CN109439751A - It is a kind of for detecting the fluorescence in situ hybridization probe kit of KIAA1549-BRAF fusion - Google Patents
It is a kind of for detecting the fluorescence in situ hybridization probe kit of KIAA1549-BRAF fusion Download PDFInfo
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Abstract
The invention belongs to molecular biology fields, and in particular to a kind of for detecting the fluorescence in situ hybridization probe kit of KIAA1549-BRAF fusion.The fluorescence in situ hybridization probe includes: BRAF gene hybridization probe library 1, and the initial position of probe is designed within the scope of each 50Kb of BRAF gene breaking point or so, and probe extends to centromere direction, and the length of probe is 150~300Kb；The initial position in BRAF gene hybridization probe library 2, probe is designed within the scope of each 50Kb of BRAF gene breaking point or so, and probe extends to telomere direction, and the length of probe is 150~300Kb；Two probe spacings are from for 0~100Kb.Hybridization probe of the present invention realizes on same chromosome two and detects apart from closer fusion, being capable of the very intuitive Fusion Strain of two genes of reaction on a cellular level；The fluorescence in situ hybridization probe hybridization time is short, has high signal-to-noise ratio, very high specificity.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of for detecting KIAA1549-BRAF fusion
Fluorescence in situ hybridization probe kit.
Background technique
Pilocytic astrocytoma (PA) is the encephalic primary tumo(u)r that a kind of WHO is classified as I grade, in children's neurosurgery
Most commonly seen glioma, count its account for about brain tumor 25%.KIAA1549 gene and BRAF gene are respectively positioned on 7
Number region chromosome q34, the tandem sequence repeats of BRAF gene result in KIAA1549/BRAF Gene Fusion, in pilocytic star
(60%~80%) high-incidence in cytoma.The position merged into each other according to KIAA1549 and BRAF gene is different, has reported 5 a few days ago
The different fusion variants of kind, are respectively as follows: KIAA1549ex16-BRAF ex9, KIAA1549ex15-BRAF ex9, and
KIAA1549ex16-BRAF ex11, KIAA1549ex18-BRAF ex10 and KIAA1549ex19-BRAF ex9；First three according to
Secondary to account for whole variance purposes 45%, 28% and 5%, latter two is relatively fewer.Although generating the inherent mechanism mesh of above-mentioned fusion
It is preceding still not very clear, but different types of KIAA1549-BRAF fusion mutation is considered to have identical function.
So far, there is not very specific conclusion yet for the molecular mechanism in PA occurrence and development, in the dye of PA patient
Detect that abnormal gene copy, the high throughput sequencing technologies of full-length genome show mitogen-activated protein kinase in colour solid 7q34
(MAPK) access is abnormal, and the exception of the access merges into each other related to KIAA1549 gene and BRAF gene.The text increasingly arrived
Report is offered, KIAA1549:BRAF fusion has been used as the diagnosis marker of pilocytic astrocytoma.Furthermore 2015 year
The detection of KIAA1549:BRAF fusion is recommended to be used as capillary in " Chinese central nervous system diagnosis of glioma and treatment guidelines "
The diagnosis marker of born of the same parents' astrocytoma.
The detection method of the fusion has 4 kinds at present, genome sequencing (WGS), transcript profile sequencing (RNA-seq),
Real-time fluorescence quantitative PCR (qRT-PCR) and fluorescence in situ hybridization (FISH), since first two method is at high cost, requires sample
Height makes it be difficult to carry out extensively in most of laboratories；Latter two method be also " Chinese central nervous system diagnosis of glioma with
The detection method recommended in treatment guidelines ", since KIAA1549:BRAF Gene Fusion is there are many form, round pcr detection is opposite
It is relatively difficult and can not detect the amalgamation mode that do not report.Detection of fluorescence in situ hybridization (FISH) technology for fusion
The Fusion Strain that two kinds of genes can intuitively be reacted very much, for the amalgamation mode having reported different fused types and not reported
It can detect, overcome the deficiency of round pcr.FISH technology should be for the detection of KIAA1549:BRAF Gene Fusion
First choice, but since two genes are respectively positioned on No. 7 regions chromosome q34, and chromosome location is closer, therefore routine FISH is glimmering
Light probe is difficult to detect the fusion.At present for the fusion there are mainly two types of detection probe: BRAF be broken probe and
KIAA1549/BRAF fusion probe.Since existing BRAF fracture probe design section is respectively in the BRAF gene region of fracture
Each 550Kb range (Fig. 2) in left and right, after BRAF gene fracture, distance is the region 1.4Mb between signaling point, this apart from closer,
It cannot be distinguished to cut-off and split signaling point；And KIAA1549/BRAF fusion probe design section melts in two genes respectively
Region is closed, has more a fusion signal among two signaling points after fracture fusion, but distance is about 1.0Mb between three signaling points
Region, same indistinguishable cut-off split signaling point.Therefore, routine BRAF fracture probe and KIAA1549/BRAF merge base at present
Because probe cannot distinguish positive and negative sample completely.
