CN106222250A - A kind of preparation method of FISH probe - Google Patents

A kind of preparation method of FISH probe Download PDF

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Publication number
CN106222250A
CN106222250A CN201610584503.7A CN201610584503A CN106222250A CN 106222250 A CN106222250 A CN 106222250A CN 201610584503 A CN201610584503 A CN 201610584503A CN 106222250 A CN106222250 A CN 106222250A
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probe
sequence
cot
mankind
1dna
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Inventor
卢皇彬
林清华
阮力
唐郑华
林煌艺
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Amoy Diagnostics Co Ltd
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Amoy Diagnostics Co Ltd
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Priority to CN201610584503.7A priority Critical patent/CN106222250A/en
Publication of CN106222250A publication Critical patent/CN106222250A/en
Priority to PCT/CN2017/093516 priority patent/WO2018014845A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Abstract

The invention discloses the preparation method of a kind of FISH probe, it comprises the steps: 1) before prepared by probe, purpose fragment is carried out sequence analysis, the Species specific sequence that in composition sequence, ratio is higher；2) one in following scheme is selected to prepare probe: Species specific sequence and mankind Cot 1 DNA of synthesis to be screened removal repetitive sequence simultaneously, obtains specific template or probe；Or, utilize the Species specific sequence of synthesis to prepare probe after removing repetitive sequence corresponding in removing template, re-use mankind Cot 1 DNA and close；Or, utilize mankind Cot 1 DNA to prepare probe after removing repetitive sequence corresponding in removing template, the Species specific sequence re-using synthesis is closed；Or, use the Species specific sequence of synthesis and mankind Cot 1 DNA to close after probe preparation simultaneously.The present invention can improve the specificity of probe.

