CN109161542B - Fluorescent in-situ hybridization probe and preparation method and application thereof - Google Patents

Fluorescent in-situ hybridization probe and preparation method and application thereof Download PDF

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CN109161542B
CN109161542B CN201810155158.4A CN201810155158A CN109161542B CN 109161542 B CN109161542 B CN 109161542B CN 201810155158 A CN201810155158 A CN 201810155158A CN 109161542 B CN109161542 B CN 109161542B
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李怡
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Abstract

The invention discloses a fluorescent in-situ hybridization probe and a preparation method and application thereof. The preparation method of the fluorescent in-situ hybridization probe and the fluorescent in-situ hybridization probe prepared by the preparation method are specially designed for the genome non-repetitive region, so that the non-specific reaction can be reduced, and the background interference can be reduced; the constructed probe is single-stranded DNA, and compared with a traditional double-stranded probe such as BAC or PCR product and the like, the constructed probe can reduce the pairing of the probe and increase the hybridization efficiency; and the constructed single-stranded DNA probe fragment is small, can be combined with a target sequence more quickly, and reduces the hybridization time.

Description

Fluorescent in-situ hybridization probe and preparation method and application thereof
Technical Field
The invention relates to the field of molecular biology detection, in particular to a fluorescent in-situ hybridization probe and a preparation method and application thereof.
Background
Fluorescence In Situ Hybridization (FISH) is a technique that uses a Fluorescence-labeled DNA fragment as a probe to hybridize with a DNA molecule in the genome of a cell based on a known population-specific DNA sequence and detects the presence and abundance of the specific DNA. The basic principle of FISH is to label a DNA (or RNA) probe with a special nucleotide molecule, then directly hybridize the probe to a chromosome or a DNA fiber slice, and then detect qualitative, positioning and relative quantitative analysis of a DNA sequence on the chromosome or the DNA fiber slice by the specific binding of a monoclonal antibody coupled with a fluorescein molecule and the probe molecule. The FISH has the advantages of safety, rapidness, high sensitivity, long-term preservation of the probe, simultaneous display of various colors and the like, and not only can display the metaphase, but also can display interphase nuclei.
Currently, commercial FISH detection of genes such as HER2 mainly uses BAC (bacterial Artificial chromosome) or PCR products as probes to perform hybridization detection after fluorescent molecular labeling. However, BAC and PCR product probes have some non-specific sequences, the probe specificity is not high, and the signal background is high. The existing probe segments are generally large, the general length is between 200-500nt, an effective method is not available for fragmentation, the hybridization efficiency is low, and the hybridization time is long. And at present, the probes randomly participate in fluorescence when being labeled with fluorescence, so that the fluorescence number of each probe is uncertain, and the difference of each batch is large.
Disclosure of Invention
Accordingly, there is a need for a fluorescent in situ hybridization probe with high specificity, high hybridization efficiency and high fluorescence labeling intensity, and a preparation method and applications thereof.
A method for preparing a fluorescent in situ hybridization probe comprises the following steps:
constructing a probe library: aiming at a non-repetitive target gene region, continuously designing a series of single-stranded fragments which are completely complementary with the target gene region and have the length of 35nt-200nt as candidate probes, respectively adding amplification primer fragments at two ends of the candidate probes, and synthesizing by a chip to obtain a target probe library;
amplifying the library of probes of interest;
carrying out in vitro transcription on the amplified product to prepare library RNA corresponding to the target probe library;
carrying out reverse transcription on the library RNA to prepare a single-stranded DNA probe;
and carrying out fluorescence labeling on the prepared single-stranded DNA probe to obtain the DNA probe.
In one embodiment, the candidate probe is 40-50nt in length.
In one embodiment, the sequence of the amplification primer fragment is shown as SEQ ID NO.1 and SEQ ID NO. 2.
In one embodiment, the library of probes of interest is amplified using an emulsion PCR amplification method.