Summary of the invention
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of for detecting KIAA1549-BRAF fusion base
The fluorescence in situ hybridization probe kit of cause.
For achieving the above object, the technical solution adopted by the present invention are as follows:
It is a kind of for detecting the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion, which is characterized in that it is described glimmering
Light in situ hybridization probe includes two probe libraries, BRAF gene hybridization probe library 1: is designed in BRAF gene the initial position of probe
Within the scope of each 50Kb of breaking point or so, probe extends to centromere direction, and the length of probe is 150~300Kb；BRAF gene is miscellaneous
Hand over probe library 2: the initial position of probe is designed within the scope of each 50Kb of BRAF gene breaking point or so, and probe prolongs to telomere direction
It stretches, the length of probe is 150~300Kb；Distance is 0~100Kb between two probe libraries.
In above scheme, the initial position of probe described in BRAF gene hybridization probe library 1 is aobvious for 11 extra of BRAF gene
Son；The initial position of probe described in BRAF gene hybridization probe library 2 is 9 exon of BRAF gene.
In above scheme, two different colors of fluorescence dye is marked in two probe libraries of the fluorescence in situ hybridization probe
Material.
Heretofore described fluorescent dye is selected from: CY3, CY5, TAMRA, Texas Red, Rhodamine, FITC,
Biotin-aha and FAM.
Comprising above-mentioned for detecting the kit of the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion.
It is walked for detecting the preparation method of the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion, including as follows
It is rapid:
(1) BRAF gene hybridization probe library 1 and BRAF gene hybridization probe library 2 are downloaded from UCSC Genome Browser
The genome non repetitive sequence of institute overlay area, wherein 1, BRAF gene hybridization probe library overlay area are as follows: initial position exists
Within the scope of each 50Kb of BRAF gene breaking point or so, probe extends to centromere direction, and the length of probe is 150~300Kb,
2, BRAF gene hybridization probe library overlay area are as follows: initial position is within the scope of each 50Kb of BRAF gene breaking point or so, Xiang Duan
Grain direction development length is 150~300Kb；Distance is 0~100Kb between two probe libraries.
(2) the genome non repetitive sequence for 1, the BRAF gene hybridization probe library overlay area for obtaining step (1) uses
Perl plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported into OligoArray software
Probe design, probe screening are carried out, the probe filtered out exports in Microsoft Excel, obtains a series of BRAF gene probe sequences
Column 1；
(3) the genome non repetitive sequence for 2, the BRAF gene hybridization probe library overlay area for obtaining step (1) uses
Perl plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported into OligoArray software
Probe design, probe screening are carried out, the probe filtered out exports in Microsoft Excel, obtains a series of BRAF gene probe sequences
Column 2；
(4) chemical synthesis is carried out to a series of BRAF gene probe sequences 1 obtained by step (2) using DNA synthesizer, will changed
The probe for learning synthesis is mixed, and BRAF gene hybridization probe library 1 is prepared；Similarly, using DNA synthesizer to step (3)
A series of BRAF gene probe sequences 2 of gained carry out chemical synthesis, and chemically synthesized probe is mixed, BRAF is prepared
Gene recombination probe library 2；
(5) using the universal primer with different fluorophors respectively to BRAF gene hybridization probe library 1 and BRAF gene
Hybridization probe library 2 carries out amplification label reaction, and the BRAF gene hybridization probe library 1 and BRAF gene hybridization for obtaining fluorescent marker are visited
Needle library 2, the BRAF gene hybridization probe library 1 and BRAF gene hybridization probe library 2 of the fluorescent marker are constituted for detecting
The fluorescence in situ hybridization probe of KIAA1549-BRAF fusion.
