TOP2A gene detection probe and preparation method and application thereof
阅读说明：本技术 一种top2a基因检测探针及其制备方法和应用 (TOP2A gene detection probe and preparation method and application thereof ) 是由 李怡 赵浩 于 2019-09-25 设计创作，主要内容包括：本发明提供一种TOP2A基因检测探针及其制备方法和应用。分别选择TOP2A基因区域与17号染色体着丝粒区域作为目标基因,根据目标基因设计并筛选得到目的探针,对目的探针进行扩增得到扩增产物,扩增产物经转录、反转录后得到单链DNA探针,单链DNA探针进行荧光标记得到针对TOP2A基因区域的TOP2A单链探针与针对17号染色体着丝粒区域的对照单链探针。制得的TOP2A单链探针与对照单链探针为线性的单链DNA,单链DNA能够减少探针自身的配对,可以在保证特异性的情况下更加快速的与靶向序列结合,缩短杂交时间。(The invention provides a TOP2A gene detection probe and a preparation method and application thereof. Respectively selecting a TOP2A gene region and a No.17 chromosome centromere region as target genes, designing and screening according to the target genes to obtain target probes, amplifying the target probes to obtain amplification products, transcribing and reversely transcribing the amplification products to obtain single-stranded DNA probes, and carrying out fluorescent labeling on the single-stranded DNA probes to obtain a TOP2A single-stranded probe aiming at the TOP2A gene region and a control single-stranded probe aiming at the No.17 chromosome centromere region. The prepared TOP2A single-stranded probe and the contrast single-stranded probe are linear single-stranded DNA, the single-stranded DNA can reduce the pairing of the probes, and can be combined with a target sequence more quickly under the condition of ensuring specificity, so that the hybridization time is shortened.)
1. A preparation method of TOP2A gene detection probe is characterized in that the TOP2A gene detection probe comprises TOP2A single-chain probe aiming at TOP2A gene region and contrast single-chain probe aiming at 17 # chromosome centromere region;
the preparation method of the TOP2A single-stranded probe comprises the following steps:
selecting a TOP2A gene region as a TOP2A target gene, designing a single-stranded fragment with the length of 100nt-300nt as a TOP2A single-stranded candidate probe aiming at the TOP2A target gene, removing the TOP2A single-stranded candidate probe which is non-specifically hybridized and overlapped with the TOP2A target gene to obtain a TOP2A target probe, and designing a TOP2A amplification primer fragment on the upstream and downstream of the TOP2A target probe obtained by screening;
amplifying the TOP 2A-purpose probe to obtain a TOP2A amplification product, wherein the TOP2A amplification product forms a TOP 2A-purpose probe library;
in vitro transcription is carried out on the TOP2A amplification product to prepare a TOP2A RNA library corresponding to the TOP2A target probe library;
carrying out reverse transcription on the TOP2A RNA library to prepare a TOP2A single-stranded DNA probe, and carrying out fluorescence labeling on the TOP2A single-stranded DNA probe to obtain a fluorescently-labeled TOP2A single-stranded probe;
the preparation method of the control single-stranded probe comprises the following steps:
selecting a centromere repetitive region of No.17 chromosome as a control gene, designing a single-stranded fragment with the length of 206nt as a control target probe aiming at the control gene, and designing control amplification primer fragments on the upstream and downstream of the control target probe;
amplifying the control target probe to obtain a control amplification product, wherein the control amplification product forms a control probe;
in vitro transcription is carried out on the control amplification product to prepare control probe RNA corresponding to the control probe;
and carrying out reverse transcription on the control probe RNA to prepare a control single-stranded DNA probe, and carrying out fluorescent labeling on the control single-stranded DNA probe to obtain a fluorescent-labeled control single-stranded probe.
2. The method for preparing the TOP2A gene detection probe according to claim 1, wherein the amplification of the TOP 2A-mesh probe to obtain the TOP2A amplification product specifically comprises the following steps:
amplifying the TOP 2A-purpose probe by using a PCR amplification method;
connecting the amplified TOP 2A-purpose probe to a plasmid;
amplifying by taking a plasmid as a template to obtain the TOP2A amplification product;
the method for amplifying the control target probe to obtain the control amplification product specifically comprises the following steps:
amplifying the control target probe by using a PCR amplification method;
ligating the amplified control target probe to a plasmid;
and amplifying by taking the plasmid as a template to obtain the control amplification product.
