit the invention discloses molecular beacon probe and containing the molecular beacon probe, the molecular beacon probe include tetra- kinds of Probe K34, Probe K35, Probe K38 and Probe K183 probes, and their base sequence is respectively：Probe K34：5'‑CCCTACCCTAGTTGGAGCThGTGGCGTAGGCA GGGTAGGG‑3'；Probe K35：5'‑CCCTACCCTAGTTGG AGCTGhTGGCGTAGGCAGGGTAGGG‑3'；Probe K38:5'‑CCCTACCCTTGGAGCTGGTGaCGTAGGCAAGAGG GTAGGG‑3'；ProbeK183:5'‑CCCTACCCACAGC AGGTCAcGAGGAGTACAGTGGGTAGGG‑3'；The 5' ends of every kind of probe are marked with fluorophor, and 3' ends are marked with quenching group.Molecular beacon probe and kit of the present invention can detect to KRAS gene mutation, and detection sensitivity is high, accuracy in detection is high.

Description

A kind of molecular beacon probe and kit for detecting KRAS gene mutation
Technical field
The present invention relates to the biology field of detection KRAS gene mutation, and in particular to one kind detection KRAS genes are dashed forward The molecular beacon probe and kit of change.
Background technology
KRAS genes are oncogenes, the point mutation of KRAS genes be concentrated mainly on specific amino acid codes (the 12nd, 13rd, 61 codon) on, account for more than the 90% of all mutation.KRAS gene mutation can cause cell evasion apoptosis, this in pancreas Incidence in gland cancer, colorectal cancer, lung cancer is higher.In cancer of pancreas.KRAS point mutation incidences are up to more than 90%. KRAS gene mutation detection is significant to early diagnosis of tumor, tumor individual therapy etc..At present, KRAS gene mutation Detection method mainly have gene sequencing method, and the detection method based on quantitative fluorescent PCR.The detection of gene sequencing method is sensitive Spend not high enough, the detection method accuracy based on quantitative fluorescent PCR is not high enough.
The content of the invention
The technical problem to be solved in the present invention is the defects of overcoming prior art, there is provided a kind of detection KRAS gene mutation Molecular beacon probe and kit, molecular beacon probe of the invention can be direct by fluorescence microscope by hybridization reaction The fluorescence signal of crossbred is observed, to judge to whether there is the mutant of KRAS genes into the cell.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
A kind of molecular beacon probe, the molecular beacon probe include Probe K34, Probe K35, Probe K38 and Tetra- kinds of probes of Probe K183, their base sequence are respectively：
Probe K34：
5'-CCCTACCCTAGTTGGAGCThGTGGCGTAGGCAGGGTAGGG-3'；
Probe K35：
5'-CCCTACCCTAGTTGGAGCTGhTGGCGTAGGCAGGGTAGGG-3'；
Probe K38：
5'-CCCTACCCTTGGAGCTGGTGaCGTAGGCAAGAGGGTAGGG-3'；
Probe K183：
5'-CCCTACCCACAGCAGGTCAcGAGGAGTACAGTGGGTAGGG-3'；
The 5' ends of every kind of probe are marked with fluorophor, and 3' ends are marked with quenching group.
Preferably, the fluorophor is selected from：6-FAM、Fluorescein、JOE、TET、HEX、Cyanine3、ROX、 Texas Red, Cyanine 5 or Cyanine 5.5, the quenching group are selected from：BHQ-1, BHQ-2, BHQ-2 or DABCYL.
It is further preferred that when 5' ends are labeled as 6-FAM (or FAM), when 3' ends are marked with BHQ-1 or DABCYL, fluorescence The excitation wavelength of group is 494nm, launch wavelength 515nm.When 5' ends are labeled as Fluorescein, 3' ends are marked with BHQ-1 Or during DABCYL, the excitation wavelength of fluorophor is 495nm, launch wavelength 520nm.When 5' ends are labeled as JOE, 3' ends mark When having BHQ-1 or DABCYL, the excitation wavelength of fluorophor is 520nm, launch wavelength 548nm.When 5' ends are labeled as TET, When 3' ends are marked with BHQ-1 or DABCYL, the excitation wavelength of fluorophor is 521nm, launch wavelength 536nm.When 5' ends are marked HEX is designated as, when 3' ends are marked with BHQ-1 or DABCYL, the excitation wavelength of fluorophor is 535nm, launch wavelength 555nm. When 5' ends are labeled as Cyanine 3, when 3' ends are marked with BHQ-2 or DABCYL, the excitation wavelength of fluorophor is 550nm, hair The a length of 570nm of ejected wave.When 5' ends are labeled as ROX, when 3' ends are marked with BHQ-2 or DABCYL, the excitation wavelength of fluorophor is 573nm, launch wavelength 602nm.When 5' ends are marked with BHQ-2 or DABCYL labeled as Texas Red, 3' ends, fluorescent base The excitation wavelength of group is 583nm, launch wavelength 603nm.When 5' ends are labeled as Cyanine5,3' ends be marked with BHQ-3 or During DABCYL, the excitation wavelength of fluorophor is 751nm, launch wavelength 674nm.When 5' ends are labeled as Cyanine 5.5,3' When end is marked with BHQ-3 or DABCYL, the excitation wavelength of fluorophor is 675nm, launch wavelength 694nm.