In above scheme, the condition of the screening of probe described in step (2) and step (3) are as follows: probe length 50bp, TM value 85
~99 DEG C, GC is free of TTTT/GGGG/AAAA/CCCC, probe minimum interval 5bp than 40~80%.
In above scheme, the every probe 5 ' end and 3 ' ends of a series of BRAF gene probe sequences 1 obtained by step (2) add
The sequence label of upper 18bp；The every probe 5 ' end and 3 ' ends of a series of BRAF gene probe sequences 2 obtained by step (3) add
The sequence label of 18bp；The base sequence of the sequence label is as follows:
5 ' end sequence labels are as follows: TGTAAAACGACGGCCAG；3 ' end sequence labels are as follows: GGTCATAGCTGTTTCCTG.
In above scheme, the sequence of the universal primer is as follows:
M13-F TGTAAAACGACGGCCAGT；
M13-R CAGGAAACAGCTATGACC。
In above scheme, the PCR reaction system of step (5) the amplification label reaction is as follows:
In above scheme, the PCR reaction condition of step (5) the amplification label reaction is as follows: 95 DEG C of 5min；94 DEG C of 30s,
58 DEG C of 30s, 72 DEG C of 30s carry out 35 circulations altogether；72℃10min.
Mentioned reagent box is in preparation for detecting the application in KIAA1549-BRAF fusion product, concrete application side
Method includes the following steps:
(1) sample process: taking paraffin section sample of the thickness for having baked piece at 4~5 microns, immerses 65 DEG C of dewaxings of preheating
10~15 minutes in agent I；It is immersed after taking-up slide in 65 DEG C of dewaxing agent II of preheating 10~15 minutes；In room temperature after taking-up slide
Under successively immerse 100% ethyl alcohol, 85% ethyl alcohol, each 2~3 minutes in 70% ethyl alcohol；65 DEG C that preheating is immersed after taking-up slide are gone
3~5 minutes in ionized water；It is immersed in 95 DEG C of deionized waters 30~40 minutes after taking out slide, Hydro pre-cooling slide 1min；It takes out
It is immersed after slide in 37 DEG C of protease working solutions of preheating 20~40 minutes；It is soaked in eluent after taking-up slide and rinses two
It is secondary, 5 minutes every time；70%, 85% and 100% each 2~3min of graded ethanol is immersed respectively after taking out slide；Take out slide rear chamber
Temperature is dried, spare；
(2) preparation of the quick detection probe hybridization mixture of KIAA1549-BRAF fusion: by the BRAF of fluorescent marker
Gene recombination probe library 1, the BRAF gene hybridization probe library 2 of fluorescent marker and hybridization buffer are mixed according to the volume ratio of 1:1:9
It closes；
(3) probe sample co-variation: every sample is fast using step (2) mixing gained KIAA1549-BRAF fusion
Fast detection probe hybridization mixture is hybridized, using 22X22mm coverslip cover plate, using rubber glue mounting, by glass after mounting
Piece is placed in 85 DEG C of denaturation 5min, 42 DEG C of hybridization 2h in hybridization instrument；
(4) post-hybridization washing: wait throw off coverslip after the completion of hybridizing, slide is placed in the washing lotion of 68 DEG C of preheatings to wash and is gone
Except unbonded probe；
(5) redye and microscopy: anti-cancellation mountant mounting be added dropwise in the slide dried, then fluorescence microscopy under the microscope
Results of hybridization.
In above scheme, the component of hybridization buffer described in step (2) are as follows: 10wt% deionized formamide, 10wt%
Dextran sulfate, 1ugRNaseA, 0.3M sodium chloride, 10mM citrate buffer solution.
In above scheme, washing lotion described in step (3) are as follows: sodium chloride containing 0.3M, 0.03M sodium citrate and 0.1%
Tween-20。
Beneficial effects of the present invention are as follows: 1) present invention is simultaneously near BRAF gene breaking point to centromere direction region
And BRAF breaking point nearby designs probe to telomere direction region, since two genes of KIAA1549 and BRAF are contaminated in same
On colour solid and apart from closer, (insertion for not having segment) two kinds of fluorescence signals are adjacent when not merging, when merging
When two genes among be inserted into one section of 2Mb segment (segment includes BRAF green probe region), it is more compared to when not merging
A signaling point out, the differentiation fusion type that can be will be apparent that；The present invention breaks intrinsic thinking, is broken by BRAF gene
Probe realizes on same chromosome two and detects apart from closer fusion, can be very straight on a cellular level
The Fusion Strain of two genes of reaction of sight；2) probe of the present invention not only can detecte KIAA1549/BRAF fusion,
It can also detect that BRAF is broken simultaneously, the analysis for other fused types；3) two probe libraries of the present invention can be marked
Remember use all fluorescent dyes relevant to FISH detection, range of choice is extensive；4) designed distance between two probe libraries of the invention
It is small, KIAA1549/BRAF fusion sample can be clearly distinguished, and the fluorescence in situ hybridization probe hybridization time is short, has
High signal-to-noise ratio, very high specificity and sample recall rate.
