WO2017114010A1 - Top2a gene detection probe, preparation method therefor, and test kit - Google Patents

Abstract

Disclosed are a TOP2A gene detection probe, and a preparation method therefor, the method comprising the following steps: selecting a BAC clone to be at least one of RP11-1152A10 and CTD-3217C5, or selecting the BAC clone to be at least one of RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5; respectively performing plasmid extraction on the clones, obtaining plasmid DNA, and quantifying; labelling with fluorescein. Also disclosed is a test kit including the TOP2A gene detection probe.

Description

TOP2A gene detection probe and preparation method and kit thereof Technical field

The invention belongs to the biotechnology, in particular to a TOP2A gene detection probe and a preparation method and kit thereof.

Background technique

The TOP2A gene is a gene encoding DNA topoisomerase II alpha (TOPIIα), the biological role of DNA topoisomerase, and the first is to regulate the supercoiled state of DNA and the ligated or unknotted DNA. State, which indirectly affects the process of nucleic acid metabolism in cells; second, it is directly involved in the process of cellular processes, DNA recombination, repair, transcription and replication that need to be interrupted and reattached. Patients with abnormal TOP2A gene have poor prognosis and shortened recurrence-free survival, especially in patients with TOP2A gene deletion. Gene amplification suggests a recurrence of the tumor, or a long-term decline in efficacy.

Anthracyclines are the cornerstone of postoperative adjuvant chemotherapy for breast cancer, and studies have confirmed that TOP2A gene is associated with anthracycline efficacy [Brase, JC, Schmidt, M, Fischbach, T, Sultmann, H, Bojar, H, Koelbl, H (2010) ERBB2and TOP2A in breast cancer: a comprehensive analysis of gene amplification, RNA levels, and protein expression and their influence on prognosis and prediction. Clin Cancer Res 16:pp.2391-2401, Fountzilas, G, Christodoulou, C, Bobos, M , Kotoula, V, Eleftheraki, AG, Xanthakis, I (2012) Topoisomerase II alpha gene amplification is a favorable prognostic factor in patients with HER2-positive metastatic breast cancer treated with trastuzumab.J Transl Med 10: pp. 212, Bartlett J M S, McConkey C C, Munro A F, et al. Predicting Anthracycline Benefit: TOP2A and CEP17—Not Only but Also[J].Journal of Clinical Oncology, 2015: JCO.2013.54. 7869.], TOP2A gene status changes are associated with the risk of tumor recurrence and patient survival.

Therefore, it is important to correctly detect and assess the TOP2A status of breast cancer. The TOP2A gene is located in the 17q21 region of chromosome 17, and the abnormalities of TOP2A are classified into two cases of amplification and deletion. FISH detection can make a clear judgment for these two cases.

Fluorescence in situ hybridization (FISH) is a non-radioactive in situ hybridization technique developed on the basis of the original radioactive in situ hybridization technique in the late 1980s. At present, this technology has been widely used in animal and plant genomic structure research, chromosome fine structure variation analysis, viral infection analysis, human prenatal diagnosis, tumor genetics and genome evolution research in many fields. The basic principle of FISH is to use a known labeled nucleic acid as a probe to heterologously bind to an unknown single-stranded nucleic acid in a material to be tested according to the principle of base complementation to form a hybrid double-stranded nucleic acid which can be detected. Since the DNA molecules are linearly arranged along the longitudinal axis of the chromosome on the chromosome, the probe can be directly hybridized to the chromosome to localize the specific gene on the chromosome. Compared with traditional radiolabeled in situ hybridization, fluorescence in situ hybridization has the characteristics of rapid detection signal, high hybridization specificity and multi-staining, so it has received widespread attention in the field of molecular cytogenetics.

The probes used for hybridization can be roughly classified into three categories: 1) chromosome-specific repeat probes, such as alpha satellites, satellite class III probes, which often have a hybrid target of more than 1 Mb, do not contain scattered repeats, and bind tightly to the target. Strong hybridization signal, easy to detect; 2) whole chromosome or chromosomal region-specific probe consisting of a very different nucleotide fragment on a chromosome or a segment of a chromosome, which can be cloned into phage and plasmid A chromosome-specific large fragment is obtained; 3) a specific position probe consisting of one or several cloned sequences.

