Preparation method for in-situ hybridization probe

Abstract

A preparation method for in-situ hybridization probes as follows: fragmenting objective DNAs, recovering 150-600 bp fragments, and after an enzyme modification, ligating, at intervals, the fragments with DNA adaptors containing restriction enzyme site sequences to large DNA loops and long chains; obtaining and labeling a large amount of DNAs in step A or B: A. isothermal amplifying, adding a single nucleotide substrate with a marker when amplifying, to obtain a DNA product with a marker; or B. isothermal amplifying, doping a single nucleotide substrate with a marker to the obtained product with a nick translation or random primer method, to obtain a DNA product with a marker; and digesting the DNA product with the marker by using corresponding restriction enzyme, to obtain in-situ hybridization probes with lengths of 150-600 bp. The method of the present invention accurately controls length range of the probes, reduces production cost, and improves product quality.

Claims

1. A preparation method for in-situ hybridization probes, comprising the following steps: fragmenting target DNAs, and recovering 150-600 bp small fragments; performing an enzyme modification processing on two ends of each of the 150-600 bp small fragments, to make the two ends blunt, then adding phosphate to 5′ end and dA to 3′ end of each single strand; ligating the small fragments after processed and DNA adaptors alternately to form large DNA loops and long chains, wherein the adaptor has restriction enzyme site sequences, phosphate at the 5′ end and dT at the 3′ end of each single strand; obtaining and labeling the DNAs by: A, isothermal amplifying, mixing a single nucleotide substrate with a marker when amplifying, to obtain a DNA product with the marker; or B, isothermal amplifying, and then mixing a nucleotide substrate with a marker to the obtained DNA product by performing a nick translation method or a random primer PCR method, to obtain a DNA product with the marker; in the step A or B, the marker is a direct marker or an indirect marker; digesting the DNA product with the marker by using a DNA restriction enzyme corresponding to a base sequence of the above DNA adaptor, wherein a length range of the enzyme digested product is 150-600 bp; and obtaining a probe, wherein if the marker in the DNA product with the marker is the direct marker, the obtained enzyme digested product being the in-situ hybridization probe; and wherein if the marker in the DNA product with the marker is the indirect marker, the obtained enzyme digested product being coupled with the direct marker having corresponding reactivity, thereby obtaining the in-situ hybridization probe.

2. The preparation method for the in-situ hybridization probes of claim 1, wherein the obtained in-situ hybridization probe is further purified and quantified.

3. The preparation method for the in-situ hybridization probes of claim 1, wherein the target DNAs are purified target DNAs.

4. The preparation method for the in-situ hybridization probes of claim 1, wherein in the step A or B, a primer used when isothermal amplifying is an oligonucleotide of an adaptor sequence containing restriction enzyme sites or oligonucleotides of random sequences.

5. The preparation method for the in-situ hybridization probes of claim 1, wherein in the step A or B, an enzyme used when isothermal amplifying is a DNA polymerase having chain replacement activity.

6. The preparation method for the in-situ hybridization probes of claim 5, wherein an enzyme used when isothermal amplifying is Phi29 DNA polymerase or Bst DNA polymerase large fragments.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is an agarose gel electrophoresis diagram of an HER2 gene detection FISH probe in Embodiment 2;

(2) FIG. 2 is a FISH experiment result diagram of the HER2 gene detection FISH probe in Embodiment 2 and CEN 17 in Embodiment 1 on a metaphase cell chromosome of a normal person;

(3) FIG. 3 is a FISH experiment result diagram of the HER2 gene detection FISH probe in Embodiment 2 and CEN 17 in Embodiment 1 on an interphase cell chromosome of a normal person; and

(4) FIG. 4 is a FISH experiment result diagram of a chromosome 13 specific FISH probe in Embodiment 3 on a metaphase cell chromosome of a normal person.

DETAILED DESCRIPTION OF EMBODIMENTS

(5) Embodiments of the present invention will be described in details below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference signs indicate the same or similar components or components having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention, but cannot be understood as limitations to the present invention. If specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in the art or according to the product instructions are performed. The used reagents or instruments that are not indicated with manufacturers are all conventional products commercially available.