Present invention also offers a kind of kit, the kit includes above-mentioned at least one molecular beacon probe.
Preferably, mentioned reagent box also includes hybridization solution, and hybridization solution described in per 100ml includes following components：25mL 50* Denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon sperm DNA, 10g dextran sulfate.
When being diluted with above-mentioned hybridization solution to molecular beacon probe, it is preferred that the concentration of the molecular beacon probe is 5pmol/ul。
The method that KRAS gene mutation detection is carried out using above-mentioned molecular beacon probe or/and kit is：
(1) according to the various mutant sequence engineers of KRAS genes and synthetic molecules beacon probe；
The base sequence of KRAS gene mutation body corresponding to above-mentioned four kinds of molecular beacon probes is respectively：
34G>T/A/C
ATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCT[G>T/A/C] GTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATT
35G>T/A/C
ATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTG[G>T/A/C] TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATT
38G>A
ATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G>A] CGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATT
183A>C
TTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTC [A>C]AGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGG
(2) probe is diluted to certain concentration with above-mentioned hybridization solution,
(3) tumor tissues frozen section, 3-8 μm of thickness are prepared；
(4) tumor tissues frozen section is sprawled and slide
(5) with 10% formalin (i.e. 1 part of formalin adds 9 parts of water to be formulated) fixer fixing organization in slide.
(6) molecular beacon probe through 95 DEG C of thermal denaturations is added dropwise to the position of the tumor tissues on slide, capping slide is simultaneously Closing, is then incubated 15 minutes at 42 DEG C；
(7) in fluorescence microscopy Microscopic observation result.
The beneficial effect that the present invention is reached：
Molecular beacon probe and kit of the present invention can detect to KRAS gene mutation, and detection sensitivity height, Accuracy in detection is high.
Embodiment
With reference to specific embodiment, the invention will be further described.Following examples are only used for clearly illustrating Technical scheme, and can not be limited the scope of the invention with this.
In DNA hybridization liquid used in following examples, DNA hybridization liquid described in per 100ml includes following components：25mL 50* Denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon sperm DNA, 10g dextran sulfate.
Embodiment 1
First according to the base sequence (34G of KRAS gene mutation body>T/A/C synthetic molecules beacon probe Probe) is designed K34, the base sequence of the probe are：5'FAM-CCCTACCCTAGTTGGAGCThGTGGCGTAGGCAGGGTAGGG-BHQ1 3'.The molecular beacon is that wherein that 5' ends mark is FAM, 3' ends mark by the distinguished sequence of the neck ring structure of base composition It is BHQ1, the excitation wavelength of fluorophor is 494nm, launch wavelength 515nm.According to the base sequence of KRAS gene mutation body (34G>T/A/C synthesizing fluorescently labeled oligonucleotide probe Probe K34c) are designed：5'-FAM- TAGTTGGAGCThGTGGCGTAGGCA-3'；
Common fluorescent labeled oligonucleotide probe experimental procedure:
(1) tumor tissue section is placed on slide with drying slide after (- 20 DEG C) fixations of ice acetone at room temperature.
(2) it is fluorescence labeling oligonucleotide probe is dilute with hybridization solution (2xSSC, 50% formamide, 10% dextran sulfate) Release to final concentration of 5pmol/ul, 75 DEG C of deformation 5min, the position of the tumor tissues on slide be added dropwise, is capped slide and closes, Then it is incubated 30~45 minutes at 50 DEG C；
(3) by slide successively with washing lotion A (2xSSC, 50% formamide), washing lotion B (10mM PBS pH8.0,0.1% Nonidet P-40) respectively cleaning 2 times.