The fluorescein labeling of the probe can be performed by direct and indirect labeling. Indirect labeling is performed by biotin-labeled DNA probes, which are detected by fluorescein avidin or streptavidin after hybridization. At the same time, the avidin-biotin-fluorescein complex can be used to amplify the fluorescent signal, so that a fragment of about 500 bp can be detected. The direct labeling method is to directly bind fluorescein to the probe nucleotide or the pentose phosphate backbone, or to incorporate fluorescein nucleoside triphosphate in the nick translation labeling probe. The direct labeling method has simple steps in detection and is convenient for clinical use.

At present, there is a lack of a highly specific detection kit for the detection of the TOP2A gene FISH method.

Summary of the invention

One of the objects of the present invention is to provide a TOP2A gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting the TOP2A gene state, that is, detecting the finger copy number change of the TOP2A gene, and has good specificity.

The technical solution for achieving the above object is as follows.

A method for preparing a TOP2A gene detection probe comprises the following steps:

(1) selecting a BAC clone as at least one of RP11-1152A10 and CTD-3217C5, or selecting a BAC clone as at least one of RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5;

(2) separately extracting a plasmid from the clone to obtain a plasmid DNA, and quantifying;

(3) The plasmid DNA is labeled with fluorescein, and the fluorescein labeled by the plasmid DNA of different sources is the same, that is, it is obtained.

In one embodiment, the BAC clones are RP11-1152A10 and CTD-3217C5.

In one embodiment, the BAC clones are RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5.

In one embodiment, the labeled fluorescein selects a fluorescent dye known in the art, preferably fluorescein is Alexa

FITC, Alexa

Rhodamine, Texas Red, pacific

DEAC.

In one embodiment, the labeling of the gene probe can be performed by labeling the corresponding fluorescein to the double-stranded nucleic acid by methods in the prior art, including but not limited to: random primer method, nick translation, etc., marker gene probe The needle can be a commercially available nick translation labeling kit and/or a random primer labeling kit, preferably abbott and/or Roche's Nick Translation Kit. In the step (3) of the present invention, the plasmid DNA is preferably subjected to fluorescein labeling by a random primer method or a nick translation method.

In one embodiment, the label has a temperature of from 24 ° C to 26 ° C and a marked time of from 4 to 6 hours.

Another object of the present invention is to provide a TOP2A gene detecting kit.

The technical solution for achieving this purpose is as follows.

A TOP2A gene detection kit comprising the above TOP2A gene detection probe.

In one embodiment, a chromosome 17 discrimination probe (CSP17) probe for internal control is included, the identification probe being different in color from the fluorescein labeled by the TOP2A gene detection probe.

In one embodiment, there is also a COT Human DNA for blocking the repeat sequence, and a DAPI counterstain.

The invention has the following beneficial effects:

1. The present invention detects the TOP2A gene copy number by FISH (Fluorescence In-Situ Hybridization) method by screening the optimal TOP2A gene detection probe and its combination.

2. The preferred clone of the present invention has good detection specificity and high sensitivity. By adjusting the label temperature and time, the length probe is limited to about 500 bp, which improves the hybridization efficiency and reduces the hybridization background.

3. The signal counting line is accurate and fast, and the result is reproducible. The lack of clinical detection of TOP2A mutation is beneficial to screening more patients who benefit from targeted drugs and improving the survival rate and overall survival of breast cancer patients.

4. Through the TOP2A kit of the present invention, the TOP2A state change is known from the gene level, various The signal type shows the genetic diversity of tumor cells in solid tissues, which can be applied in the fields of tumor biology and cytogenetics. It can help comprehensively evaluate each molecular marker and assist in the selection of clinical targeted therapy drugs and treatment options for breast cancer.