(6) A first preparation method for an in-situ hybridization probe of the present invention is as follows:

(7) I. fragmenting and processing a purified target DNA to small fragments with a length of about 300 bp;

(8) II. recovering small DNA fragments within the length range of 150-600 bp;

(9) III. performing an enzyme modification processing on two ends of small DNA fragments recovered in step II, to make the two ends blunt, then the 5′ end phosphate added and the 3′ end dA added;

(10) IV. under the action of DNA ligase, ligating the small DNA fragments (subjected to the enzyme modification in step III) and DNA adaptors (which have restriction enzyme site sequence, phosphate at 5′ end and dA at 3′ end) at intervals to form large DNA loops and long chains;

(11) V. by adopting oligonucleotide of the adaptor sequence as a primer, performing LAMP and MDA on the large DNA loops and long chains obtained in step IV, and doping deoxyribonucleotide with a marker simultaneously, such as dUTP-fluorescein or aa-dUTP, to obtain DNA with the marker after amplification;

(12) VI. performing an enzyme digesting on the above obtained DNA with the marker after amplification, by using a restriction enzyme corresponding to the inserted adaptor sequence, to obtain an enzyme digested product with the length range of 150-600 bp; and

(13) VII. purifying and quantifying the above obtained enzyme digested product to obtain the probe, completing preparation of the DNA hybridization probe.

(14) The target DNA in step I is a double-stranded DNA, may be linear or circular, may be in a supercoil state, and may be continuous or may be nicked, and a source includes but is not limited to plasmid, BAC, YAC, PCR products, RT-PCR products, DNA enzyme digested products or ligated products, artificial amplification products and artificial synthetic products.

(15) The ligation satisfies complementation between dT of the 3′ end on the DNA adaptor and dA of a 3′ end of the small DNA fragment.

(16) In step V, if the doped marker is an indirect maker aa, it further needs to add a coupling step between steps VI and VII, and the direct marker is labeled on it.

(17) A second preparation method for an in-situ hybridization probe of the present invention is the same as the above method except that the primer in the above step V is a random primer.

(18) A third preparation method for an in-situ hybridization probe of the present invention is the same as the above two methods except for the following steps:

(19) Step V. not doping the deoxyribonucleotide with the marker when performing isothermal amplifying;

(20) Step VI. doping a single nucleotide substrate with a marker to the DNA obtained in step V with the nick translation method, labeling the DNA;

(21) Step VII. digesting the marked DNA product by adopting a restriction enzyme corresponding to the enzyme digesting site on the adaptor, to obtain an enzyme digested product with the length range of 150-600 bp; and

(22) Step VIII. purifying and quantifying the above obtained enzyme digested product to obtain the probe, completing preparation of the DNA in-situ hybridization probe.

(23) A fourth preparation method for an in-situ hybridization probe of the present invention is the same as the third preparation method for the in-situ hybridization probe except for the following step:

(24) Step VI. doping the single nucleotide substrate with the marker to the DNA obtained in step V with random primer method, labeling the DNA.

(25) A fifth preparation method for an in-situ hybridization probe of the present invention is the same as the fourth preparation method for the in-situ hybridization probe except for the following step:

(26) Step VI. for the DNA obtained in step V, adopting oligonucleotide of the adaptor sequence as a primer to replace the random primer, labeling the DNA.

Embodiment 1: Preparation of Human Chromosome 17 Enumeration FISH Probe (CEN17)

(27) I. Genomic DNA of a normal person (which is commercially available) is used as a template, a 0.85 kb PCR product of chromosome 17 centromeric α satellite repetitive sequence is obtained according to a method described in the article of Warburton et al. (Peter E. Warburton, Gillian M. Greig, Thomas Haaf, and Huntington F. Willard. PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis. GENOMICS 11: 324-333 (1991)).

(28) The PCR product is purified with the QIAquick™ PCR purification kit.