(4) in fluorescence microscopy Microscopic observation result.Molecular beacon and common fluorescent labeled oligonucleotide probe experimental result Criterion identical be：More visual field observations and the cell to sending fluorescence in every visual field are carried out to sample under 20 times of object lens Counted and added up, by following criterion：
Sample feminine gender (-)：40 different visuals field of Continuous Observation, find no the cell for sending fluorescence；The sample positive (+)： 40 different visuals field of Continuous Observation, the cell number for sending fluorescence are more than 1/40 visuals field, carry out experimental result judgement.
Known 40, the KRAS gene mutation rectum cancer tissue sample that is not present now is taken KRAS genes (34G to be present with known>T) 40, the Colorectal Carcinoma sample of mutation, frozen section, thickness 3um are then respectively.Tumor tissues frozen section is sprawled With slide, with 10% formalin (i.e. 1 part of formalin adds 9 parts of water to be formulated) fixer fixing organization, with 2xSSC 65 DEG C develop a film, the molecular beacon probe Probe K34 through 95 DEG C of thermal denaturations are diluted to 5pmol/ul with 1xDNA hybridization solutions, be added dropwise The position of 20ul tumor tissues on slide, it is capped slide and closes, 42 DEG C is incubated 15 minutes.Directly seen under fluorescence microscope Examine and take a picture, count fluorescence developing cell.With fluorescence labeling oligonucleotide probe Probe K34c few core is marked by common fluorescent Thuja acid probe experimental procedure is tested.Judged by above-mentioned criterion, experimental result such as following table：
Key sample (goldstandard) Molecular beacon probe Fluorogenic oligonucleotide probe Sample number
+ + + 36
+ + - 4
- - - 37
- - + 3
Note：In above-mentioned list, it is known that sample (goldstandard)：It is known that KRAS gene mutation rectum cancer tissue sample 40 is not present Example is used as negative goldstandard, it is known that KRAS genes (34G be present>T) 40, the Colorectal Carcinoma sample of mutation is as positive gold Standard；“-”：Represent negative and KRAS gene mutation is not present；“+”：Represent positive and KRAS genes (34G be present>T) it is mutated.
Negative, positive as goldstandard using known rectum cancer tissue's sample, the positive rate of molecular beacon is (36+ 4)/(36+4) X100%=100%, the positive rate of fluorescence labeling oligonucleotide probe is 36/ (36+4) X100%= 90%；The false positive recall rate of molecular beacon is 0/ (37+3) X100%=0%, the false positive of fluorescence labeling oligonucleotide probe Recall rate is 3/ (37+3) X100%=7.5%.From experimental result, molecular beacon probe is visited better than fluorogenic oligonucleotides Pin.
Embodiment 2
First according to the base sequence (35G of KRAS gene mutation body>T/A/C synthetic molecules beacon probe Probe) is designed K35, the base sequence of the probe are：5'JOE-CCCTACCCTAGTTGGAGCTGhTGGCGTAGGCAGGGTAGGG-BHQ-1 3'；.The molecular beacon is that wherein that 5' ends mark is JOE, 3' ends mark by the distinguished sequence of the neck ring structure of base composition It is BHQ1, the excitation wavelength of fluorophor is 520nm, launch wavelength 548nm.According to the base sequence of KRAS gene mutation body (35G>T/A/C synthesizing fluorescently labeled oligonucleotide probe Probe K35c) are designed：5'-FAM- TAGTTGGAGCTGhTGGCGTAGGCA-3'；
Known 40, the KRAS gene mutation rectum cancer tissue sample that is not present now is taken KRAS genes (35G to be present with known>T/ A/C) 60, the Colorectal Carcinoma sample of mutation, then cooks frozen section, thickness 6um respectively.By tumor tissues frozen section Sprawl and slide, with 10% formalin (i.e. 1 part of formalin adds 9 parts of water to be formulated) fixer fixing organization, with 2xSSC 65 DEG C are developed a film, and the molecular beacon probe Probe K35 through 95 DEG C of thermal denaturations are diluted into 5pmol/ul with 1xDNA hybridization solutions, drop Add the position of 20ul tumor tissues on slide, be capped slide and close, 42 DEG C are incubated 15 minutes.Directly under fluorescence microscope Observe and take a picture, count fluorescence developing cell.
