CN109161542A - fluorescence in situ hybridization probe and its preparation method and application - Google Patents

fluorescence in situ hybridization probe and its preparation method and application Download PDF

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CN109161542A
CN109161542A CN201810155158.4A CN201810155158A CN109161542A CN 109161542 A CN109161542 A CN 109161542A CN 201810155158 A CN201810155158 A CN 201810155158A CN 109161542 A CN109161542 A CN 109161542A
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李怡
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Guangzhou Manual Biotechnology Co Ltd
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Abstract

The invention discloses a kind of fluorescence in situ hybridization probe and its preparation method and application.The preparation method of fluorescence in situ hybridization probe of the invention and the fluorescence in situ hybridization probe being prepared using the preparation method are designed specifically for the non-duplicate area of genome, can reduce non-specific responding, reduce background interference；The probe of building is single stranded DNA, compared with double-chain probes such as traditional BAC or PCR product, it is possible to reduce the pairing of probe and itself increases hybridization efficiency；And the ssDNA probe segment constructed is small, can reduce hybridization time more quickly in conjunction with targeting sequence.

Description

Fluorescence in situ hybridization probe and its preparation method and application
Technical field
The present invention relates to molecular Biological Detection fields, more particularly, to a kind of fluorescence in situ hybridization probe and its preparation side Method and application.
Background technique
Fluorescence in situ hybridization technique (Fluorescence in situ hybridization, FISH) is according to known kind The special DNA sequence dna of group, to hybridize with DNA molecular in cellular genome using the DNA fragmentation of fluorescent marker as probe, inspection Survey the presence and abundance of the specific DNA.The basic principle of FISH is by the special nucleic acid molecule mark of DNA (or RNA) probe Note, then by probe direct cross to chromosome or DNA fiber slice, then with the monoclonal antibody being coupled with fluorescein molecule Qualitative, positioning, relative quantification of the DNA sequence dna on chromosome or DNA fiber slice are detected with probe molecule specific binding Analysis.FISH have many advantages, such as safety, quickly, high sensitivity, probe energy long-term preservation, can show multiple color simultaneously, not only It can show division phases, moreover it is possible to be shown in interphase nucleus.
The genes FISH detection such as current commercialized HER2 mainly uses BAC (Bacterial Artificial Chromosome) or PCR product as probe carries out hybridization check after fluorescent molecule marks.However, BAC and PCR is produced Physical prospecting needle is there are some non-specific sequences, and probe specificity is not high, and signal background is higher.Existing probe fragment is generally bigger than normal, and one As length between 200-500nt, lack effective ways carry out small fragment, hybridization efficiency is relatively low, and hybridization time is long.And it is existing It is to participate in fluorescence at random, therefore each fluorescence probe number is uncertain, causes each batch when probe is in mark fluorescent Diversity ratio is larger.
Summary of the invention
Based on this, it is necessary to it is miscellaneous to provide the fluorescent in situ that a species specificity is high, hybridization efficiency is high and Fluorescence labeling intensity is high Hand over probe and its preparation method and application.
A kind of preparation method of fluorescence in situ hybridization probe, includes the following steps:
It constructs Probe Library: being directed to non-repetitive target gene regions, continuously design a series of complete with target gene regions The single-chain fragment that complete complementary length is 35nt-200nt adds amplification at the both ends of candidate probe respectively and draws as candidate probe Object segment synthesizes to obtain target probe library by chip；
Expand the target probe library；
Amplified production is transcribed in vitro, library RNA corresponding with the target probe library is prepared；
Reverse transcription is carried out to the library RNA, prepares ssDNA probe；
To the ssDNA probe of preparation carry out fluorescent marker to get.
The length of the candidate probe is 40-50nt in one of the embodiments,.
The sequence of the amplimer segment such as SEQ ID NO.1 and SEQ ID NO.2 institute in one of the embodiments, Show.
The target probe library is expanded using the method that emulsion-based PCR expands in one of the embodiments,.