(29) II. 1 μg of DNAs obtained in the above step are fragmented to a length of about 300 bp by using a focused ultrasonic method. Through 2% agarose gel electrophoresis with 100 bp DNA ladder as a reference, DNA fragments within the length range of 150-600 bp are cut from the gel and recovered with a gel extraction kit (QIAquick™ Gel Extraction Kit, Qiagen).

(30) III. Enzyme modification processing: by adopting NEBNext® Ultra End Repair/dA-Tailing Module of the New England Biolabs company, the ends of the DNA fragments recovered in step II are blunted according to operation steps of the instruction, the 5′ ends thereof are phosphorylated, and dA is added at the 3′ ends, and then the modified DNA fragments are recovered with a PCR Purification Kit (QIAquick™ PCR Purification Kit, Qiagen).

(31) IV. Ligating into large loops and long chains: the adaptor, as below, containing DNA restriction enzyme Sac II site sequences is ligated with the modified DNA fragments recovered in step III.

(32) The adaptor is double-stranded, and the 5′ ends thereof are all phosphorylated, and the sequences are shown as follows:

(33) TABLE-US-00001 5′-pCCGCGGT-3′ 3′-TGGCGCCp-5′.

(34) Steps of the ligation with the adaptor are as follows:

(35) The following ligating system is adopted, a microcentrifuge tube is placed on an ice, 50 μl of the following reaction solution (a reaction volume may be expanded in proportion) is added, gently mixed by using a pipettor, briefly centrifuged, and then placed at 16° C. overnight.

(36) TABLE-US-00002 10 times of T4 DNA ligase reaction buffer solution 5 μl; DNA fragments recovered in step VI 100 ng; Adaptor 3 ng; T4 DNA ligase 50 u; Deionized water filling up to 50 μl.

(37) Purify and recover the ligated product with the QIAamp™ DNA Blood Kit of Qiagen. Non-ligated adaptor is removed. Measure the OD value of the purified DNA. A large-loop and long-chain DNA probe template is obtained.

(38) V. Obtaining of a large amount of marked DNAs: the probe template DNAs obtained in step VI are amplified in a large amount while doping a marker dUTP-FITC (fluorescein isothiocyanate) (which may also be an indirect marker aa-dUTP. If it is so, it needs to add a coupling reaction with FITC SE after step VI. Please refer to the embodiments described later for details), the inserted sequence CCGCGGT is used as a primer, and the reaction system and procedure are as follows (the reaction volume may be increased in proportion):

(39) TABLE-US-00003 DNA probe template 1 ng; 10 times of buffer solution 1.5 μl Primer 100 μM (final concentration in 15 μl) Deionized water adding up to 15 μl;

(40) Put it at 96° C. for 2 minutes and then at a room temperature for 10 minutes. Thereafter, it is placed on ice, the following components are then added so that the final volume is 50 μl, and they are mixed well:

(41) TABLE-US-00004 10 times of buffer solution 5 μl DMSO 2 μl BSA 2 μg dNTP (dUTP-FITC:dTTP = 3:1) 400 μM (final concentration in 50 μl of the reaction system) Bst DNA polymerase, 20 μ large fragments Deionized water adding up to 50 μl

(42) Then, they are placed at 60° C. for 6 hours, and then at 80° C. for 15 minutes.

(43) The QIAamp™ DNA Blood Kit of Qiagen is adopted to perform purifying and recovering on the product. OD value is measured.

(44) VI. Enzyme digesting: the produced obtained in step V is digested by the DNA restriction enzyme Sac II to obtain an enzyme digested product with a length range of 150-600 bp.

(45) The enzyme digesting reaction system is (the reaction volume may be increased or reduced in proportion) as follows:

(46) TABLE-US-00005 Product obtained in step V 100 μg Sac II 1000 u (unit) Deionized water reaching up to 1000 μl

(47) The above enzyme digesting reaction system is placed at 37° C. for 16 hours;

(48) VII. The enzyme digested product is purified with a PCR purification kit (QIAquick™ PCR Purification Kit, Qiagen). Measure the OD value. A chromosome 17 enumeration FISH probe (CEN 17) labeled with green fluorescein FITC is obtained.