Entered with fluorescence labeling oligonucleotide probe Probe K35c by common fluorescent labeled oligonucleotide probe experimental procedure Row experiment.Judged by above-mentioned criterion, experimental result such as following table：
Key sample (goldstandard) Molecular beacon probe Fluorogenic oligonucleotide probe Sample number
+ + + 51
+ + - 9
- - - 35
- - + 5
Note：In above-mentioned list, it is known that sample (goldstandard)：It is known that KRAS gene mutation rectum cancer tissue sample 40 is not present Example is used as negative goldstandard, it is known that KRAS genes (35G be present>T/A/C) 60, the Colorectal Carcinoma sample of mutation is as sun Property goldstandard；“-”：Represent negative and KRAS gene mutation is not present；“+”：Represent positive and KRAS genes (35G be present>T/A/ C) it is mutated.
Negative, positive as goldstandard using known rectum cancer tissue's sample, the positive rate of molecular beacon is (51+ 9)/(51+9) X100%=100%, the positive rate of fluorescence labeling oligonucleotide probe is 51/ (51+9) X100%= 85%；The false positive recall rate of molecular beacon is 0/ (35+5) X100%=0%, the false positive of fluorescence labeling oligonucleotide probe Recall rate is 5/ (35+5) X100%=12.5%.From experimental result, molecular beacon probe is visited better than fluorogenic oligonucleotides Pin.
Embodiment 3
First according to the base sequence (38G of KRAS gene mutation body>A synthetic molecules beacon probe Probe K38) are designed, should The base sequence of probe is：5'FAM-CCCTACCCTTGGAGCTGGTGaCGTAGGCAAGAGGGTAGGG-DABCYL 3'；.Should Molecular beacon is that wherein that 5' ends mark is FAM, and 3' ends mark is by the distinguished sequence of the neck ring structure of base composition DABCYL, the excitation wavelength of fluorophor is 494nm, launch wavelength 515nm.According to the base sequence of KRAS gene mutation body (38G>A synthesizing fluorescently labeled oligonucleotide probe Probe K38c) are designed:5'-FAM- TTGGAGCTGGTGaCGTAGGCAAGA-3'；
Known 40, the KRAS gene mutation rectum cancer tissue sample that is not present now is taken KRAS genes (38G to be present with known>A) 40, the Colorectal Carcinoma sample of mutation, frozen section, thickness 3um are then respectively.Tumor tissues frozen section is sprawled With slide, with 10% formalin (i.e. 1 part of formalin adds 9 parts of water to be formulated) fixer fixing organization, with 2xSSC 65 DEG C develop a film, the molecular beacon probe Probe K38 through 95 DEG C of thermal denaturations are diluted to 5pmol/ul with 1xDNA hybridization solutions, be added dropwise The position of 20ul tumor tissues on slide, it is capped slide and closes, 42 DEG C is incubated 15 minutes.Directly seen under fluorescence microscope Examine and take a picture, count fluorescence developing cell.
Entered with fluorescence labeling oligonucleotide probe Probe K38c by common fluorescent labeled oligonucleotide probe experimental procedure Row experiment.Judged by above-mentioned criterion, experimental result such as following table：
Key sample (goldstandard) Molecular beacon probe Fluorogenic oligonucleotide probe Sample number
+ + + 34
+ + - 6
- - - 35
- - + 5
Note：In above-mentioned list, it is known that sample (goldstandard)：It is known that KRAS gene mutation rectum cancer tissue sample 40 is not present Example is used as negative goldstandard, it is known that KRAS genes (38G be present>A) 40, the Colorectal Carcinoma sample of mutation is as positive gold Standard；“-”：Represent negative and KRAS gene mutation is not present；“+”：Represent positive and KRAS genes (38G be present>A) it is mutated.
Negative, positive as goldstandard using known rectum cancer tissue's sample, the positive rate of molecular beacon is (34+ 6)/(34+6) X100%=100%, the positive rate of fluorescence labeling oligonucleotide probe is 34/ (34+6) X100%= 85%；The false positive recall rate of molecular beacon is 0/ (35+5) X100%=0%, the false positive of fluorescence labeling oligonucleotide probe Recall rate is 5/ (35+5) X100%=12.5%.From experimental result, molecular beacon probe is visited better than fluorogenic oligonucleotides Pin.