3. The method for preparing the TOP2A gene detection probe according to claim 1, characterized in that, in the step of preparing the TOP2A single-stranded DNA probe by reverse transcription of the TOP2ARNA library, the method comprises the step of adding amino dNTP into a reaction system for probe labeling, and the obtained TOP2A single-stranded DNA probe is labeled with amino;
the step of preparing the control single-stranded DNA probe by reverse transcription of the control probe RNA comprises a step of adding amino dNTP into a reaction system for probe labeling, and the obtained control single-stranded DNA probe is labeled with amino.
4. The method for preparing the TOP2A gene detection probe according to claim 1, wherein the TOP2A amplification primer fragment comprises a TOP2A upstream and downstream amplification primer pair, and the sequences of the TOP2A upstream and downstream amplification primer pair comprise SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.32 and SEQ ID NO.34, SEQ ID NO.32 and SEQ ID NO.32, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.39 and SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.51 and SEQ ID NO. 52.
5. The method for preparing the TOP2A gene detection probe according to claim 1, wherein the control amplification primer fragment comprises a control upstream and downstream amplification primer pair, and the sequences of the control upstream and downstream amplification primer pair comprise the sequence shown in SEQ ID No.53 and the sequence shown in SEQ ID No. 54.
6. The method for preparing the TOP2A gene detection probe according to claim 3, wherein in the step of fluorescently labeling the TOP2A single-stranded DNA probe to obtain the TOP2A single-stranded probe, N-hydroxysuccinimide ester labeled with a first fluorescent dye is reacted with the TOP2A single-stranded DNA probe labeled with an amino group, so as to fluorescently label the TOP2A single-stranded DNA probe to obtain the fluorescently labeled TOP2A single-stranded probe.
7. The method for preparing TOP2A gene detection probe according to claim 3, wherein in the step of fluorescently labeling the control single stranded DNA probe to obtain a control single stranded probe, N-hydroxysuccinimide ester labeled with a second fluorescent dye is reacted with the amino group-labeled control single stranded DNA probe to fluorescently label the control single stranded DNA probe to obtain a fluorescently labeled control single stranded probe.
8. A TOP2A gene detection probe, characterized in that it is prepared by the method for preparing TOP2A gene detection probe as described in any one of claims 1-7.
9. A TOP2A gene detection chip, characterized by containing TOP2A gene detection probe as claimed in claim 8.
10. A TOP2A gene detection kit, characterized by comprising the TOP2A gene detection probe of claim 8.
Technical Field
The invention relates to the technical field of biology, in particular to a TOP2A gene detection probe and a preparation method and application thereof.
Background
Breast cancer is one of common malignant tumors in women, and in recent years, the incidence rate in China is on a straight-line rising trend. Clinical studies have shown that Recurrence-free survival (RFS) is shortened in breast cancer patients with TOP2A gene abnormalities, including either TOP2A gene amplification or TOP2A gene deletion, with poor prognosis, especially in TOP2A gene-deleted breast cancer patients. However, patients with abnormal TOP2A gene received anthracycline chemotherapy more effectively than patients with normal TOP2A gene. Patients with the TOP2A gene amplification treated with the Cyclophosphamide + Epirubicin + Fluorouracil (Cyclophosphamide + Epirubicin + fluorouricil, CEF) regimen reduced the risk of relapse by 61% and the risk of death by 51%, whereas patients with no TOP2A gene amplification were only able to reduce the risk of relapse by 6% and the risk of death by 10% with the CEF regimen. Therefore, the detection of the state of the TOP2A gene in a breast cancer patient is helpful for judging prognosis and guiding clinical rational medication, and is of significant help for treating breast cancer.
Currently commercialized TOP2A gene Fluorescence In Situ Hybridization (FISH) detection probes mainly use Bacterial Artificial Chromosomes (BAC) or PCR products labeled by fluorescent molecules as probes. Because BAC and PCR product probes have some non-specific sequences, the specificity of the fluorescent in situ hybridization probe is not high, the signal background is higher, and the fragments of the existing fluorescent in situ hybridization probe are usually larger, so that the hybridization efficiency is lower and the hybridization time is long.