Description

A kind of preparation method of FISH probe
Technical field
The present invention relates to the preparation method of a kind of FISH probe.
Background technology
FISH technology, as on-radiation detection system, has the advantage that 1, fluorometric reagent and probe economy, safety； 2, probe steady, can use in two years after a labelling；3, experimental period is short, can rapidly be result, specificity is good, location Accurately；4, FISH can position length in the DNA sequence of 1kb, its sensitivity is suitable with radioactive probe；5, polychrome FISH passes through Same core showing, different colors can detect multiple sequence simultaneously；6, metaphase chromosome quantity can both have been shown on slide Or the change of structure, it is also possible in suspension, show the structure of interphase chromosome DNA.
The probe sequence that FISH uses derives from genome or BAC (bacterial artificial chromosome) DNA, comprises many repetition sequences Row.Tandem repetitive sequence (Tandem repeat), as being present in centric α satellite DNA (Satellite DNA)；It is dispersed in weight Complex sequences (Interspersed repeat), such as Alu family.These repetitive sequences in probe, easy when with genomic hybridization Produce nonspecific signals, how to remove the difficult problem become in probe preparation process.
Mankind Cot-1DNA is to be commonly used to one of closing and the instrument removing repetitive sequence in hybridization.Before probe preparation, Mankind Cot-1DNA can be used to filter out repetitive sequence, obtain specific template and prepare probe again.
But, mankind Cot-1DNA only comprises a part for repetitive sequence in purpose fragment, therefore, uses Cot-1DNA also Can not effectively remove non-specific signals.
Summary of the invention
The invention provides the preparation method of a kind of FISH probe, which overcome the preparation side of FISH probe in background technology Deficiency existing for method.
The present invention solves being the technical scheme is that of its technical problem
The preparation method of a kind of FISH probe, it comprises the steps:
1) before prepared by probe, purpose fragment is carried out repetitive sequences analysis, and synthesizes accordingly in purpose fragment sequence The Species specific sequence that ratio is higher；
2) one in following scheme is selected to prepare probe:
Species specific sequence and the mankind Cot-1DNA of synthesis are screened removal repetitive sequence simultaneously, obtains specificity mould Plate or probe；
Or, utilize the Species specific sequence of synthesis to prepare probe after removing corresponding repetitive sequence, re-use the mankind Cot-1DNA closes；
Or, utilize mankind Cot-1DNA screening to prepare probe after removing corresponding repetitive sequence, re-use the special of synthesis Property repetitive sequence is closed；
Or, use the Species specific sequence of synthesis and mankind Cot-1DNA to close after probe preparation simultaneously.
The method of screening repetitive sequence, special including the mankind Cot-1DNA of biotin coupling and/or biotin coupling Property repetitive sequence by affinity chromatograph remove purpose fragment repetitive sequence；Or mankind Cot-1DNA and/or specificity repeat Sequence is combined into double-strand, with double-stranded specific nuclease excision repetitive sequence with the preferential renaturation of repetitive sequence in purpose fragment.
In a preferred embodiment, step 1) use Genomic information research institute (base Because of information research institute) or the online sequence analysis of institute for systems biology (Inst Systems Biology) soft Part RepeatMasker carries out repetitive sequences analysis to purpose fragment.
In step 1) in ratio is higher in composition sequence Species specific sequence, term " repetitive sequence " is such as background technology Described, including tandem repetitive sequence (Tandem repeat), as being present in centric α satellite DNA (Satellite DNA)； Interspersed repeat sequence (Interspersed repeat), such as Alu family.
The definition of term " ratio is higher " be in sequence number of repetition more than 10 or sequence length accounting more than 5%.
Described synthesis can use prior art.
The method using the present invention, can be greatly improved the specificity of probe, and background noise is few, and signal to noise ratio is good.
Accompanying drawing explanation
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is the schematic flow sheet of the present invention.
Fig. 2 is the FISH probe testing result figure of embodiment 2；
Up three figures are a sample, and descending three figures are another sample.
Fig. 3 is the probe prepared of traditional method (cot-1 closing) and the probe of embodiment 2 preparation hybridization effect on cell Fruit contrast.
Detailed description of the invention
Embodiment 1
As a example by HER-2 gene, use Genomic information research institute (Giri) online Sequence analysis software RepeatMasking carries out repetitive sequences analysis, and synthesis purpose fragment sequence accordingly to purpose fragment The Species specific sequence that middle ratio is higher.
1, on Giri website, click on RepeatMasking, input purpose fragment sequence, candidate type and analysis classes After type, submit to and analyze.
2, analysis result divides 5 parts, and Part I is " Map of Hits ", enumerates the repetitive sequence that all comparisons are arrived Title, type and comparison are to the positional information in purpose fragment, and Part II is " Masked sequence ", comparison is arrived Repetitive sequence is hidden, and only shows special target sequence, and Part III is " Local alignments ", set forth in detail each The repetitive sequence that individual comparison is arrived and the match condition of purpose fragment, Part IV is " Masked regions ", only shows repetition Sequence, Part V is " Summary tables ", summarizes comparison number of times and the bases longs of repetitive sequence type and correspondence, As shown in the table:
3, according to analysis result, choose the sequence that multiplicity is higher, such as L1 or SINE1, closed by the method for chemosynthesis Become this partial sequence.
Embodiment 2
As a example by HER-2 gene, the mankind Cot-1DNA of biotin coupling is used to remove the corresponding repetitive sequence in removing template, The probe obtained after labelling uses Species specific sequence to close.
1, HER-2BAC DNA fragmentation