DRAWINGS

Figure 1 is a schematic illustration of the detection probe sequence in Example 1.

Fig. 2 is a graph showing the results of FISH detection of human peripheral blood cultured cell sheets in Example 1.

Fig. 3 is a graph showing the results of FISH detection of breast cancer tissue samples in Example 4, wherein the detection signal type is 2R2G, and the TOP2A gene is not amplified.

4 is a diagram showing the results of FISH detection of breast cancer tissue samples in Example 4, wherein the detection signal type is 6-12R2G, and the TOP2A gene is amplified.

detailed description

In order to facilitate the understanding of the present invention, the present invention will be described more fully hereinafter. The invention can be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the understanding of the present disclosure will be more fully understood.

Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning The terms used in the description of the present invention are for the purpose of describing the specific embodiments and are not intended to limit the invention. The term "and / or "includes any and all combinations of one or more related listed items.

Example 1 Preparation of TOP2A gene detection probe

The preparation method of the TOP2A detection probe of the present embodiment includes the following steps.

(1) A clone containing the TOP2A gene of interest and the sequences at both ends was selected, as shown in FIG.

GSP TOP2A includes two groups, including a first probe, a second probe, a third probe, and a fourth probe, respectively. Specifically, the following table is purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries. The following two sets of detection probes were separately prepared.

TOP2A chr17: 38, 544, 773-38, 574, 202, 29, 430 bp

Probe set 1	BAC	Insert start and end position
First probe	RP11-1152A10	Chr17:38376940...38546337(169Kb)
Second probe	CTD-3217C5	Chr17:38521566...38730400(209Kb)

Probe set 2	BAC	Insert start and end position
Third probe	RP11-737D6	Chr17:38257742...38440899(183Kb)
Fourth probe	CTD-3087O22	Chr17:38430841...38564776(134Kb)
Fifth probe	RP11-48O10	Chr17:38564771...38724970(160Kb)
Sixth probe	CTD-2134K5	Chr17:38716958...38834072(117Kb)

(2) Preparation of GSP TOP2A gene detection probe: Ultra-low copy plasmid DNA extraction was performed on different BAC clones using Qiagen's Plasmid Maxi Kit according to the instructions, and the plasmid DNA was quantified by measuring the absorbance at 260 nm and 280 nm. Diluted to 200 ng/ul with auto-sterilized ultrapure water, and dispensed in a 1.5 ml centrifuge tube. Finally, the obtained two or four kinds of plasmid DNA were mixed and sealed at -20 °C.

(3) The plasmid DNA mixture was fluorescently labeled by a nick translation method, and each probe labeled fluorescein was Spectrum-Orange. Using abbott's Nick Translation Kit, strictly as protected from light The PCR reaction system was prepared on ice under the conditions.

After mixing, mix and shake, mark at 25 ° C for 5 hours, then incubate at 80 ° C for 10 minutes to inactivate the enzyme. Take 5 ul of 2% agarose gel for electrophoresis, and there is a diffuse band around 500 bp.

The labeled product was subjected to ethanol precipitation and concentration, and sodium acetate and absolute ethanol were sequentially added to a 1.5 ml centrifuge tube in the following manner, and protected from light and ice:

Labeled product 45ul

Sodium acetate (3mol/L) 5ul

Anhydrous ethanol 125ul

After mixing, put in a -70 ° C refrigerator for at least 2 hours, centrifuge at 13000 rpm for 30 minutes at 4 ° C, carefully remove the supernatant, do not stir the precipitate, add 1 ml of 70% ethanol, centrifuge at 13000 rpm for 15 minutes at 4 ° C, carefully go Clear, do not stir the sediment, dry away from light. The precipitate was dissolved in 1 ul of purified water to obtain a GSP TOP2A gene probe, which was stored at -20 ° C in the dark.