Disclosure of Invention
The invention aims to provide a TOP2A gene detection probe and a preparation method and application thereof, and the prepared TOP2A gene detection probe can be used for detecting the condition of TOP2A gene abnormality with high specificity and high accuracy.
In order to achieve the above object, the first aspect of the present invention provides a method for preparing a TOP2A gene detection probe, wherein the TOP2A gene detection probe comprises a TOP2A single-stranded probe for a TOP2A gene region and a control single-stranded probe for a number 17 chromosome centromere region;
the preparation method of the TOP2A single-stranded probe comprises the following steps:
selecting a TOP2A gene region as a TOP2A target gene, designing a single-stranded fragment with the length of 100nt-300nt as a TOP2A single-stranded candidate probe aiming at the TOP2A target gene, removing the TOP2A single-stranded candidate probe which is non-specifically hybridized and overlapped with the TOP2A target gene to obtain a TOP2A target probe, and designing a TOP2A amplification primer fragment on the upstream and downstream of the TOP2A target probe obtained by screening;
amplifying the TOP 2A-purpose probe to obtain a TOP2A amplification product, wherein the TOP2A amplification product forms a TOP 2A-purpose probe library;
in vitro transcription is carried out on the TOP2A amplification product to prepare a TOP2ARNA library corresponding to the TOP2A target probe library;
carrying out reverse transcription on the TOP2A RNA library to prepare a TOP2A single-stranded DNA probe, and carrying out fluorescence labeling on the TOP2A single-stranded DNA probe to obtain a fluorescently-labeled TOP2A single-stranded probe;
the preparation method of the control single-stranded probe comprises the following steps:
selecting a centromere repetitive region of No.17 chromosome as a control gene, designing a single-stranded fragment with the length of 206nt as a control target probe aiming at the control gene, and designing control amplification primer fragments on the upstream and downstream of the control target probe;
amplifying the control target probe to obtain a control amplification product, wherein the control amplification product forms a control probe;
in vitro transcription is carried out on the control amplification product to prepare control probe RNA corresponding to the control probe;
and carrying out reverse transcription on the control probe RNA to prepare a control single-stranded DNA probe, and carrying out fluorescent labeling on the control single-stranded DNA probe to obtain a fluorescent-labeled control single-stranded probe.
As a further improvement of the technical scheme, the amplification of the TOP 2A-purpose probe to obtain the TOP2A amplification product specifically comprises the following steps:
amplifying the TOP 2A-purpose probe by using a PCR amplification method;
connecting the amplified TOP 2A-purpose probe to a plasmid;
amplifying by taking a plasmid as a template to obtain the TOP2A amplification product;
the method for amplifying the control target probe to obtain the control amplification product specifically comprises the following steps:
amplifying the control target probe by using a PCR amplification method;
ligating the amplified control target probe to a plasmid;
and amplifying by taking the plasmid as a template to obtain the control amplification product.
As a further improvement of the technical proposal, the step of preparing the TOP2A single-stranded DNA probe by reverse transcription of the TOP2A RNA library comprises the step of adding amino dNTP into a reaction system for probe labeling, and the obtained TOP2A single-stranded DNA probe is labeled with amino;
the step of preparing the control single-stranded DNA probe by reverse transcription of the control probe RNA comprises a step of adding amino dNTP into a reaction system for probe labeling, and the obtained control single-stranded DNA probe is labeled with amino.
As a further improvement of the above technical solution, the TOP2A amplification primer fragment comprises a TOP2A upstream and downstream amplification primer pair, the sequence of the TOP2A upstream and downstream amplification primer pair comprises SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.32, SEQ ID NO.32 and SEQ ID NO.32, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.39 and SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.51 and SEQ ID NO. 52.
As a further improvement of the technical scheme, the control amplification primer segment comprises a control upstream and downstream amplification primer pair, and the sequences of the control upstream and downstream amplification primer pair comprise a sequence shown as SEQ ID NO.53 and a sequence shown as SEQ ID NO. 54.
As a further improvement of the technical scheme, in the step of carrying out fluorescent labeling on the TOP2A single-stranded DNA probe to obtain the TOP2A single-stranded probe, N-hydroxysuccinimide ester labeled by a first fluorescent dye is reacted with the TOP2A single-stranded DNA probe labeled by an amino group, so that the TOP2A single-stranded DNA probe is subjected to fluorescent labeling to obtain the TOP2A single-stranded probe labeled by fluorescence.