In one of the embodiments, when emulsion-based PCR expands, the PCR reaction system of preparation is divided into more parts, and respectively PCR reaction is carried out on thermal cycler, is merged part or all of PCR product after reaction and is carried out subsequent processing.
Reverse transcription is being carried out to the library RNA in one of the embodiments, when preparing ssDNA probe, including to The step of amino dNTP carries out probe label is added in reaction system, obtained ssDNA probe is marked with amino.
It is using fluorescent dye in one of the embodiments, when carrying out fluorescent marker to the ssDNA probe of preparation The N-hydroxy-succinamide ester of label is reacted with the ssDNA probe of amino labeled carries out fluorescence to the ssDNA probe Label.
A kind of fluorescence in situ hybridization probe, using the preparation side of fluorescence in situ hybridization probe described in any of the above-described embodiment Method is prepared.
A kind of fluorescence in situ hybridization detection chip, contains above-mentioned fluorescence in situ hybridization probe.
A kind of fluorescence in situ hybridization detection kit containing above-mentioned fluorescence in situ hybridization probe or contains above-mentioned fluorescence original Position hybridization check chip.
The preparation method of above-mentioned fluorescence in situ hybridization probe and the fluorescent in situ for using the preparation method to be prepared are miscellaneous Probe is handed over, is designed specifically for the non-duplicate area of genome, non-specific responding can be reduced, reduce background interference；The probe of building It is single stranded DNA, compared with double-chain probes such as traditional BAC or PCR product, it is possible to reduce the pairing of probe and itself increases hybridization Efficiency；And the ssDNA probe segment constructed is small, can reduce hybridization time more quickly in conjunction with targeting sequence.
And the proposition of the invention carries out single amplification, amplified production transcription building text by using emulsion-based PCR Library RNA, then construct to obtain ssDNA probe by library RNA reverse transcription direct carries out PCR expansion by Probe Library compared with traditional Increasing obtains double chain DNA probe, and transcription and reverse transcription can be made full use of to obtain linear single stranded DNA, expanded in Probe Library Cheng Zhong, there is no nonspecific amplifications, reduce skewed popularity amplification, the homogeneity of probe can be improved, and then be conducive to mention The accuracy of high subsequent in situ hybridization.
In addition, mixing amino-dNTP by using reverse transcription, it is then coupled fluorescent dye, each probe mark can be increased The fluorescence number of note, and then fluorescence intensity when being conducive to increase fluorescence in situ hybridization improve the accuracy of testing result and reliable Property.
Detailed description of the invention
Fig. 1 is the testing result of the agarose gel electrophoresis of emulsion-based PCR amplified production；
Fig. 2 is the result that the RNA obtained is transcribed in vitro in Nanodrop detection；
Fig. 3 is the result of Nanodrop detection probe concentrations；
Fig. 4 is the microscopic examination result to the fluorescence in situ hybridization of normal cell.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough Comprehensively.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool The purpose of the embodiment of body, it is not intended that in the limitation present invention.
The preparation method of the fluorescence in situ hybridization probe of one embodiment, includes the following steps:
Step 1: building Probe Library: non-repetitive target gene regions are directed to, a series of and target gene is continuously designed The length of region complete complementary is the single-chain fragment of 35nt-200nt as candidate probe, is added respectively at the both ends of candidate probe Amplimer segment synthesizes to obtain target probe library by chip.
The target gene regions of present embodiment are the non-duplicate area of genome, by designing candidate visit to non-duplicate area Needle can reduce nonspecific reaction, reduce background interference.
In the present embodiment, it for different target gene regions, is spaced preset length and constructs candidate probe, it such as can be with The length of 1-10bp is spaced to construct candidate probe, can be interfered with each other to avoid between probe, is conducive to improve testing result Accuracy.
Preferably, the length of candidate probe is preferably between 35-100nt, further preferably in 45nt or so, such as in 40- Between 50nt.The ssDNA probe segment of present embodiment building is small, can reduce more quickly in conjunction with targeting sequence Hybridization time.