Detailed description of the invention
Fig. 1 is KIAA1549-BRAF Gene Fusion mode.
Fig. 2 is that common BRAF gene is broken probe face principle.
Fig. 3 is of the present invention for detecting the design of the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion
Thinking and working principle.
Fig. 4 is to be sentenced using the result of fluorescence in situ hybridization probe of the present invention detection KIAA1549-BRAF fusion
It reads.
Fig. 5 is that fluorescence in situ hybridization probe of the present invention and common BRAF gene are broken probe in detecting KIAA1549-
The testing result of BRAF fusion compares.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
The preparation of the 1 quick detection probe of KIAA1549-BRAF fusion of embodiment
The preparation of the quick detection probe of KIAA1549-BRAF fusion of the present invention, includes the following steps:
(1) BRAF gene hybridization probe 1 is downloaded from UCSC Genome Browser and BRAF gene hybridization probe 2 is covered
The genome non repetitive sequence of cover area, wherein 1 overlay area of BRAF gene hybridization probe are as follows: chr7 140,581,693-
140,781,692 (positions be 11 exon of BRAF gene to the centromere direction region 200kb)；BRAF gene hybridization probe
2, library overlay area are as follows: (play position is 9 exon of BRAF gene to telomere to chr7 140,787,547-140,987,546
The direction region 200kb)；Acquired sequence saves as fasta format, and repetitive sequence region replaces with N in genome；
(2) the genome non repetitive sequence of 1 overlay area of BRAF gene hybridization probe for obtaining step (1) uses
Perl plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported into OligoArray software
Carry out probe design, probe screening, the condition of probe screening are as follows: 85~99 DEG C of value of probe length 50bp, TM, GC ratio 40-80%,
Without TTTT/GGGG/AAAA/CCCC, minimum interval 5bp between probe；Then the probe filtered out is exported into Microsoft Excel
In, then the sequence label of 18bp is added at 5 ' ends of every probe and 3 ' ends respectively；The base sequence of sequence label is as follows: 5 ends
Sequence label are as follows: TGTAAAACGACGGCCAG (M13 upstream sequence), 3 end sequence labels are as follows: GGTCATAGCTGTTTCCTG
(M13 downstream sequence)；Obtain a series of BRAF gene probe sequence 1 of tape label sequences；
(3) the genome non repetitive sequence for 2, the BRAF gene hybridization probe library overlay area for obtaining step (1) uses
Perl plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported into OligoArray software
Carry out probe design, probe screening, the condition of probe screening are as follows: 85~99 DEG C of value of probe length 50bp, TM, GC ratio 40-80%,
Without TTTT/GGGG/AAAA/CCCC, minimum interval 5bp between probe；Then the probe filtered out is exported into Microsoft Excel
In, then the sequence label of 18bp is added at 5 ' ends of every probe and 3 ' ends respectively；The same step of the base sequence of sequence label
(2)；Obtain a series of BRAF gene probe sequence 2 of tape label sequences；
(4) it is carried out using BRAF gene probe sequence 1 of the DNA synthesizer to a series of tape label sequences obtained by step (2)
Synthesis, the probe of synthesis is mixed, BRAF gene hybridization probe library 1, BRAF gene hybridization probe library 1 is prepared
The probe of sequence label is had comprising 4723；Similarly, using DNA synthesizer to a series of tape label sequences obtained by step (3)
BRAF gene probe sequence 2 synthesized, the probe of synthesis is mixed, BRAF gene hybridization probe library 2 is prepared,
BRAF gene hybridization probe library 2 includes 3641 probes for having sequence label；
(5) the M13 universal primer that 5 ' ends have green fluorescence group and Chinese red fluorophor, universal primer are respectively synthesized
Sequence are as follows: M13-F TGTAAAACGACGGCCAGT, M13-R CAGGAAACAGCTATGACC；Green fluorescence is had using 5 ' ends
The M13 universal primer of group carries out amplification label reaction to BRAF gene hybridization probe library 1；The fluorescence of Exocarpium Citri Rubrum is had using 5 ' ends
The M13 universal primer of group carries out amplification label reaction to BRAF gene hybridization probe library 2；The PCR of the amplification label reaction
Reaction system is as follows:
The PCR reaction condition of the amplification label reaction is as follows: 95 DEG C of 5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, altogether
Carry out 35 circulations；72℃10min；
(6) using acetate ethanol sodium method respectively to the amplification marked product in BRAF gene hybridization probe library 1 obtained by step (5)
It is purified with the amplification marked product in BRAF gene hybridization probe library 2, is diluted again obtain concentration as 20ng/ul's after purification
Fluorescent hybridization BRAF gene hybridization probe library 1, concentration are the fluorescent hybridization BRAF gene hybridization probe library 2 of 20ng/ul.