(4) GSP TOP2A gene probe verification: Two sets of probes were used, respectively, and the two sets of probes were verified by human normal division metaphase lymphocyte droplets as test samples (the detection method refers to the prior art and Example 3). The cultured cells contain metaphase or interphase chromosomal DNA. When fluorescent in situ hybridization, the chromosomal DNA appears as a morphologically recognizable chromosome or nucleus. The results of the two sets of probes are the same, as shown in Figure 2: Figure of FISH hybridization results of metaphase chromosomes. In the figure, the position of chromosome 17q21 can be seen to display a red fluorescent signal, and the internal control probe CSP17 (Chromosome No. 17 discriminating probe can be labeled with chromosome 17, purchased from SE17, D17Z1 (KBI-20017, KREATECH) shows green fluorescence. The TOP2A gene probe signal is bright, and the sensitivity and specificity of 100% can be observed on the metaphase chromosome in human peripheral blood cultured cell sheets; the TOP2A gene copy number can be clearly recorded by using the paraffin sample piece for hybridization detection.

Example 2: Preparation method of TOP2A gene detection kit

The TOP2A gene detection kit includes two components of a TOP2A hybridization solution and a DAPI counterstaining agent, wherein the TOP2A hybridization solution comprises the GSP TOP2A gene probe described in Example 1 (two sets of detection probes respectively, corresponding to two kits) ), CSP17 probe (Chromosome 17 probe), buffer component for hybridization environment (promoting hybridization), COT Human DNA for blocking repeats, and the like. DAPI counterstaining agent is mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, so that the nucleus shows blue fluorescence, and the counterstaining agent containing p-phenylenediamine can maintain fluorescence stability.

The specific formula is as follows:

(1) Preparation of hybridization solution

(2) DAPI counterstain preparation

10mg of p-phenylenediamine dissolved in 1ml of PBS, adjusted to pH 9.0, added 9ml of glycerin, repeated shocks Mix well and store at -20 °C. 2.5 μl of DAPI solution (0.1 mg/ml) was dissolved in 1 ml of anti-fade solution, and shaken and shaken repeatedly in the dark, and stored at -20 °C in the dark.

(3) Finished product assembly

Component name	Specification/10test	Quantity
Hybrid solution	100μl / tube	1 tube
DAPI counterstain	100μl / tube	1 tube
Instruction manual		1 serving

Example 3: Detection method of TOP2A gene detection kit

1, slide pretreatment

1.1 slides are placed in a 65±5°C incubator for overnight baking;

1.2 Remove the slide, put it into xylene and dewax it for 15 minutes at room temperature;

1.3 Remove the slide, then put it into another cylinder of xylene and continue dewaxing for 15 minutes at room temperature;

1.4 Remove the slide, then place it in absolute ethanol for 10 minutes at room temperature to remove residual xylene;

1.5 Remove the slide, and then put it into 100%, 90%, 70% gradient ethanol room temperature for 3 minutes each time;

1.6 Remove the slide, then put it in purified water for 3 minutes at room temperature, and use the lint-free paper towel to absorb excess water;

1.7 Remove the slide and place it in purified water at 100±5 °C for 25 minutes (the slice is placed horizontally in the container with the sample side facing up);

1.8 Remove the slide and let it dry at room temperature;

1.9 Place the slide face up on the shelf, add appropriate amount of pepsin reaction solution in the sample area, and digest for 5 to 15 minutes;

1.10 remove excess liquid and place it in room temperature 2 × SSC for 5 minutes;

1.11 remove the slide and place it in another cylinder at room temperature 2 × SSC for 5 minutes;

1.12 remove the slide, and then put it into room temperature 70%, 90%, 100% gradient ethanol dehydration for 3 minutes each;

1.13 Remove the slide and allow to dry at room temperature.

The reaction time of pepsin needs to be determined by preliminary tests. Samples prepared in the same batch can be pre-tested as described, usually at intervals of 5 minutes. For example, the digestion time is 5 minutes, 10 minutes, and 15 minutes, respectively. After the "slide pretreatment" is completed, the tissue digestion state can be observed under a bright field using a 10× or 20× objective lens; or DAPI counterstaining can be directly performed. The digestion state is judged.