In the present embodiment, after obtaining candidate probe, further include being compared by BLAST, obtain Non-specific hybridization Tm value removes non-specific Tm value and is more than the candidate probe of preset value, and selects non-overlap probe as final candidate probe Step.
Amplimer segment can be various general amplimers, such as in a specific embodiment, amplimer The sequence of segment such as SEQ ID NO.1 (5 '-GGAGGCCGGAGAATTGTAATACGACTCACTATAGGGAGA-3 ') and SEQ Shown in ID NO.2 (5 '-CGTGGTCGCGTCTCA-3 ').
Probe Library constructed by present embodiment is by the target probe library of building using chip synthetic technology in gene It is synthesized on chip.
Step 2: the target probe library is expanded.
Specifically, present embodiment is to expand the target probe library using the method that emulsion-based PCR expands.And in cream When liquid PCR amplification, the PCR reaction system of preparation is divided into more parts, and carries out PCR reaction on thermal cycler respectively, reaction knot Merge part or all of PCR product after beam and carries out subsequent processing.PCR reaction can carry out but be not limited to 25-35 circulation, can root According to specific probe length situations such as depending on.
Step 3: being transcribed in vitro amplified production, prepares library RNA corresponding with the target probe library.
Step 4: reverse transcription is carried out to the library RNA, prepares ssDNA probe.
In the present embodiment, reverse transcription is being carried out to the library RNA, when preparing ssDNA probe, including to reaction The step of amino (Amino) dNTP carries out probe label is added in system, obtained ssDNA probe is marked with amino.One It is to carry out probe label using amino dUTP (Amino-dUTP) to be not limited in other embodiments in a specific embodiment It is marked using amino dUTP, amino dATP, amino dCTP etc. also can be used and be marked.
Single amplification is carried out by using emulsion-based PCR, amplified production transcription constructs library RNA, then by library RNA reverse transcription Building obtains ssDNA probe, direct carry out PCR amplification compared with traditional by Probe Library and obtains double chain DNA probe, Ke Yichong Divide and obtain linear single stranded DNA using transcription and reverse transcription, in Probe Library amplification procedure, there is no nonspecific expansions Increase, reduces skewed popularity amplification, the homogeneity of probe can be improved, and then be conducive to improve the accuracy of subsequent in situ hybridization.
Step 5: to the ssDNA probe of preparation carry out fluorescent marker to get.
It is corresponding, it is the N- hydroxyl using fluorochrome label when carrying out fluorescent marker to the ssDNA probe of preparation Succinimide ester (NHS ester) is reacted with the ssDNA probe of amino labeled carries out fluorescence mark to the ssDNA probe Note.Fluorescent dye can be but not limited to Green fluorescent dye Alexa Fluor 488.
Amino-dNTP is mixed by using reverse transcription, is then coupled fluorescent dye, the glimmering of each probe label can be increased Light number, and then be conducive to fluorescence intensity when increase fluorescence in situ hybridization, improve the accuracy and reliability of testing result.
In addition, present embodiment additionally provides a kind of fluorescence in situ hybridization probe, use described in any of the above-described embodiment The preparation method of fluorescence in situ hybridization probe be prepared, and further provide a kind of fluorescence in situ hybridization detection chip or Detection kit contains above-mentioned fluorescence in situ hybridization probe.
The preparation method of above-mentioned fluorescence in situ hybridization probe and the fluorescent in situ for using the preparation method to be prepared are miscellaneous Handing over probe is single stranded DNA, compared with double-chain probes such as traditional BAC or PCR product, it is possible to reduce the pairing of probe and itself increases Add hybridization efficiency；And the ssDNA probe segment constructed is small, can reduce hybridization more quickly in conjunction with targeting sequence Time.
Fluorescence in situ hybridization probe of the invention and its construction method are made below by way of a specific embodiment further detailed Thin explanation, it is appreciated that fluorescence in situ hybridization probe and its construction method of the invention is not limited to following specific embodiments.