Embodiment 2: Preparation of Human Epidermal Growth Factor Receptor 2 (HER2) Gene Detection FISH Probe

(49) Embodiment 2 is different from the above Embodiment 1 in that the DNA is from BAC, the amplification and labeling are performed separately, a 6-base random primer (included in the kit used) is used when amplifying, after amplification, a marker is doped using nick translation method, and the doped marker is a direct maker or an indirect marker.

(50) I. According to the position of HER2 gene on chromosome, a bacterial artificial chromosome (Bacterial Artificial Chromosome, BAC) clone: RP11-909L6 is purchased from the ThermoFisher Scientific company, wherein the HER2 gene and its adjacent chromosome DNAs are included, and the length thereof is about 185 kb.

(51) An end sequencing method is adopted to sequence hundreds of bases at two ends of the BAC clone insertion. The sequencing result is compared with the known sequence in NCBI data base for verification.

(52) II. 1 μg of BAC DNA is fragmented by using a focused ultrasonic method and the 150-600 bp DNA fragments are recovered, wherein the method is the same as that in Embodiment 1.

(53) III. Enzyme modification processing: the method is the same as that in Embodiment 1.

(54) IV. Ligating the DNA fragments recovered in step II and adaptor containing a DNA restriction enzyme Sma I site sequence;

(55) The adaptor is double-stranded, and the 5′ ends thereof are all phosphorylated. The sequences are shown as follows:

(56) TABLE-US-00006 5′-pCCCGGGT-3′ 3′-TGGGCCCp-5′;

(57) Ligating with the adaptor: the method is the same as that in Embodiment 1.

(58) V. Obtaining of a large amount of DNAs: the DNA obtained in step IV is used as a template for a large amount of DNA amplification, the REPLI-g Single Cell Kit of Qiagen (the principle thereof being LAMP and MDA, adopting Phi29 DNA polymerase, and adopting 6-base oligonucleotides of random sequences as primer) is adopted to operate according to the instruction, and the amplified product is purified and recovered by adopting the QIAamp™ DNA Blood Kit of Qiagen.

(59) VI. Labeling of the DNA: the amplified product obtained in step V is used as a template, and a direct marker dUTP-TA MRA™ (carboxytetramethylrhodamine) or an indirect marker aa-dUTP is doped by nick translation, wherein the method is as follows:

(60) TABLE-US-00007 Purified amplified product 100 μg dNTP 0.2 mM (final (dUTP-FITC:dTTP = 3:1) concentration in the reaction system) DNA polymerase I 500 u DNA enzyme I 0.001 u Deionized water reaching up to 1000 μl

(61) The above mixture is placed at 30° C. for 16 hours; and

(62) Purify and recover the labeled products with the QIAamp™ DNA Blood Kits of Qiagen. Measure the OD value of the purified DNA.

(63) VII. Enzyme digesting: the DNAs obtained in step VI are enzyme digested by restriction enzyme Sma I, to obtain an enzyme digested product with a length range of 150-600 bp.

(64) The enzyme digesting reaction system is as follows:

(65) TABLE-US-00008 DNA 100 μg Sma I 1000 u (unit) Deionized water reaching up to 1000 μl

(66) Incubate at 37° C. for 16 hours.

(67) VIII. The PCR purification kit of Qiagen is adopted to purify the enzyme digested product obtained in step VII and measure the OD value. If the direct marker is doped in step VI, then the HER2 probe labeled with the orange-red fluorescent TAMRA™ is obtained; and if the indirect marker is doped in step VI, and then a step of IX is needed.

(68) IX. Coupling between the aa doped in the DNA and TAMRA™ SE, and referring to the following paper or product instruction (the reaction volume may be correspondingly expanded in proportion):

(69) W. Gregory Cox and Victoria L. Singer. Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling. BIOTECHNIQUES 36: 114-122 (January, 2004).