The ultrasonic instrument that interrupts is used to be interrupted by HER-2BAC DNA so that it is master tape is positioned at 250-1000bp.
2, using random primering to prepare the mankind Cot-1DNA of biotin coupling, labelling system is as follows:
System 100μL 250μL
Human Cot-1DNA N1(0.4-0.8μg) N1(1-2μg)
2.5×Buffer 40μL 100μL
Biotin-N8 N2(8-10μg) N2(20-25μg)
dH2O 26-N1-N2 65-N1-N2
dNTP 32μL 80μL
Klenow Fragment(5U/μL) 2μL 5μL
Concrete operations are as follows:
1. a step (on ice)
After piping and druming mixing, 95 DEG C of degeneration 10min in PCR instrument, it is immediately placed on 5min on ice, then brief centrifugation.
Two steps (on ice)
After piping and druming mixing, 37 DEG C of reaction more than 3h in PCR instrument,
Add 2.5 μ L Stop Buffer and terminate reaction 2-3min, use sodium acetate, ethanol precipitation and concentrate, being resuspended in Appropriate ddH2In O.
3, the mankind Cot-1DNA of biotin coupling and the HER-2BAC DNA hybridization of fragmentation
The HER-2BAC DNA of 100ng fragmentation and the mankind Cot-1DNA hybridization of 2.5 μ g biotin couplings, cumulative volume 15 μL。
95 DEG C of degeneration 10min, 65 DEG C of hybridized overnight, after adding 15 μ L washings in productMyOneTM Streptavidin C1 (Invitrogen 65002) magnetic bead, room temperature combines 30min, and magnetic frame absorption 3min, transfer supernatant is extremely New EP pipe.
Supernatant uses DNA Purification Kit, obtains the HER-2DNA template of a duplicate removal.
4, using random primering to prepare HER-2 probe, labelling system is as follows:
System 10μL 20μL 25μL
The HER-2DNA of duplicate removal N(40-80ng) N(80-160ng) N(100-200ng)
2.5×Buffer 4μL 8μL 10μL
dH2O 1-N 2-N 2.5-N
Lack C dNTP 1.6μL 3.2μL 4μL
Cy3-dCTP 3.2μL 6.4μL 8μL
Klenow Fragment(5U/μL) 0.2μL 0.4μL 0.5μL
Concrete operations are as follows:
1. a step (on ice)
After piping and druming mixing, 95 DEG C of degeneration 10min in PCR instrument, it is immediately placed on 5min on ice, then brief centrifugation.
Two steps (on ice)
Note: fluorescein finally adds, remaining fluorescein keeps in Dark Place in-20 DEG C.
After piping and druming mixing, 37 DEG C of reaction more than 3h in PCR instrument, add 2.5 μ L Stop Buffer and terminate reaction 2- 3min, uses sodium acetate, ethanol precipitation and concentrates, being resuspended in 5 μ L ddH2In O.After brief centrifugation, take 5 μ LHER-2 probes, 5 μ L centromeric probe (preparation method is similar with HER-2 probe), 5 μ L 1mg/mL Species specific sequences, 85 μ L hybridization solution (ratios For 1:1:1:17), HER2/CSP17 double-color probe.
Be used for the detection of breast carcinoma FFPE sample with obtained probe, result is shown in Fig. 3.
Embodiment 3
As a example by HER-2 gene, first use random priming label probe, re-use the mankind Cot-of biotin coupling The biotin labeled special repetitive sequence that carries of 1DNA and synthesis removes the repetitive probe in probe, obtains specificity and visits Pin.
1, using random primering to prepare HER-2 probe, labelling system is as follows:
System 10μL 20μL 25μL
HER-2DNA N(40-80ng) N(80-160ng) N(100-200ng)
2.5×Buffer 4μL 8μL 10μL
dH2O 1-N 2-N 2.5-N
Lack C dNTP 1.6μL 3.2μL 4μL
Cy3-dCTP 3.2μL 6.4μL 8μL
Klenow Fragment(5U/μL) 0.2μL 0.4μL 0.5μL
Concrete operations are as follows:
1. a step (on ice)
After piping and druming mixing, 95 DEG C of degeneration 10min in PCR instrument, it is immediately placed on 5min on ice, then brief centrifugation.
Two steps (on ice)
Note: fluorescein finally adds, remaining fluorescein keeps in Dark Place in-20 DEG C.
After piping and druming mixing, 37 DEG C of reaction more than 3h in PCR instrument, add 2.5 μ L Stop Buffer and terminate reaction 2- 3min, stand-by.
2, using random primering to prepare the mankind Cot-1DNA of biotin coupling, labelling system is as follows:
System 100μL 250μL
Human Cot-1 DNA N1(0.4-0.8μg) N1(1-2μg)
2.5×Buffer 40μL 100μL
Biotin-N8 N2(8-10μg) N2(20-25μg)
dH2O 26-N1-N2 65-N1-N2
dNTP 32μL 80μL
Klenow Fragment(5U/μL) 2μL 5μL
Concrete operations are as follows:
1. a step (on ice)
After piping and druming mixing, 95 DEG C of degeneration 10min in PCR instrument, it is immediately placed on 5min on ice, then brief centrifugation.
Two steps (on ice)
After piping and druming mixing, 37 DEG C of reaction more than 3h in PCR instrument,
Add 2.5 μ L Stop Buffer and terminate reaction 2-3min, use sodium acetate, ethanol precipitation and concentrate, being resuspended in Appropriate ddH2In O.
3, the special repetitive sequence of the biotin modification of the mankind Cot-1DNA of biotin coupling, synthesis and labelling obtain HER-2 probe hybridizes
In HER-2 probe labelling system add the mankind Cot-1DNA of 25 times of (mol ratio) biotin couplings, 25 times (rub You than) the special repetitive sequence of biotin modification that synthesizes and hybridization solution, cumulative volume 50 μ L.
95 DEG C of degeneration 10min, 46 DEG C of hybridized overnight, after adding 15 μ L washings in productMyOneTM Streptavidin C1 (Invitrogen 65002) magnetic bead, room temperature combines 30min, is placed on magnetic frame absorption 3min, transfer Supernatant is to new EP pipe.Use sodium acetate, ethanol precipitation and concentrate, being resuspended in appropriate ddH2In O.The probe now obtained is HER-2 specific probe.
Embodiment 4
As a example by HER-2 gene, first use random priming label probe, re-use the spy of mankind Cot-1DNA and synthesis Repetitive sequence is closed by different repetitive sequence.
2, using random primering to prepare HER-2 probe, labelling system is as follows:
System 10μL 20μL 25μL
HER-2DNA N(40-80ng) N(80-160ng) N(100-200ng)
2.5×Buffer 4μL 8μL 10μL
dH2O 1-N 2-N 2.5-N
Lack C dNTP 1.6μL 3.2μL 4μL
Cy3-dCTP 3.2μL 6.4μL 8μL
Klenow Fragment(5U/μL) 0.2μL 0.4μL 0.5μL
Concrete operations are as follows:
1. a step (on ice)
After piping and druming mixing, 95 DEG C of degeneration 10min in PCR instrument, it is immediately placed on 5min on ice, then brief centrifugation.
Two steps (on ice)
Note: fluorescein finally adds, remaining fluorescein keeps in Dark Place in-20 DEG C.
After piping and druming mixing, 37 DEG C of reaction more than 3h in PCR instrument, add 2.5 μ L Stop Buffer and terminate reaction 2- 3min, uses sodium acetate, ethanol precipitation and concentrates, being resuspended in appropriate ddH2In O, HER-2 probe.
2,5 μ L HER-2 probes, 5 μ L centromeric probes (preparation method is similar with HER-2 probe), 5 μ L 2mg/mL people are taken Class Cot-1DNA, 5 μ L 1mg/mL Species specific sequences, 80 μ L hybridization solutions (ratio is 1:1:1:1:16), HER2/ CSP17 double-color probe.