2. Simultaneous denaturation of samples and probes (light protection operation)

2.1 The hybridization solution in the test kit described in Example 2 was taken out from the refrigerator at -20 ± 5 ° C, shaken and mixed, and centrifuged instantaneously;

2.2 Add 10 μl of the hybridization solution to the hybridization area, quickly cover the 18×18mm coverslip, and gently press to make the hybridization solution evenly distributed to avoid air bubbles;

2.3 Use rubber rubber to seal the edge of the cover glass to completely cover the contact between the cover glass and the slide;

2.4 Place the slide into the hybridization apparatus, wet the humidity strip of the in situ hybridization instrument, insert the wet strip, cover the top of the hybridization apparatus, set the “Denat&Hyb” program, denature at 85 ° C for 5 minutes, and hybridize at 37 ° C for 10 to 18 hours. (If there is no hybrid instrument, you can use alternative instruments, such as constant temperature hot stage for denaturation, electric heating oven / or water bath for hybridization, pay attention to accurate temperature and maintain hybrid humidity).

3. Washing and counterstaining after hybridization (protection from light)

3.1 30 minutes before washing, the prepared lotion I, lotion II, placed in a water bath of 37 ± 1 ° C, measured to ensure that the temperature is appropriate;

3.2 Turn off the power of the hybridization instrument, take out the slide, gently remove the rubber glue, and remove the cover slip. (If the cover slip is difficult to remove, it can be shaken in the wash solution I to facilitate its falling off;

3.3 slides were placed in 37 ± 1 ° C lotion I (2 × SSC) for 10 minutes;

3.4 remove the slide, and then put it into 37 ± 1 ° C lotion II (0.1% NP-40/2 × SSC) for 5 minutes;

3.5 Remove the slides and let them stand in 70% ethanol for 3 minutes at room temperature;

3.6 Remove the slide and naturally dry the slide in the dark;

3.7 Room temperature, add 10μl DAPI counterstain to 22×22mm coverslip, the target area of the slide is facing down, put it on the cover slip, gently press to avoid air bubbles, store in the dark, to be observed.

The above listed reagents were all prepared in a circular dyeing tank (40 ml each), and up to 5 slices per dyeing tank. For non-room temperature solutions, pre-heat the reagents to the specified temperature before starting the operation. During the washing process, the dyeing tank can be gently shaken at intervals of 2 to 3 minutes to improve the washing effect.

4, the result analysis

The relevant fluorescence and DAPI need to be observed with a suitable filter block. Among them, the CSP17 probe shows a green signal; the GSP TOP2A probe is a red signal.

4.1 Use a suitable filter, look under a 40× objective, and count under a 100× objective;

4.2 Adjust the appropriate focal length, have a clear concept of signal and background; the signal point is located in the cell; when there is a fluorescent signal point outside the cell, it should be distinguished from the intracellular signal point, it is best to avoid the area for counting;

4.3 Sweeping several tumor cell areas, selecting at least 4 areas with good nuclear boundaries, requiring complete nuclear boundary, DAPI staining uniform, no nuclear overlap, CSP17 probe (green signal point) signal clear;

4.4 Start the analysis from the upper left corner of the selection area, scan from left to right, and observe multiple fields of view;

4.5 Organizational Counting Requirements:

a. Count only tumor tissue (use of HE stained tablets for control observation before FISH detection)

b. Avoid counting in areas with necrotic areas and unclear nuclear boundaries

c. Subjective discrimination is not counted

d. skip the weak signal and the core count without a specific signal or high background;

4.6 Go to the 100× objective lens, adjust the focal length, and find all signal points at different levels of the core;

4.7 Count signal points in each core; focus to find all signal points in each core, count two signals in one region, count only one or more FISH signals for each color, no signal or only The core of a color signal is not counted; the total number of cells observed (signal normal and abnormal) is recorded;

4.8 counting method

A total of 40 to 100 tumor cell nuclear GSP TOP2A (red) and CSP17 (green) signals were counted in five clear tumor regions, and the number of GSP TOP2A and CSP 17 signals in individual nuclei was counted.