(70) X. After the product is purified through ethanol precipitation, the OD value is measured, and the HER2 probe labeled with the orange-red fluorescent TAMRA™ is obtained.

(71) Explanation about Embodiment 2: the characteristics of the nick translation method lie in rapidness, convenience, homogeneity of labeling, high specificity, and higher labeling rate of probe than that of probe labeled with the Bst DNA polymerase; and the indirect labeling saves cost in comparison with the direct labeling.

Embodiment 3: Preparation of Human Chromosome 13 Specific FISH Probe

(72) Embodiment 3 is different from the above Embodiments 1 and 2 in that: the target DNA is from the entire chromosome, random primer is adopted, isothermal amplifying and marking are performed simultaneously, and the doped marker is an indirect marker.

(73) I. Purchase or self-provide (the method is omitted) peripheral blood metaphase lymphocytes of a normal person.

(74) II. The cytogenetic chromosome banding technique is adopted to identify and number chromosomes under a microscope, and ten complete chromosomes 13 are scraped and collected from a slide.

(75) III. Obtaining of a large amount of DNAs: the chromosomes obtained in step II are used as a DNA source, the REPLI-g Single Cell Kit of Qiagen is adopted to perform a large amount of DNA amplification according to the instruction, and the amplified product is purified and recovered by the QIAamp™ DNA Blood Kit of Qiagen.

(76) IV. 1 μg of DNAs obtained in the above step is fragmented by using a focused ultrasonic method and 150-600 bp DNA fragments are recovered. The method is the same as that in Embodiment 1.

(77) V. Enzyme modification processing: the method is the same as that in Embodiment 1.

(78) VI. An adaptor containing a DNA restriction enzyme site sequence is ligated with the recovered DNA fragments after the modification in step V to obtain a DNA probe template. The method is the same as that in Embodiment 1.

(79) VII. Obtaining of a large amount of marked DNAs: the DNA template obtained in step VI is used to perform a large amount of isothermal amplifications while doping the indirect marker aa-dUTP simultaneously, and the 6-base random oligonucleotide (the sequence is NNNNNN, wherein N denotes A or T or G or C) is used as a primer. The reaction system and procedure are as follows (the reaction volume may be increased in proportion):

(80) TABLE-US-00009 DNA probe template 1 ng; 10 times of buffer solution 1.5 μl Primer 50 μM (final concentration in 15 μl) Deionized water adding up to 15 μl;

(81) Incubate at 96° C. for 2 minutes, and then at a room temperature for 10 minutes. Thereafter, it is placed on ice, the following components are then added so that the final volume is 50 μl, and mixed well:

(82) TABLE-US-00010 10 times of buffer solution 5 μl DMSO 2 μl BSA 2 μg dNTP 400 μM (final (aa-dUTP:dTTP = 3:1) concentration in 50 μl of the reaction system) Bst DNA polymerase 20 μ large fragments Deionized water adding up to 50 μl

(83) Incubate at 60° C. for 6 hours, and then at 80° C. for 15 minutes.

(84) Purify and recover the products with the QIAamp™ DNA Blood Kits of Qiagen. Measure the OD value of the purified DNA.

(85) VIII. The DNAs obtained in step VII are enzyme digested by using a restriction enzyme to obtain 150-600 bp fragments. The method is the same as that in Embodiment 1.

(86) IX. The QIAquick™ PCR Purification Kit of Qiagen is adopted to purify the enzyme digested product. Measure the OD of the purified.

(87) X. Coupling between the aa doped in the DNA and TAMRA™ SE: the method is the same as that in Embodiment 2.

(88) XI. After the product is purified through ethanol precipitation, the OD value is measured and the chromosome 13 specific FISH probe labeled with the orange-red fluorescent TAMRA™ is obtained.