1. design and synthetic oligonucleotide probe library
Design oligonucleotide probe library: selection includes No. 17 chromosome chr17 of human genome of HER2 gene: The region about 230kb of 37758215-37988632, the region are HER2 gene duplicate region in cancer cell.It is set every 3bp Count the candidate probe of one with the length 45nt of target area complete complementary or so.It is compared by BLAST, obtains Non-specific hybridization Tm value.The probe that non-specific Tm value is greater than 45 DEG C is removed by marking.The candidate probe of non-overlap is selected, obtains 1630 altogether Qualified probe sequence, position in the genome are as shown in the table:
Using chip synthesising probing needle library, obtained by primer amplification.
Primer sequence is as follows:
Primer 1 5-’GGAGGCCGGAGAATTGTAATACGACTCACTATAGGGAGA-3’
Primer 2 5-’CGTGGTCGCGTCTCA-3’
2. emulsion-based PCR amplification probe library
Emulsion-based PCR expansion is carried out using Micellulia DNA Emulsion&Purification kit (EURX company) Increase Probe Library.
Specific step is as follows:
1) lotion is established:
Emulsion components 1 220μl
Emulsion components 2 20μl
Emulsion components 3 60μl
Total volume 300μl
Concussion mixes, and is placed in stand-by on ice.
2) PCR reaction is established on ice:
3) carry out lotion reaction: 50 μ l water phases of mixing and 300 μ l oil water mixtures mix well, are dispensed into 7 0.2ml In PCR pipe.PCR reaction is carried out according to following condition on thermal cycler.
4) purified pcr product: merging the emulsion-based PCR product in each PCR pipe, and 1ml butanol is added, and mixes.Add 400 μ l Orange-DX is mixed.Centrifugation two minutes.Upper organic phase is removed, water phase crosses pillar combination.After washing lotion washing three times, make It is eluted with 100 μ l elutriants.Use Nanodrop quantitative PCR product.
5) result verification
As shown in Figure 1, PCR product detects on 2% gel, there can be specific band at 100bp, illustrate lotion PCR amplification success.
Library RNA is generated 3. being transcribed in vitro
A) following in-vitro transcription reaction system (MEGAscript is establishedTMT7 Transcription Kit, ThermoFisher Scientific company), it carries out that generation RNA is transcribed in vitro:
Ingredient Volume (μ l)
PCR product 480ng -
10×buffer 4
10mM rNTP pool 16
T7enzyme mix(50Unit/μl) 4
Nuclease free water Up to 40
It is reacted 4 hours for 37 DEG C in PCR instrument.
B) Qiagen RNeasy Mini kits RNA is used.
42 μ g RNA are obtained altogether for reacting in next step.
C) result verification
10 times of 1 μ l RNA dilutions are taken, obtain RNA using Nanodrop detection Transcription In vitro transcription, as a result as shown in Figure 2.
4. using SuperScriptTM III First-Strand Synthesis System(ThermoFisher Scientific company) carry out reverse transcription generation ssDNA probe.
A) prepare following reaction on ice:
It mixes, is placed in PCR instrument and carries out 65 DEG C of denaturation 5 minutes, place rapidly on ice.
B) prepare following reaction mixture:
Ingredient Volume (μ l)
10×RT buffer 10
25mM MgCl2 20
0.1M DTT 10
SUPERaseOUT 5
SuperScript III RT 2.5
50 DEG C are reacted 2 hours, are added into 2.5 μ l SuperScript III RT, the reaction was continued 2 hours.11 μ l are added Exonuclease I buffer and 2 μ l Exonuclease I is suspended 5 seconds, is centrifuged 5 seconds.37 DEG C digest 15 minutes.It is added 12 μ l 0.5M EDTA is mixed.85 DEG C of 5 minutes inactivation reverse transcriptase.Reaction is placed on ice.