As a further improvement of the above technical solution, in the step of obtaining the control single-stranded probe by fluorescently labeling the control single-stranded DNA probe, N-hydroxysuccinimide ester labeled with a second fluorescent dye is reacted with the amino group-labeled control single-stranded DNA probe, so that the control single-stranded DNA probe is fluorescently labeled to obtain the fluorescently-labeled control single-stranded probe.
The second aspect of the invention provides a TOP2A gene detection probe, which is prepared by using the preparation method of the TOP2A gene detection probe.
The third aspect of the invention provides a TOP2A gene detection chip, which contains the TOP2A gene detection probe.
The fourth aspect of the invention provides a TOP2A gene detection kit, which contains the TOP2A gene detection probe.
The invention has the beneficial effects that:
the invention provides a TOP2A gene detection probe and a preparation method and application thereof. The target probe is obtained by designing a probe sequence aiming at the genome non-repetitive region and screening, so that the non-specific reaction can be reduced, and the background signal interference can be reduced; the single-stranded DNA probe is obtained after the target probe is amplified, transcribed and reversely transcribed, the single-stranded probe prepared by the single-stranded DNA probe through fluorescent labeling is linear small-segment single-stranded DNA, the small-segment single-stranded DNA can reduce the pairing of the probe, the probe can be more quickly combined with a target sequence under the condition of ensuring the specificity, and the hybridization time is shortened.
Drawings
To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of the present invention.
FIG. 1 is a schematic diagram of the detection result of TOP2A gene detection probe for detecting TOP2A gene abnormality provided by the embodiment of the invention.
Detailed Description
The terms as used herein:
"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 ~ 5" is disclosed, the ranges described should be construed to include the ranges "1 ~ 4", "1 ~ 3", "1 ~ 2 and 4 ~ 5", "1 ~ 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
The invention provides a preparation method of a TOP2A gene detection probe, and the prepared TOP2A gene detection probe comprises a TOP2A single-chain probe aiming at a TOP2A gene region and a contrast single-chain probe aiming at a No.17 chromosome centromere region.
One embodiment of the present invention provides a method for preparing a TOP2A single-stranded probe, comprising the following steps:
step 1: selecting a TOP2A gene region as a TOP2A target gene, designing a TOP2A single-stranded candidate probe according to the TOP2A target gene, removing the TOP2A single-stranded candidate probe which is non-specifically hybridized and overlapped with the TOP2A target gene to obtain a TOP 2A-purpose probe, and designing a TOP2A amplification primer segment on the upstream and downstream of the TOP 2A-purpose probe obtained by screening.
In the step 1, the TOP2A gene region is selected as the TOP2A target gene, and the specific steps of designing the TOP2A single-stranded candidate probe according to the TOP2A target gene are as follows: the TOP2A gene region is selected as TOP2A target gene, 40 single-stranded fragments with the length of 100nt-300nt are designed for TOP2A target gene as TOP2A single-stranded candidate probes, and the length of each TOP2A single-stranded candidate probe can be 100nt, 200nt, 300nt and the like.
As mentioned above, the TOP2A single-stranded candidate probes are designed by the interval preset length, so that mutual interference among the probes can be avoided, and the accuracy of the detection result can be improved. The fragment of each TOP2A single-stranded candidate probe is small, and can be combined with a target sequence more quickly under the condition of ensuring specificity, so that the hybridization time is shortened.
The specific steps of removing the TOP2A single-stranded candidate probe which is non-specifically hybridized and overlapped with the TOP2A target gene to obtain the TOP2A target probe are as follows: removing TOP2A single-stranded candidate probes located in the TOP2A gene repetitive region by BLAST comparison, removing TOP2A single-stranded candidate probes with the non-specific Tm value between 75-85 ℃ by scoring, and selecting non-overlapping TOP2A single-stranded candidate probes as TOP 2A-purpose probes to finally obtain 26 TOP 2A-purpose probes.
In the step of designing TOP2A amplification primer segments on the upstream and downstream of the TOP 2A-purpose probe obtained by screening, the TOP2A amplification primer segments comprise TOP2A upstream and downstream amplification primer pairs, wherein the upstream amplification primer is designed with a T7 promoter sequence, and the downstream amplification primer is designed with a specific RT primer binding sequence.