The application method of the 2 quick detection probe of KIAA1549-BRAF fusion of embodiment
The preparation of the embodiment 1 gained quick detection probe of KIAA1549-BRAF fusion is melted in detection KIAA1549-BRAF
Application method when gene is closed, is specifically comprised the following steps:
(1) sample process: taking paraffin section sample of the thickness for having baked piece at 4~5 microns, immerses 65 DEG C of dewaxings of preheating
10~15 minutes in agent I；It is immersed after taking-up slide in 65 DEG C of dewaxing agent II of preheating 10~15 minutes；In room temperature after taking-up slide
Under successively immerse 100% ethyl alcohol, 85% ethyl alcohol, each 2~3 minutes in 70% ethyl alcohol；65 DEG C that preheating is immersed after taking-up slide are gone
3~5 minutes in ionized water；It is immersed in 95 DEG C of deionized waters 30~40 minutes after taking out slide, Hydro pre-cooling slide 1min；It takes out
It is immersed after slide in 37 DEG C of protease working solutions of preheating 20~40 minutes；It is soaked in eluent after taking-up slide and rinses two
It is secondary, 5 minutes every time；70%, 85% and 100% each 2-3min of graded ethanol is immersed respectively after taking out slide；Take out slide rear chamber
Temperature is dried, spare；
(2) embodiment 1 preparation of the quick detection probe hybridization mixture of KIAA1549-BRAF fusion: is prepared into institute
Concentration be 20ng/ul BRAF gene hybridization probe library 1, concentration be 20ng/ul BRAF gene hybridization probe library 2 and hybridization
Buffer is mixed according to the volume ratio of 1:1:9；The component of the hybridization buffer are as follows: 50wt% deionized formamide, 10wt%
Dextran sulfate, 1ugRNaseA, 0.4M sodium chloride, 10mM citrate buffer solution；
(3) probe sample co-variation: every sample mixes gained KIAA1549-BRAF using 10ul step (2) and merges base
Because of quick detection probe hybridization mixture, using 22X22mm coverslip cover plate, using rubber glue mounting, slide is set after mounting
85 DEG C of denaturation 5min, 42 DEG C of hybridization 2h in hybridization instrument；
(4) wait throw off coverslip after the completion of hybridizing, slide post-hybridization washing: is placed in the washing lotion of 68 DEG C of preheatings (containing 0.3M
Sodium chloride, 0.03M sodium citrate and 0.1%Tween-20) in washing remove unbonded probe；
(5) it redyes and microscopy: 10ul anti-cancellation mountant mounting is added dropwise in the slide dried, then under fluorescence microscope
Observe results of hybridization.
Detection side described in the quick detection probe of KIAA1549-BRAF fusion and embodiment 2 prepared using embodiment 1
Method, the time that hybridization is arranged is 2h；Sample is carried out in strict accordance with the specification that it is provided using common BRAF fracture probe simultaneously
For detecting KIAA1549-BRAF fusion, hybridization time is set as 16h for processing and crossover operation.Testing result such as Fig. 5 institute
Show, as can be seen from Figure 5: when merging since insertion 2Mb segment includes green probe region therefore compared to not merging
When have more a green point (signaling point is closer apart from BRAF gene)；KIAA1549-BRAF prepared by the present invention
The differentiation positive and negative sample that the quick detection probe of fusion can be perfectly clear in shorter hybridization time, positive sample
(three signaling points, each yellow signal point of BRAF and allele region, Insert Fragment include a green point for this；
That is yellow green three signaling points of positive sample two), (two signaling points are not inserted into segment, BRAF and allele area to negative sample
Each yellow signal point, that is, negative sample in domain is two yellow signal points).And common BRAF as a comparison case is broken probe,
Because probe area and probe spacing are bigger from designing, more after one segment of insertion among KIAA1549 and BRAF gene
A signaling point opens (i.e. two signaling points are still adjacent) at a distance of closer visually cannot be distinguished of original signaling point out, therefore
Negative and positive sample cannot be distinguished.Illustrate KIAA1549-BRAF fusion fluorogenic hybridization probe design of the present invention
Rationally, not only hybridization time is short, but also has high signal-to-noise ratio, high specificity and sample recall rate.