In one embodiment, during emulsion PCR amplification, the prepared PCR reaction system is divided into multiple parts, PCR reactions are respectively carried out on a thermal cycler, and after the reaction is finished, part or all of the PCR products are combined for subsequent treatment.
In one embodiment, the preparation of the single-stranded DNA probe by reverse transcription of the library RNA comprises a step of adding amino dNTPs to the reaction system for probe labeling, and the obtained single-stranded DNA probe is labeled with an amino group.
In one embodiment, the prepared single-stranded DNA probe is fluorescently labeled by reacting N-hydroxysuccinimide ester labeled with a fluorescent dye with an amino-labeled single-stranded DNA probe.
A fluorescent in situ hybridization probe is prepared by adopting the preparation method of the fluorescent in situ hybridization probe in any embodiment.
A fluorescence in situ hybridization detection chip contains the fluorescence in situ hybridization probe.
A fluorescence in situ hybridization detection kit contains the fluorescence in situ hybridization probe or the fluorescence in situ hybridization detection chip.
The preparation method of the fluorescence in situ hybridization probe and the fluorescence in situ hybridization probe prepared by the preparation method are specially designed for the genome non-repetitive region, so that the non-specific reaction can be reduced, and the background interference can be reduced; the constructed probe is single-stranded DNA, and compared with a traditional double-stranded probe such as BAC or PCR product and the like, the constructed probe can reduce the pairing of the probe and increase the hybridization efficiency; and the constructed single-stranded DNA probe fragment is small, can be combined with a target sequence more quickly, and reduces the hybridization time.
The invention creatively provides that single amplification is carried out by using emulsion PCR, the amplified product is transcribed to construct library RNA, then the library RNA is reversely transcribed to construct a single-stranded DNA probe, compared with the traditional method of directly carrying out PCR amplification by using a probe library to obtain a double-stranded DNA probe, the invention can fully utilize transcription and reverse transcription to obtain linear single-stranded DNA, and during the amplification process of the probe library, non-specific amplification does not exist, the biased amplification is reduced, the uniformity of the probe can be improved, and the accuracy of subsequent in-situ hybridization is further improved.
In addition, by adopting reverse transcription to mix amino-dNTP and then coupling fluorescent dye, the fluorescence number marked by each probe can be increased, thereby being beneficial to increasing the fluorescence intensity during fluorescence in-situ hybridization and improving the accuracy and reliability of a detection result.
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FIG. 1 shows the result of agarose gel electrophoresis of the emulsion PCR amplification product;
FIG. 2 shows the results of Nanodrop assays for RNA obtained by in vitro transcription;
FIG. 3 shows the results of the concentration of the Nanodrop detection probe;
FIG. 4 shows microscopic examination of fluorescence in situ hybridization of normal cells.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The method for preparing a fluorescent in situ hybridization probe according to an embodiment includes the steps of:
the method comprises the following steps: constructing a probe library: aiming at a non-repetitive target gene region, a series of single-stranded fragments which are completely complementary with the target gene region and have the length of 35nt-200nt are continuously designed to be used as candidate probes, amplification primer fragments are respectively added at two ends of the candidate probes, and a target probe library is obtained through chip synthesis.
The target gene region of the embodiment is a non-repetitive region of a genome, and by designing a candidate probe for the non-repetitive region, non-specific reaction can be reduced, and background interference can be reduced.
In the embodiment, the candidate probes are constructed at preset intervals according to different target gene regions, for example, the candidate probes can be constructed at intervals of 1-10bp, so that mutual interference among the probes can be avoided, and the accuracy of the detection result can be improved.
Preferably, the length of the candidate probe is preferably between 35nt and 100nt, more preferably around 45nt, such as between 40 nt and 50 nt. The single-stranded DNA probe constructed by the embodiment has small fragment, can be combined with a target sequence more quickly, and reduces the hybridization time.
In this embodiment, after obtaining the candidate probes, the method further includes a step of obtaining a Tm value for non-specific hybridization by BLAST comparison, removing the candidate probes having a non-specific Tm value exceeding a preset value, and selecting non-overlapping probes as final candidate probes.