Embodiment 4: Effect Verification Experiment

(89) Agarose gel electrophoresis is performed for the HER2 gene detection FISH probe labeled with orange-red fluorescein TAMRA™ obtained in Embodiment 2, and the result is shown in FIG. 1. FIG. 1 is the result of agarose gel electrophoresis of the obtained HER2 probe, wherein lane 1 is a DNA ladder of 100 bp, and lane 2 is the probe. It shows that the probe length is within the design range of 150-600 bp, meeting the design requirements.

(90) Preparation of HER2 Gene Detection FISH Probe Hybridization Solution

(91) The components of the HER2 gene detection FISH probe hybridization solution per 10 μl are as follows: 20 ng of the HER2 gene detection FISH probe labeled with orange-red fluorescein TAMRA™ obtained in Embodiment 2, 10 ng of chromosome 17 enumeration probe CEN17 obtained in Embodiment 1, 1 μg of human COT-1 DNA (purchased from ThermoFisher Scientific), 50% volume ratio of the deionized formamide, 2×SSC, and 10% weight and volume ratio of dextran sulfate.

(92) Preparation of Chromosome 13 Specific FISH Probe Hybridization Solution

(93) The components of the chromosome 13 specific FISH probe hybridization solution per 10 μl are as follows: 20 ng of the chromosome 13 specific FISH probe labeled with orange-red fluorescein TAMRA™ obtained in Embodiment 3, 1 μg of human COT-1 DNA (purchased from ThermoFisher Scientific), 50% volume ratio of the deionized formamide, 2×SSC, and 10% weight and volume ratio of dextran sulfate.

(94) In-Situ Hybridization:

(95) Chromosome slides are conventionally prepared from metaphase cell suspension. The slide is dehydrated and dried in serial ethanol and digested with pepsin. 10 μl of the above HER2 gene detection FISH probe hybridization solution or chromosome 13 specific FISH probe hybridization solution is added to the hybridization area on slide. It is covered with a coverslip and sealed with rubber water, and is denatured at 78° C. for 5 minutes, and hybridized at 37° C. overnight in a wet box. Then, the slide is washed according to the following sequence: in 4×SSC solution, shaking and rinsing for 5 minutes; in 2×SSC and 0.1% volume ratio of Tween-20 solution, shaking and rinsing for 4×2.5 minutes; in 0.1×SSC solution, shaking and rinsing for 5 minutes; dehydrating and drying in serial ethanol. 20 μl DAPI counterstain is added by droplets to the hybridization area, and it is immediately covered with a coverslip. A suitable filter is selected to observe, photograph and record the results under a fluorescence microscope.

(96) The experiment results are shown in FIGS. 2, 3 and 4. FIG. 2 is a FISH experiment result of the HER2 gene detection FISH probe on chromosome of a metaphase cell (from peripheral blood mononuclear cells) cultured in vitro from a normal person; wherein 1 denotes a signal generated by the HER2 gene detection FISH probe, and 2 denotes a signal generated by the chromosome 17 enumeration in-situ hybridization probe CEN17.

(97) FIG. 3 is a FISH experiment result diagram of the HER2 gene detection FISH probe on an interphase cell (from peripheral blood mononuclear cells) cultured in vitro from a normal person; wherein 1 denotes a signal generated by the HER2 gene detection FISH probe, and 2 denotes a signal generated by the chromosome 17 enumeration in-situ hybridization probe CEN17.

(98) It can be seen from FIGS. 2 and 3 that the CEN17 SIGNAL is located at a centromere of the chromosome 17, and the HER2 signal is located near the centromere of the chromosome 17 (zone 1 and band 2, 17q12). There are two signals in normal cells, meeting the design requirements and expected result.

(99) FIG. 4 is a FISH experiment result of the chromosome 13 specific FISH probe on chromosome of a metaphase cell (from peripheral blood mononuclear cells) cultured in vitro from a normal person, and a pair of the whole chromosomes 13 show fluorescent signals, meeting the design requirements and expected result.

(100) Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be understood as limitations to the present invention or as limitations to the application scope of probe products obtained according to the present invention. Those skilled in the field can make changes, modifications, replacements and transformations to the above embodiments within the scope of the present invention without departing from the principle and purpose of the present invention.