C) it digests RNA: the RNaseA of the RNaseH and 4 μ l 10mg/ml of 4 μ l 5U/ μ l is added, is carried out according to following procedure Reaction:
Temperature Time
37℃ 120min
70℃ 20min
50℃ 60min
95℃ 5min
It is slowly decreased to 50 DEG C 0.1℃/sec
50℃ 60min
4℃ Heat preservation
D) it is reacted using Zymo Quick-RNA Miniprep kits.
Single strand oligonucleotide probes are detected using Nanodrop, primary first-order equation can obtain 20 μ g.
5. probe mark fluorescent
A) 5 μ l 3M NaAc are added in single stranded DNA to 50 μ l after purification, mix, 130 μ l, 100% ethyl alcohol is added, mix, set Overnight in -20 DEG C.
B) ethanol precipitation: 4 DEG C of centrifugation 30min remove supernatant, primary using 70% ethanol washing.
C) fluorescence probe coupling reaction: drying precipitated DNA is dissolved in 5 μ l water.
Reagent Dosage
The single stranded DNA (about 20 μ g) of amino labeled 5μl
0.2M NaCO3 3μl
Alexa Fluor 488NHS ester 2μl
Room temperature is protected from light 2 hours.
D) 40 μ l pure water are added, are reacted using Zymo Quick-RNA Miniprep kits.
E) Nanodrop detection probe concentrations and fluorescence are used, as a result sees Fig. 3.Acquisition concentration and probe concentration is 170ng/ μ l, AlexaFluor488 is 57.4pmol/ μ l.Average each probe marks 5-6 fluorescence.It dispenses spare.
6. fluorescence in situ hybridization
Fluorescence in situ hybridization detection HER2 gene is carried out using the HER2 probe of fluorescent marker.Specific step is as follows: 1) will Cell or tissue is fixed on slide, is pre-processed；2) by containing probe hybridization solution be added slide, carry out denaturation and it is miscellaneous It hands over；3) it is sufficiently washed, removes background；4) carry out redying simultaneously mounting, microscopy.
The step 1) of above scheme according to circumstances has certain difference to the pretreatment of measuring samples slide, used here as cell It is carried out for sample, specific as follows: cell fixes 30 minutes by methanol/glacial acetic acid of 3:1 or -20 DEG C are stayed overnight, and centrifugation is gone Supernatant；Cell is resuspended using fixer and has obtained appropriate cell density；Take drop cell suspension drop in slide, gas is dry；At 65 DEG C Aging slide 2 minutes；2min is washed in room temperature with 2 × SSC, 70%, 90% is sequentially placed into, is dehydrated each 2min in 100% alcohol, Room temperature dries slide；Slide is put into the pepsin solution (0.25%) of heating water bath to 37 DEG C and handles 5min, taken immediately Slide is successively put into the alcohol of 2 × SSC, 70%, 90%, 100 and respectively handles 2 minutes at room temperature by slide out, and gas is dry Slide.
Step 2) is specific as follows: being added dropwise on 12 hybridization solutions to slide of the μ l containing probe, coverslip mounting；8 are denaturalized at 67 DEG C Minute, pay attention to being protected from light；Hybridize 2 hours at 42 DEG C.
Step 3) is specific as follows: removing coverslip.Be protected from light be placed in 37 DEG C of preheating washing buffers (2 × SSC, 0.1% NP40 it) washs 15 minutes；It is protected from light and is placed in 2 × SSC of room temperature washing 15 minutes；Repeating room temperature washed once；It is fast in 0.2 × SSC Speed washed once；It is air-dried.
Step 4) is specific as follows: 15 μ l are added dropwise containing DAPI and redye liquid, covers slide, uses nail sheet for oil seal.Microscopy.