The amplification primer fragments can be a variety of common amplification primers, such as in one specific example, the amplification primer fragments have the sequences shown in SEQ ID NO.1 (5'-GGAGGCCGGAGAATTGTAATACGACTCACTATAGGGAGA-3') and SEQ ID NO.2 (5'-CGTGGTCGCGTCTCA-3').
The probe library constructed in this embodiment is synthesized on a gene chip by using a chip synthesis technique.
Step two: amplifying the library of probes of interest.
Specifically, the present embodiment is to amplify the target probe library by using an emulsion PCR amplification method. And during emulsion PCR amplification, dividing the prepared PCR reaction system into multiple parts, respectively carrying out PCR reaction on a thermal cycler, and combining part or all of PCR products for subsequent treatment after the reaction is finished. The PCR reaction can be performed for, but is not limited to, 25 to 35 cycles, depending on the length of the specific probe, etc.
Step three: and carrying out in vitro transcription on the amplified product to prepare library RNA corresponding to the target probe library.
Step four: and carrying out reverse transcription on the library RNA to prepare a single-stranded DNA probe.
In this embodiment, the preparation of a single-stranded DNA probe by reverse transcription of the library RNA comprises a step of adding an Amino (Amino) dNTP to the reaction system to label the probe, and the resulting single-stranded DNA probe is labeled with an Amino group. In one specific example, the probe is labeled with Amino-dUTP (Amino-dUTP), but in other examples, the probe is not limited to being labeled with Amino-dUTP, and may be labeled with Amino-dATP, Amino-dCTP, or the like.
The single-strand amplification is carried out by using emulsion PCR, the amplified product is transcribed to construct library RNA, then the library RNA is reversely transcribed to construct a single-strand DNA probe, compared with the traditional method of directly carrying out PCR amplification by using a probe library to obtain a double-strand DNA probe, the transcription and reverse transcription can be fully utilized to obtain linear single-strand DNA, non-specific amplification does not exist in the amplification process of the probe library, the biased amplification is reduced, the uniformity of the probe can be improved, and the accuracy of subsequent in-situ hybridization is favorably improved.
Step five: and carrying out fluorescence labeling on the prepared single-stranded DNA probe to obtain the DNA probe.
Correspondingly, when the prepared single-stranded DNA probe is fluorescently labeled, the single-stranded DNA probe is fluorescently labeled by reacting N-hydroxysuccinimide ester (NHS ester) labeled with a fluorescent dye with the amino group-labeled single-stranded DNA probe. The fluorescent dye may be, but is not limited to, the green fluorescent dye Alexa Fluor 488.
By adopting reverse transcription to mix amino-dNTP and then coupling fluorescent dye, the fluorescence number marked by each probe can be increased, thereby being beneficial to increasing the fluorescence intensity during fluorescence in-situ hybridization and improving the accuracy and reliability of a detection result.
In addition, the present embodiment further provides a fluorescence in situ hybridization probe, which is prepared by the method for preparing a fluorescence in situ hybridization probe according to any of the embodiments, and further provides a fluorescence in situ hybridization detection chip or detection kit, which contains the fluorescence in situ hybridization probe.
The preparation method of the fluorescent in-situ hybridization probe and the fluorescent in-situ hybridization probe prepared by the preparation method are single-stranded DNA, and compared with the traditional double-stranded probes such as BAC or PCR products, the method can reduce the pairing of the probe and increase the hybridization efficiency; and the constructed single-stranded DNA probe fragment is small, can be combined with a target sequence more quickly, and reduces the hybridization time.
The fluorescent in situ hybridization probe and the method for constructing the same according to the present invention will be described in further detail with reference to a specific example, but it is understood that the fluorescent in situ hybridization probe and the method for constructing the same according to the present invention are not limited to the specific example described below.