Example 4: Clinical evaluation of TOP2A gene detection kit

Using the two sets of detection probes described in Example 1, the test kit described in Example 2 was tested on 20 clinical samples (which were confirmed by pathological examination, see the table below). According to the detection method of Example 3, the detection was repeated 3 times, the results were consistent, and the repeatability of the detection results was good; the detection consistency of the two probe combinations was good. Compared with commercially available commercial reagents, the test results are completely consistent, and the specificity and sensitivity of the reagents are high. 3 and 4 are graphs showing the results of the probe set 1. The results of probe set 2 were the same as those of probe set 1, and the figures are omitted. Figure 3 shows the results of the negative sample test. The typical signal type is 2R2G. The result is that the TOP2A gene is not amplified. Figure 4 shows the positive sample detection result. The signal type is 2~6R/2G. The result is judged as TOP2A gene amplification. The red signal in the figure shows GSP TOP2A, and the green signal shows CSP 17 (used to locate the centromere probe on chromosome 17).

In the present invention, the detection can also be achieved by using one of the TOP2A genes, respectively, and the specific results are omitted. However, the use of the combined probes and the detection signal are better with respect to the combination of the probes. Theoretically, the longer the length of the probe, the brighter the fluorescence signal obtained during actual detection, but because more gene sequences may be involved, the complexity of the resulting signal is increased, and the difficulty of detection is also enhanced. The total lengths of the BAC clones of the detection probes of Group 1 and Group 2 for the TOP2A gene of the present invention are: 353 Kb and 576 Kb, respectively, which are nucleic acid mixtures comprising the TOP2A gene and its both ends.

The inventors found in the verification of the probe of the present invention that the longer detection probe did obtain a stronger fluorescent signal, and the same result was obtained in the verification of the clinical sample. Therefore, in the design of the fluorescent probe, the signal brightness can be increased by appropriately extending the length of the fluorescent probe, but how to use it in combination, there are certain technical difficulties, and a good detection result is to be achieved, in addition to the experience in design. It is also necessary to verify the difference in signal type through clinical sample verification.

The technical features of the embodiments may be combined in any combination. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, It should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (9)

1. A method for preparing a TOP2A gene detecting probe, comprising the steps of:

   (1) selecting a BAC clone as at least one of RP11-1152A10 and CTD-3217C5, or selecting a BAC clone as at least one of RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5;

   (2) extracting plasmids from BAC clones respectively, obtaining plasmid DNA, and quantifying;

   (3) The plasmid DNA is labeled with fluorescein, and the fluorescein labeled by the plasmid DNA of different sources is the same, that is, it is obtained.
2. The method for preparing a TOP2A gene detecting probe according to claim 1, wherein the BAC clone is RP11-1152A10, and CTD-3217C5; or the BAC clone is RP11-737D6, CTD-3087O22, RP11-48O10 , and CTD-2134K5.
3. The method for preparing a TOP2A gene detecting probe according to claim 1, wherein the fluorescein is Alexa

   

   FITC, Alexa

   

   Rhodamine, Texas Red, pacific

   

   Or DEAC.
4. The method for preparing a TOP2A gene detecting probe according to any one of claims 1 to 3, wherein in step (3), the plasmid DNA is subjected to fluorescein labeling by a random primer method or a nick translation method.
5. The method for producing a TOP2A gene detecting probe according to claim 4, wherein the label has a temperature of 24 ° C to 26 ° C and a time of 4 to 6 hours.
6. The TOP2A gene detecting probe obtained by the production method according to any one of claims 1 to 5.
7. A TOP2A gene detecting kit comprising the TOP2A gene detecting probe of claim 6.
8. The TOP2A gene detecting kit according to claim 7, further comprising a chromosome 17 discrimination probe for internal control, the identification probe being different in color from the fluorescein labeled by the TOP2A gene detecting probe.
9. The TOP2A gene detecting kit according to claim 7 or 8, further comprising COT Human DNA for blocking the repeat sequence, and DAPI counterstaining agent.

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