Embodiment 4
First according to the base sequence (183A of KRAS gene mutation body>C synthetic molecules beacon probe Probe K183) are designed, The base sequence of the probe is：5'HEX-CCCTACCCACAGCAGGTCAcGAGGAGTACAGTGGGTAGGG-BHQ1 3'.Should Molecular beacon is that wherein that 5' ends mark is HEX, and 3' ends mark is BHQ- by the distinguished sequence of the neck ring structure of base composition 1, the excitation wavelength of fluorophor is 535nm, launch wavelength 555nm.According to the base sequence (183A of KRAS gene mutation body >C synthesizing fluorescently labeled oligonucleotide probe Probe K183c) are designed:5'-FAM-ACAGCAGGTCAcGAGGAGTACAGT- 3'；
Known 40, the RAS gene mutations rectum cancer tissue sample that is not present now is taken KRAS genes (183A to be present with known>C) 40, the Colorectal Carcinoma sample of mutation, frozen section, every thickness 3um, every example 5 are then done respectively.By tumor tissues Frozen section is sprawled and slide, with 10% formalin (i.e. 1 part of formalin adds 9 parts of water to be formulated) fixer fixation group Knit, developed a film for 65 DEG C with 2xSSC, the molecular beacon probe Probe K183 through 95 DEG C of thermal denaturations are diluted to 1xDNA hybridization solutions 5pmol/ul, the position of 20ul tumor tissues on slide is added dropwise, is capped slide and closes, 42 DEG C are incubated 15 minutes.Directly exist Fluorescence microscopy Microscopic observation is simultaneously taken a picture, and counts fluorescence developing cell.
Entered with fluorescence labeling oligonucleotide probe Probe K183c by common fluorescent labeled oligonucleotide probe experimental procedure Row experiment.Judged by above-mentioned criterion, experimental result such as following table：
Key sample (goldstandard) Molecular beacon probe Fluorogenic oligonucleotide probe Sample number
+ + + 37
+ + - 3
- - - 33
- - + 7
Note：In above-mentioned list, it is known that sample (goldstandard)：It is known that KRAS gene mutation rectum cancer tissue sample 40 is not present Example is used as negative goldstandard, it is known that KRAS genes (183A be present>C) 40, the Colorectal Carcinoma sample of mutation is as positive gold Standard；“-”：Represent negative and KRAS gene mutation is not present；“+”：Represent positive and KRAS genes (183A be present>C) it is mutated.
Negative, positive as goldstandard using known rectum cancer tissue's sample, the positive rate of molecular beacon is (37+ 3)/(37+3) X100%=100%, the positive rate of fluorescence labeling oligonucleotide probe is 37/ (37+3) X100%= 92.5%；The false positive recall rate of molecular beacon is 0/ (33+7) X100%=0%, and the vacation of fluorescence labeling oligonucleotide probe is positive Property recall rate is 7/ (33+7) X100%=17.5%.From experimental result, molecular beacon probe is better than fluorogenic oligonucleotides Probe.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, some improvement and deformation can also be made, these are improved and deformation Also it should be regarded as protection scope of the present invention.

Claims (5)

  1. A kind of 1. molecular beacon probe, it is characterised in that the molecular beacon probe include Probe K34, Probe K35, Tetra- kinds of probes of Probe K38 and Probe K183, their base sequence are respectively：
    Probe K34：5'-CCCTACCCTAGTTGGAGCThGTGGCGTAGGCAGGGTAGGG-3'；
    Probe K35：5'-CCCTACCCTAGTTGGAGCTGhTGGCGTAGGCAGGGTAGGG-3'；
    Probe K38：5'-CCCTACCCTTGGAGCTGGTGaCGTAGGCAAGAGGGTAGGG-3'；
    Probe K183：5'-CCCTACCCACAGCAGGTCAcGAGGAGTACAGTGGGTAGGG-3'；
    The 5' ends of every kind of probe are marked with fluorophor, and 3' ends are marked with quenching group.
  2. 2. a kind of molecular beacon probe according to claim 1, it is characterised in that the fluorophor is selected from：6-FAM、 Fluorescein, JOE, TET, HEX, Cyanine 3, ROX, Texas Red, Cyanine 5 or Cyanine 5.5, it is described to quench The group that goes out is selected from：BHQ-1, BHQ-2, BHQ-2 or DABCYL.
  3. 3. a kind of kit, it is characterised in that including at least one molecular beacon probe described in claim 1 or 2.
  4. A kind of 4. kit according to claim 3, it is characterised in that also including hybridization solution, hybridization solution described in per 100ml Including following components：25mL 50*denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon Smart DNA, 10g dextran sulfate.
  5. 5. a kind of kit according to claim 4, the concentration of the molecular beacon probe is 5pmol/ul.
CN201710449386.8A 2017-06-14 2017-06-14 A kind of molecular beacon probe and kit for detecting KRAS gene mutation Pending CN107385016A (en)

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