1. Design and Synthesis of oligonucleotide Probe libraries
Design of oligonucleotide probe libraries: the approximately 230kb region of chromosome 17 chr17:37758215-37988632 of the human genome comprising the HER2 gene was selected, which is the region in which the HER2 gene repeats in cancer cells. Every 3bp, a candidate probe with a length of about 45nt, which is completely complementary to the target region, is designed. The Tm values of the non-specific hybrids were obtained by BLAST alignment. Probes with non-specific Tm values greater than 45 ℃ were removed by scoring. Non-overlapping candidate probes were selected to obtain a total of 1630 qualified probe sequences whose positions on the genome are shown in the following table:
Figure BDA0001581084600000051
Figure BDA0001581084600000061
Figure BDA0001581084600000071
Figure BDA0001581084600000081
Figure BDA0001581084600000091
Figure BDA0001581084600000101
Figure BDA0001581084600000111
Figure BDA0001581084600000121
Figure BDA0001581084600000131
Figure BDA0001581084600000141
Figure BDA0001581084600000151
Figure BDA0001581084600000161
Figure BDA0001581084600000171
Figure BDA0001581084600000181
Figure BDA0001581084600000191
Figure BDA0001581084600000201
Figure BDA0001581084600000211
the probe library was synthesized using a chip and obtained by primer amplification.
The primer sequences are as follows:
Primer 1 5-’GGAGGCCGGAGAATTGTAATACGACTCACTATAGGGAGA-3’
Primer 2 5-’CGTGGTCGCGTCTCA-3’
2. emulsion PCR amplification probe library
The probe library was amplified by Emulsion PCR using Micellulia DNA Emulsion & Purification kit (EURX Co.).
The method comprises the following specific steps:
1) establishing an emulsion:
emulsion component 1 220μl
Emulsion component 2 20μl
Emulsion component 3 60μl
Total volume 300μl
Shaking and mixing evenly, and placing on ice for standby.
2) PCR reactions were set up on ice:
Figure BDA0001581084600000212
Figure BDA0001581084600000221
3) carrying out emulsion reaction: mix 50. mu.l of the aqueous phase and 300. mu.l of the oil-water mixture, mix well, and dispense into 7 0.2ml PCR tubes. The PCR reaction was carried out on a thermal cycler according to the following conditions.
Figure BDA0001581084600000222
4) And (3) purifying a PCR product: and mixing the emulsion PCR products in each PCR tube, adding 1ml of butanol, and uniformly mixing. Then 400. mu.l of Orange-DX was added and mixed well. Centrifuge for two minutes. The upper organic phase was removed and the aqueous phase was combined through the column. After washing three times with a washing solution, 100. mu.l of the eluent was used for elution. PCR products were quantified using Nanodrop.
5) Result verification
As shown in FIG. 1, the PCR product was detected on 2% gel, and there was a specific band at 100bp, indicating that the emulsion PCR amplification was successful.
3. In vitro transcription to generate library RNA
a) The following in vitro transcription reaction system (MEGAscript) was establishedTMT7 Transcription Kit, thermo fisher Scientific), to produce RNA by in vitro Transcription:
composition (I) Volume (μ l)
480ng of PCR product -
10×buffer 4
10mM rNTP pool 16
T7enzyme mix(50Unit/μl) 4
Nuclease free water Up to 40
The reaction was carried out on a PCR instrument at 37 ℃ for 4 hours.
b) RNA was purified using Qiagen RNeasy Mini kit.
A total of 42. mu.g of RNA was obtained for the next reaction.
c) Result verification
Mu.l of RNA was diluted 10-fold, and RNA was obtained by detecting in vitro transcription of the transcript using Nanodrop, and the results are shown in FIG. 2.