Visible to the microscopic examination result of normal cell 95% or more cell is shown in Fig. 4 there are two clearly hybridization signal.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
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Claims (10)

1. a kind of preparation method of fluorescence in situ hybridization probe, which comprises the steps of:
It constructs Probe Library: being directed to non-repetitive target gene regions, continuously design a series of completely mutual with target gene regions The length of benefit is the single-chain fragment of 35nt-200nt as candidate probe, adds amplimer piece respectively at the both ends of candidate probe Section, synthesizes to obtain target probe library by chip；
Expand the target probe library；
Amplified production is transcribed in vitro, library RNA corresponding with the target probe library is prepared；
Reverse transcription is carried out to the library RNA, prepares ssDNA probe；
To the ssDNA probe of preparation carry out fluorescent marker to get.
2. the preparation method of fluorescence in situ hybridization probe as described in claim 1, which is characterized in that the length of the candidate probe Degree is 40nt-50nt.
3. the preparation method of fluorescence in situ hybridization probe as described in claim 1, which is characterized in that the amplimer segment Sequence as shown in SEQ ID NO.1 and SEQ ID NO.2.
4. the preparation method of fluorescence in situ hybridization probe according to any one of claims 1 to 3, which is characterized in that use The method of emulsion-based PCR amplification expands the target probe library.
5. the preparation method of fluorescence in situ hybridization probe as claimed in claim 4, which is characterized in that in emulsion-based PCR amplification, The PCR reaction system of preparation is divided into more parts, and carries out PCR reaction on thermal cycler respectively, merges part after reaction Or whole PCR products carry out subsequent processing.
6. such as the preparation method of fluorescence in situ hybridization probe according to any one of claims 1 to 5, which is characterized in that right The library RNA carries out reverse transcription, when preparing ssDNA probe, including amino dNTP is added into reaction system and carries out probe The step of label, obtained ssDNA probe are marked with amino.
7. the preparation method of fluorescence in situ hybridization probe as claimed in claim 6, which is characterized in that the single-stranded of preparation DNA probe carry out fluorescent marker when, be using fluorochrome label N-hydroxy-succinamide ester and amino labeled it is single-stranded DNA probe reaction carries out fluorescent marker to the ssDNA probe.
8. a kind of fluorescence in situ hybridization probe, which is characterized in that using such as fluorescence original according to any one of claims 1 to 7 The preparation method of position hybridization probe is prepared.
9. a kind of fluorescence in situ hybridization detection chip, which is characterized in that visited containing fluorescence in situ hybridization as claimed in claim 8 Needle.
10. a kind of fluorescence in situ hybridization detection kit, which is characterized in that miscellaneous containing fluorescent in situ as claimed in claim 8 It hands over probe or contains fluorescence in situ hybridization detection chip as claimed in claim 9.
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CN110205357A (en) * 2019-06-22 2019-09-06 福建农林大学 A kind of preparation method of more primer oligo probes
CN110628875A (en) * 2019-09-25 2019-12-31 广州简册生物技术有限公司 Probe, kit and detection method for detecting Top2A gene
CN110699430A (en) * 2019-09-25 2020-01-17 广州简册生物技术有限公司 TOP2A gene detection probe and preparation method and application thereof
CN112266947A (en) * 2020-11-04 2021-01-26 南偲生物科技(南京)有限公司 Fluorescent in-situ hybridization probe and preparation method and application thereof
CN112795649A (en) * 2021-01-05 2021-05-14 武汉友芝友医疗科技股份有限公司 Probe set for detecting HER2 gene amplification level and application thereof
CN112921074A (en) * 2021-02-03 2021-06-08 鲲羽生物科技(江门)有限公司 In-situ hybridization method for simultaneously detecting multiple loci of genome through combined colors
CN113025611A (en) * 2021-03-16 2021-06-25 华中农业大学 Pi-FISH (fluorescence in situ hybridization) single molecule probe composition and application thereof in nucleic acid in-situ detection
CN114196788A (en) * 2021-12-23 2022-03-18 华中农业大学 Method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology
CN114381496A (en) * 2021-12-30 2022-04-22 广州安必平医药科技股份有限公司 In-situ hybridization probe and preparation method and application thereof

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