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection scope of the invention.
Sequence table
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<211> 17bp
<212> DNA
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400 > 1 of <
tgtaaaacga cggccag 17
<210> 2
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<212> DNA
<213>artificial sequence
400 > 2 of <
ggtcatagct gtttcctg 18
<210> 3
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400 > 3 of <
tgtaaaacga cggccagt 18
<210> 4
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<212> DNA
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caggaaacag ctatgacc 18
Claims (10)
1. a kind of for detecting the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion, which is characterized in that the fluorescence
In situ hybridization probe includes two probe libraries, and BRAF gene hybridization probe library 1: the initial position design of probe is disconnected in BRAF gene
Within the scope of each 50Kb of knick point or so, probe extends to centromere direction, and the length of probe is 150 ~ 300Kb；BRAF gene hybridization
Probe library 2: the initial position of probe is designed within the scope of each 50Kb of BRAF gene breaking point or so, and probe prolongs to telomere direction
It stretches, the length of probe is 150 ~ 300Kb；Distance is 0 ~ 100Kb between two probe libraries.
2. it is according to claim 1 for detecting the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion, it is special
Sign is that the initial position of probe described in BRAF gene hybridization probe library 1 is 11 exon of BRAF gene；BRAF gene
The initial position of probe described in hybridization probe library 2 is 9 exon of BRAF gene.
3. it is according to claim 1 for detecting the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion, it is special
Sign is that two different colors of fluorescent dye is marked in two probe libraries of the fluorescence in situ hybridization probe.
4. any described for detecting the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion comprising claim 1 ~ 3
Kit.
5. claim 1 ~ 3 is any described for detecting the system of the fluorescence in situ hybridization probe of KIAA1549-BRAF fusion
Preparation Method, which comprises the steps of:
(1) from UCSC Genome Browser downloading 2 overlay areas of BRAF gene probe library 1 and BRAF gene probe library
Genome non repetitive sequence, wherein 1, BRAF gene hybridization probe library overlay area are as follows: initial position is in BRAF gene breaking point
Within the scope of each 50Kb in left and right, probe extends to centromere direction, and the length of probe is 150 ~ 300Kb, BRAF gene hybridization probe
2, library overlay area are as follows: initial position is within the scope of each 50Kb of BRAF gene breaking point or so, to telomere direction development length
150~300Kb；Distance is 0 ~ 100Kb between two probe libraries；
(2) the genome non repetitive sequence for 1, the BRAF gene hybridization probe library overlay area for obtaining step (1) uses perl
Plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported OligoArray software to carry out
Probe design, probe screening, the probe filtered out export in Microsoft Excel, obtain a series of BRAF gene probe sequences 1；
(3) the genome non repetitive sequence for 2, the BRAF gene hybridization probe library overlay area for obtaining step (1) uses perl
Plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported OligoArray software to carry out
Probe design, probe screening, the probe filtered out export in Microsoft Excel, obtain a series of BRAF gene probe sequences 2；
(4) chemical synthesis is carried out to a series of BRAF gene probe sequences 1 obtained by step (2) using DNA synthesizer, chemistry is closed
At probe mixed, BRAF gene hybridization probe library 1 is prepared；Similarly, using DNA synthesizer to obtained by step (3)
A series of BRAF gene probe sequences 2 carry out chemical synthesis, and chemically synthesized probe is mixed, BRAF gene is prepared
Hybridization probe library 2；
(5) BRAF gene hybridization probe library 1 and BRAF gene are hybridized respectively using the universal primer with different fluorophors
Probe library 2 carries out amplification label reaction, obtains the BRAF gene hybridization probe library 1 and BRAF gene hybridization probe library of fluorescent marker
2, the BRAF gene hybridization probe library 1 and BRAF gene hybridization probe library 2 of the fluorescent marker are constituted for detecting
The fluorescence in situ hybridization probe of KIAA1549-BRAF fusion.