4. Using SuperScriptTMIII First-Strand Synthesis System (ThermoFisher Scientific Co.) reverse transcription was performed to generate single-stranded DNA probes.
a) The following reactions were prepared on ice:
Figure BDA0001581084600000231
mixing, denaturing at 65 deg.c for 5min in a PCR instrument, and fast setting on ice.
b) The following reaction mixture was prepared:
composition (I) Volume (μ l)
10×RT buffer 10
25mM MgCl2 20
0.1M DTT 10
SUPERaseOUT 5
SuperScript III RT 2.5
The reaction was carried out at 50 ℃ for 2 hours, supplemented with 2.5. mu.l SuperScript III RT and continued for 2 hours. Add 11. mu.l Exonuclease I buffer and 2. mu.l Exonuclease I, suspend for 5 seconds, and centrifuge for 5 seconds. Digestion was carried out at 37 ℃ for 15 minutes. Add 12. mu.l of 0.5M EDTA and mix well. Reverse transcriptase was inactivated at 85 ℃ for 5 min. The reaction was placed on ice.
c) And (3) digesting RNA: mu.l of 5U/. mu.l RNaseH and 4. mu.l of 10mg/ml RNaseA were added to carry out the reaction according to the following procedure:
temperature of Time
37℃ 120min
70℃ 20min
50℃ 60min
95℃ 5min
Slowly reduced to 50 DEG C 0.1℃/sec
50℃ 60min
4℃ Heat preservation
d) The reaction was purified using the Zymo Quick-RNA Miniprep kit.
Using the Nanodrop detection single-stranded oligonucleotide probe, 20. mu.g was obtained in one reaction.
5. Probe-labeled fluorescence
a) Mu.l of the purified single-stranded DNA was added to 5. mu.l of 3M NaAc, mixed well, added to 130. mu.l of 100% ethanol, mixed well, and left overnight at-20 ℃.
b) Ethanol precipitation: centrifuge at 4 ℃ for 30min, remove supernatant, wash once with 70% ethanol.
c) And (3) probe fluorescence coupling reaction: the precipitated DNA was dried and dissolved in 5. mu.l of water.
Reagent Dosage of
Amino-labeled Single-stranded DNA (about 20. mu.g) 5μl
0.2M NaCO3 3μl
Alexa Fluor 488NHS ester 2μl
The reaction was carried out at room temperature for 2 hours with exclusion of light.
d) Add 40. mu.l of purified water and purify the reaction using the Zymo Quick-RNA Miniprep kit.
e) Probe concentration and fluorescence were measured using Nanodrop and the results are shown in FIG. 3. The probe concentration obtained was 170 ng/. mu.l and AlexaFluor488 was 57.4 pmol/. mu.l. An average of 5-6 fluorescences were labeled per probe. And (5) subpackaging for later use.
6. Fluorescence in situ hybridization
HER2 gene was detected by fluorescent in situ hybridization using a fluorescently labeled HER2 probe. The method comprises the following specific steps: 1) fixing the cells or tissues on a glass slide for pretreatment; 2) adding hybridization solution containing the probe into the glass slide, and performing denaturation and hybridization; 3) fully washing to remove background; 4) counterstaining and mounting, and microscopic examination.
The step 1) of the above protocol has certain differences according to the conditions for pretreating the slide of the sample to be detected, and is carried out by taking a cell sample as an example, and the details are as follows: fixing the cells with 3:1 methanol/glacial acetic acid for 30 minutes or overnight at-20 ℃, and centrifuging to remove the supernatant; resuspending the cells using a fixative solution to obtain a suitable cell density; dropping a drop of cell suspension on a glass slide, and air-drying; the slides were aged at 65 ℃ for 2 minutes; washing with 2 XSSC at room temperature for 2min, sequentially adding 70%, 90%, and 100% ethanol for dehydration for 2min, and air drying the slide at room temperature; the glass slide is put into a pepsin solution (0.25%) heated to 37 ℃ in a water bath for treatment for 5min, the glass slide is immediately taken out, and the glass slide is sequentially put into 2 XSSC, 70%, 90% and 100% of alcohol for treatment for 2min at room temperature and is air-dried.
The step 2) is as follows: dripping 12 mul of hybridization solution containing the probe on a glass slide, and covering the glass slide with a cover slip; denaturation at 67 ℃ for 8 min, caution and light protection; hybridization was carried out at 42 ℃ for 2 hours.