The sequences of the TOP2A upstream and downstream amplification primer pairs include those shown as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.38 and SEQ ID NO.38, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.51 and SEQ ID NO. 52.
And 2, amplifying the TOP 2A-purpose probe to obtain a TOP2A amplification product, wherein the TOP2A amplification product forms a TOP 2A-purpose probe library.
Specifically, the present embodiment specifically includes the following steps:
amplifying a TOP 2A-purpose probe by using a PCR amplification method;
connecting the amplified TOP 2A-purpose probe to a plasmid;
and amplifying by taking the plasmid as a template to obtain a TOP2A amplification product, wherein the TOP2A amplification product forms a TOP 2A-purpose probe library.
In the embodiment, a TOP 2A-purpose probe is firstly subjected to PCR amplification, the TOP 2A-purpose probe subjected to PCR amplification is connected to a plasmid through T-A cloning, the plasmid is used as a template for amplification to obtain a TOP2A amplification product, and the TOP2A amplification product forms a TOP 2A-purpose probe library. By adopting the method, the non-specific amplification can be avoided, and the obtained TOP2A amplification product has high purity.
Step 3, carrying out in vitro transcription on the TOP2A amplification product to prepare a TOP2A RNA library corresponding to the TOP 2A-purpose probe library;
and carrying out reverse transcription on the TOP2A RNA library to prepare a TOP2A single-stranded DNA probe, and carrying out fluorescent labeling on the TOP2A single-stranded DNA probe to obtain a TOP2A single-stranded probe.
In the preparation of the TOP2A single-stranded DNA probe by reverse transcription of the TOP2A RNA library, a step of adding Amino (Amino) dNTP to the reaction system for probe labeling was included, and the obtained TOP2A single-stranded DNA probe was labeled with Amino groups. 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-dCTP, Amino-dGTP, or the like.
Correspondingly, when the prepared TOP2A single-stranded DNA probe is fluorescently labeled, N-hydroxysuccinimide ester (NHS ester) labeled with a first fluorescent dye is reacted with the TOP2A single-stranded DNA probe labeled with an amino group, so that the TOP2A single-stranded DNA probe is fluorescently labeled. In this embodiment, the first fluorescent dye is AlexaFluor 488, which is a green fluorescent dye. Of course, in other embodiments, other colors of fluorescent dyes may be used.
Alexa Fluor 488 emits bright green fluorescence, has a spectrum similar to that of fluorescein, and is stable between pH4 and pH 10. Can be excited by 488nm light to generate fluorescence with stable signals, is often used as a cell marker to be coupled to antibodies, polypeptides, proteins, tracers and other substrates, and is widely used for cell imaging and detection. The Alexa Fluor 488NHS Ester, namely the Alexa Fluor 488 containing N-Hydroxysuccinimide (N-Hydroxysuccinimide) activating group, can be directly connected with amino (-NH) on biochemical molecules2) The reaction forms a stable amide bond, primarily a primary amine, which can be used to label any primary amine-containing protein, polypeptide, amino-modified oligonucleotide, or other amino-containing biomolecule.
It can be understood that the above-mentioned method can increase the number of fluorescence labeled by each TOP2A single-stranded probe by reverse transcription incorporating amino-dNTP and then coupling with the first fluorescent dye labeled with N-hydroxysuccinimide ester, thereby increasing the fluorescence intensity during fluorescence in situ hybridization and improving the accuracy and reliability of the detection result.
The TOP 2A-purpose probe is subjected to PCR amplification firstly, the TOP 2A-purpose probe subjected to PCR amplification is connected to a plasmid through T-A cloning, the plasmid is used as a template for amplification to obtain a TOP2A amplification product, the TOP2A amplification product is transcribed to construct a TOP2A RNA library, the TOP2A RNA library is subjected to reverse transcription to obtain a TOP2A single-stranded DNA probe, and the TOP2A single-stranded DNA probe is subjected to fluorescent labeling to obtain a TOP2A single-stranded probe.
Another embodiment of the present invention provides a method for preparing a control single-stranded probe, comprising the steps of:
step 1', aiming at the centromere repetitive region of No.17 chromosome, designing a single-stranded segment with the length of 206nt as a control target probe for the control gene, and designing control amplification primer segments on the upstream and downstream of the control target probe.