6. preparation method according to claim 5, which is characterized in that the screening of probe described in step (2) and step (3)
Condition are as follows: 85 ~ 99 DEG C of value of probe length 50bp, TM, GC is free of TTTT/GGGG/AAAA/CCCC, between probe is minimum than 40 ~ 80%
Every 5bp.
7. preparation method according to claim 5, which is characterized in that a series of BRAF gene probes obtained by step (2)
The every probe 5 ' end and 3 ' ends of sequence 1 add the sequence label of 18bp；A series of BRAF gene probe sequences obtained by step (3)
The every probe 5 ' end and 3 ' ends of column 2 add the sequence label of 18bp；The base sequence of the sequence label is as follows: 5 ' ends
Sequence label are as follows: TGTAAAACGACGGCCAG；3 ' end sequence labels are as follows: GGTCATAGCTGTTTCCTG.
8. preparation method according to claim 5, which is characterized in that the sequence of the universal primer is as follows: M13-F
TGTAAAACGACGGCCAGT；M13-R CAGGAAACAGCTATGACC.
9. kit described in claim 4 is in preparation for detecting the application in KIAA1549-BRAF fusion product.
10. application according to claim 9, which is characterized in that specific application method includes the following steps:
(1) sample process: paraffin section sample of the thickness for having baked piece at 4 ~ 5 microns is taken, is immersed in 65 DEG C of dewaxing agent I of preheating
10 ~ 15 minutes；It is immersed after taking-up slide in 65 DEG C of dewaxing agent II of preheating 10 ~ 15 minutes；After taking-up slide at room temperature successively
Immerse 100% ethyl alcohol, 85% ethyl alcohol, each 2 ~ 3 minutes in 70% ethyl alcohol；It is immersed after taking-up slide 3 ~ 5 in 65 DEG C of deionized waters of preheating
Minute；It is immersed in 95 DEG C of deionized waters 30 ~ 40 minutes after taking out slide, Hydro pre-cooling slide 1min；It is immersed in advance after taking out slide
20 ~ 40 minutes in 37 DEG C of protease working solutions of heat；It is soaked in eluent and is rinsed twice after taking-up slide, 5 minutes every time；It takes
Immerse 70%, 85% and 100% each 2 ~ 3min of graded ethanol after slide respectively out；Room temperature is dried after taking out slide, spare；
(2) preparation of the quick detection probe hybridization mixture of KIAA1549-BRAF fusion: by the BRAF gene of fluorescent marker
Hybridization probe library 1, the BRAF gene hybridization probe library 2 of fluorescent marker and hybridization buffer are mixed according to the volume ratio of 1:1:9；
(3) probe sample co-variation: every sample mixes gained KIAA1549-BRAF fusion using step (2) and quickly examines
Probing needle hybridization mixture is hybridized, and using 22X22mm coverslip cover plate, using rubber glue mounting, is set slide after mounting
85 DEG C of denaturation 5min, 42 DEG C of hybridization 2h in hybridization instrument；
(4) post-hybridization washing: wait throw off coverslip after the completion of hybridizing, slide is placed in washing in the washing lotion of 68 DEG C of preheatings and is removed not
Bonding probes；
(5) it redyes and microscopy: anti-cancellation mountant mounting is added dropwise in the slide dried, then hybridize under the microscope in fluorescence microscopy
As a result.
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| CN110195098A (en) * | 2019-05-10 | 2019-09-03 | 广州安必平医药科技股份有限公司 | For detecting probe groups, kit and its application of BRAF correlation fusion gene |
| CN113136419A (en) * | 2021-06-09 | 2021-07-20 | 广州华银健康医疗集团股份有限公司 | Fluorescent quantitative PCR detection method for fusion gene mutation |
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| CN110195098A (en) * | 2019-05-10 | 2019-09-03 | 广州安必平医药科技股份有限公司 | For detecting probe groups, kit and its application of BRAF correlation fusion gene |
| CN113136419A (en) * | 2021-06-09 | 2021-07-20 | 广州华银健康医疗集团股份有限公司 | Fluorescent quantitative PCR detection method for fusion gene mutation |
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