The step 3) is as follows: the cover slip was removed. Washed away from light in prewarming wash buffer (2 XSSC, 0.1% NP40) at 37 ℃ for 15 min; washing the mixture for 15 minutes in a dark place at normal temperature by 2 XSSC; washing once at normal temperature; wash quickly in 0.2 × SSC once; and (5) air drying.
The step 4) is as follows: 15 μ l of the DAPI-containing counterstain was added dropwise, and the slides were covered with a fingernail polish. And (6) microscopic examination.
The microscopic examination of normal cells revealed that more than 95% of the cells had two clear hybridization signals, as shown in FIG. 4.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Guangzhou roster Biotechnology Limited
<120> fluorescent in-situ hybridization probe, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
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Claims (8)

1. A preparation method of a fluorescence in situ hybridization probe is characterized by comprising the following steps:
constructing a probe library: selecting a region of chromosome 17 chr17:37758215-37988632 of a human genome containing a HER2 gene, wherein the region is a region where the HER2 gene is repeated in cancer cells, designing a candidate probe which is completely complementary with a target region at intervals of 3bp and has the length between 35nt and 200nt, obtaining a Tm value of non-specific hybridization by BLAST comparison, removing probes with the non-specific Tm value being more than 45 ℃ by scoring, selecting non-overlapping candidate probes, adding amplification primer fragments at two ends of the candidate probes respectively, and obtaining a target probe library by chip synthesis; the candidate probes have 1630 probes in total, which are as follows:
Figure FDA0002824411090000011
Figure FDA0002824411090000021
Figure FDA0002824411090000031
Figure FDA0002824411090000041
Figure FDA0002824411090000051
Figure FDA0002824411090000061
Figure FDA0002824411090000071
Figure FDA0002824411090000081
Figure FDA0002824411090000091
Figure FDA0002824411090000101
Figure FDA0002824411090000111
Figure FDA0002824411090000121
Figure FDA0002824411090000131
Figure FDA0002824411090000141
Figure FDA0002824411090000151
Figure FDA0002824411090000161
amplifying the library of probes of interest;
carrying out in vitro transcription on the amplified product to prepare library RNA corresponding to the target probe library;
carrying out reverse transcription on the library RNA to prepare a single-stranded DNA probe;
carrying out fluorescence labeling on the prepared single-stranded DNA probe to obtain the DNA probe;
when the library RNA is subjected to reverse transcription to prepare a single-stranded DNA probe, the method comprises the step of adding amino dNTP into a reaction system for probe labeling, wherein the obtained single-stranded DNA probe is labeled with amino;
when the prepared single-stranded DNA probe is fluorescently labeled, the single-stranded DNA probe is fluorescently labeled by reacting N-hydroxysuccinimide ester labeled with a fluorescent dye with an amino group-labeled single-stranded DNA probe.
2. The method of claim 1, wherein the amino dNTP is amino dUTP, amino dATP, or amino dCTP.
3. The method for preparing the fluorescent in situ hybridization probe according to claim 1, wherein the sequences of the amplification primer segments are shown as SEQ ID No.1 and SEQ ID No. 2.
4. The method for preparing the fluorescent in situ hybridization probe according to any of claims 1 to 3, wherein the target probe library is amplified by an emulsion PCR amplification method.
5. The method for preparing the fluorescent in-situ hybridization probe according to claim 4, wherein during the emulsion PCR amplification, the prepared PCR reaction system is divided into a plurality of parts, PCR reactions are respectively carried out on a thermal cycler, and after the reaction is finished, part or all of the PCR products are combined for subsequent treatment.
6. A fluorescent in situ hybridization probe, which is prepared by the method for preparing the fluorescent in situ hybridization probe according to any one of claims 1 to 5.
7. A fluorescence in situ hybridization detection chip comprising the fluorescence in situ hybridization probe according to claim 6.
8. A fluorescence in situ hybridization assay kit, characterized by comprising the fluorescence in situ hybridization probe according to claim 6 or comprising the fluorescence in situ hybridization assay chip according to claim 7.
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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
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