In one embodiment, the upstream amplification primer is designed with a T7 promoter sequence, the downstream amplification primer is designed with a specific RT primer binding sequence, and the sequences of the control upstream and downstream amplification primer pairs include a sequence shown as SEQ ID No.53 and a sequence shown as SEQ ID No.54, wherein the sequence shown as SEQ ID No.53 is the sequence of the upstream amplification primer, and the sequence shown as SEQ ID No.54 is the sequence of the downstream amplification primer.
And 2' amplifying the control target probe to obtain a control amplification product, wherein the control amplification product forms a control probe.
Specifically, the present embodiment specifically includes the following steps:
amplifying the control target probe by using a PCR amplification method;
connecting the amplified control target probe to a plasmid through T-A cloning;
and amplifying by taking the plasmid as a template to obtain the control amplification product, wherein the control amplification product forms a control probe.
Step 3', carrying out in-vitro transcription on the control amplification product to prepare control probe RNA corresponding to the control probe;
and carrying out reverse transcription on the control probe RNA to prepare a control single-stranded DNA probe, and carrying out fluorescent labeling on the control single-stranded DNA probe to obtain the control single-stranded probe.
In the above, the preparation of the control single-stranded DNA probe by reverse transcription of the control probe RNA comprises a step of adding Amino (Amino) dNTP to the reaction system to label the probe, and the obtained control 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-dCTP, Amino-dGTP, or the like.
Correspondingly, when the prepared control single-stranded DNA probe is fluorescently labeled, a second fluorescent dye labeled with N-hydroxysuccinimide ester (NHS ester) is used to react with the amino-labeled control single-stranded DNA probe, thereby fluorescently labeling the control single-stranded DNA probe. In this embodiment, the second fluorescent dye is AlexaFluor 594, a red fluorescent dye. Of course, in other embodiments, the second fluorescent dye may also be a fluorescent dye with other colors and different colors from the first fluorescent dye.
The TOP2A gene detection probe prepared by the preparation method comprises a TOP2A single-stranded probe aiming at the TOP2A gene region and a control single-stranded probe aiming at the No.17 chromosome centromere region.
The prepared TOP2A single-stranded probe and the contrast single-stranded probe are both linear small-fragment single-stranded DNA, and can be combined with a target sequence more quickly under the condition of ensuring specificity, so that the hybridization time is shortened.
When the prepared TOP2A gene detection probe is used for detecting TOP2A gene abnormality, the principle is as follows: because each 17-chromosome has two sister chromatids, each sister chromatid has one TOP2A gene, the ratio of TOP2A gene to 17-chromosome centromere is 2 under the condition that the two sister chromatids are not separated, and the ratio of TOP2A gene to 17-chromosome centromere is 1 under the condition that the two sister chromatids are completely separated, the amplification and deletion conditions of TOP2A gene can be judged by the ratio of TOP2A gene to 17-chromosome centromere. The control single-stranded probe can be used as a control probe of the TOP2A single-stranded probe and is used for judging the amplification condition of the chromosome 17; the control single-stranded probe is used for judging the amplification condition of the TOP2A gene.
Because the TOP2A single-stranded probe is provided with the green fluorescent group, and the control single-stranded probe is provided with the red fluorescent group, the amplification and deletion condition of the TOP2A gene can be judged by the ratio of the green fluorescent signal to the red fluorescent signal, when the ratio of the green fluorescent signal to the red fluorescent signal is more than 2, the TOP2A gene is amplified, when the ratio of the green fluorescent signal to the red fluorescent signal is less than 1, the TOP2A gene is deleted, and when the ratio of the green fluorescent signal to the red fluorescent signal is between 1 and 2, the TOP2A gene is normal.
In addition, the invention also provides a TOP2A gene detection probe, which comprises a TOP2A single-stranded probe and a control single-stranded probe, wherein the TOP2A single-stranded probe and the control single-stranded probe are prepared by the preparation method of the TOP2A gene detection probe.
The invention further provides a TOP2A gene detection chip, which contains the TOP2A gene detection probe.
Furthermore, the invention also provides a TOP2A gene detection kit, which contains the TOP2A gene detection probe.
Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.