Claims (4)

1. a preparation method for FISH probe, it comprises the steps:
1) before prepared by probe, purpose fragment is carried out sequence analysis, the Species specific sequence that in composition sequence, ratio is higher；
2) one in following scheme is selected to prepare probe:
Species specific sequence and the mankind Cot-1DNA of synthesis are screened removal repetitive sequence simultaneously, obtain specific template or Probe；
Or, utilize the Species specific sequence of synthesis to prepare probe after removing corresponding repetitive sequence, re-use mankind Cot- 1DNA closes, and improves probe specificity；
Or, utilize mankind Cot-1DNA to prepare probe after removing corresponding repetitive sequence, the specificity re-using synthesis repeats sequence Row are closed, and improve probe specificity；
Or, use the Species specific sequence of synthesis and mankind Cot-1DNA to close after probe preparation simultaneously, improve probe Specificity.
The preparation method of a kind of FISH probe the most according to claim 1, it is characterised in that: step 1) described in sequence divide Analysis, uses online sequence analysis software RepeatMasker to carry out.
The preparation method of a kind of FISH probe the most according to claim 1, it is characterised in that: step 2) screening repetitive sequence Method, including: the mankind Cot-1DNA of biotin coupling and/or the Species specific sequence of biotin coupling by affine layer The repetitive sequence of purpose fragment is removed in analysis.
The preparation method of a kind of FISH probe the most according to claim 1, it is characterised in that: step 2) screening repetitive sequence Method, including: mankind Cot-1DNA and/or the preferential renaturation of Species specific sequence repetitive sequence in purpose fragment are combined Become double-strand, with double-stranded specific nuclease excision repetitive sequence.
CN201610584503.7A 2016-07-22 2016-07-22 A kind of preparation method of FISH probe Pending CN106222250A (en)

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Cited By (3)

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CN107099849A (en) * 2017-06-13 2017-08-29 扬州大学 A kind of oligonucleotide library of specific recognition cultivated rice Chromosome 9 whole piece galianconism and recognition methods
WO2018014845A1 (en) * 2016-07-22 2018-01-25 厦门艾德生物医药科技股份有限公司 Preparation method for fish probe
CN112553192A (en) * 2020-12-15 2021-03-26 益善生物技术股份有限公司 Purification membrane, purification column, purification kit and purification method for purifying nucleic acid probe

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WO2002093130A2 (en) * 2001-05-14 2002-11-21 Cancer Genetics, Inc. Methods of analyzing chromosomal translocations using fluorescence in situ hybridization (fish)
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WO2018014845A1 (en) * 2016-07-22 2018-01-25 厦门艾德生物医药科技股份有限公司 Preparation method for fish probe
CN107099849A (en) * 2017-06-13 2017-08-29 扬州大学 A kind of oligonucleotide library of specific recognition cultivated rice Chromosome 9 whole piece galianconism and recognition methods
CN107099849B (en) * 2017-06-13 2019-07-26 扬州大学 A kind of oligonucleotide library of the whole galianconism of specific recognition cultivated rice Chromosome 9 and recognition methods
CN112553192A (en) * 2020-12-15 2021-03-26 益善生物技术股份有限公司 Purification membrane, purification column, purification kit and purification method for purifying nucleic acid probe
CN112553192B (en) * 2020-12-15 2023-07-21 益善生物技术股份有限公司 Purification membrane, purification column, purification kit and purification method for purifying nucleic acid probe

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Application publication date: 20161214