CN111690741A - Breast cancer polygene screening probe and application thereof - Google Patents

Abstract

The invention discloses a multi-gene screening probe for breast cancer, which is prepared by the following method: aiming at high-risk mutant genes with high incidence of breast cancer, downloading coding sequences of exons of each gene, and adding 100bp flanking sequences at the 3 'end and the 5' end respectively; processing the gene sequence by using Oligowiz2.0 software to obtain an output.owz file; opening the output. owz file under a windows system by utilizing OligoWiz-2.1.0_ OFFLINE. jar software, selecting the minimum probe spacing to be 40bp, selecting the maximum probe number to be unlimited, using default values for other parameters, and deriving a probe list; adding a primer sequence of SEQ NO.1 at the 5 'end of the probe, adding a primer sequence of SEQ NO.2 at the 3' end of the probe, and then synthesizing the probe. The multi-gene screening probe for breast cancer can provide powerful medication guidance for clinical diagnosis of breast cancer patients, and facilitates accurate formulation of treatment schemes.

Description

Breast cancer polygene screening probe and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a multi-gene screening probe for breast cancer.

Background

Cancer is the leading cause of death and disability in today's world. Each tumor includes both genetic (germ line) and tumor-specific (somatic) variations. In the past decade, high throughput sequencing and bioinformatics technology has advanced tremendously, and mutation information has been collected for a large number of germline and somatic genes. Numerous clinical practices have demonstrated that treatment targeting specific proteins that mutate in cancer patients is effective. To achieve the goal of precision medicine, i.e., to apply the right drugs to the right patients, many laboratories have begun to apply second generation sequencing technologies in cancer gene testing.

Today more and more genetic variations are demonstrated in connection with various forms of tumors including breast cancer, and more targeted drugs are demonstrated to have therapeutic effects on certain types of cancer. However, because of the heterogeneity of tumor cells, the mutation of tumor cells in a tumor tissue is different, which is a key problem in realizing precise medical treatment by searching for mutant genes and using targeted drugs.

Disclosure of Invention

The invention firstly provides a multi-gene screening probe for breast cancer, which is prepared by the following method:

aiming at all high-risk mutant genes with high breast cancer incidence in the table 1 and the table 2, gene exon coding sequences are downloaded, and 100bp flanking sequences are added at the 3 'end and the 5' end respectively; (genome version selection hg19/GRCH37 in ucsc database)

Processing the gene sequence by using Oligowiz2.0 software to obtain an output.owz file;

opening the output. owz file under a windows system by utilizing OligoWiz-2.1.0_ OFFLINE. jar software, selecting the minimum probe spacing to be 40bp, selecting the maximum probe number to be unlimited, using default values for other parameters, and deriving a probe list;

adding a primer sequence of SEQ NO.1 at the 5 'end of the probe, adding a primer sequence of SEQ NO.2 at the 3' end of the probe, and then synthesizing the probe.

The specific parameters for processing the gene sequence by using Oligowiz2.0 software are set as follows:

-length: the target Oligo is preferably 80 bases in length;

-lmax and-lmin: the length of the probe is strictly limited and must be 80 bp;

-minlh: the software default value is 15;

the result of the program run is redirected to owz suffix named file;

wherein SEQ NO. 1: 5'-GAAGCGAGGA TCAACT-3', respectively;

SEQ NO.2：5’-CATTGCGTGA ACCGA-3’

the high-risk mutant genes of the high incidence of breast cancer comprise 401 mutant genes and 110 copy number variant genes, and are specifically shown in the following tables 1 and 2:

TABLE 1 Gene List for detecting mutations

Among the important genes of interest for breast cancer are: AKT1, AKT3, BRCA1, BRCA2, CDK4, CDK6, EGFR, ESR1, mTOR, PIK3CA, PTEN, TP53

TABLE 2 Gene List for copy number variation detection

The core genes of major interest for breast cancer are: ERBB2, FGFR1, FGFR2, PTEN, MYC, RB1, AKT3, CCND1, JAK2, TP53

The invention also provides a method for screening the breast cancer genes, which comprises the following steps:

extracting a DNA sample of a patient, including DNA of a blood sample, DNA of a tissue sample and cfDNA; then, the multi-gene screening probe for breast cancer is used for bioinformatics analysis of detection, and the obtained result is compared with the mutant genes and copy number variation genes listed in the tables 1 and 2 to obtain the gene diagnosis result of the patient.

Specifically, the breast cancer gene screening method comprises the following steps:

(1) DNA sample preparation

Extracting lesion tissues and paracancer or blood tissues of a patient to perform DNA extraction, and optionally embedding tissues with paraffin;

(2) DNA sample library construction

Randomly fragmenting the DNA into small fragments of hundreds of bases or shorter, and then adopting a KAPA library construction kit to prepare the library; (the main steps comprise DNA breaking, tail end filling, 3 ' end adding ' A ', joint connection, purification, library amplification and purification) the library is placed at-20 ℃ for storage after preparation;

(3) hybrid Capture and sequencing

The breast cancer polygene screening probe is applied to hybridization capture (capture of DNA fragments is realized by capture magnetic beads) with a DNA library, and sample sequencing is carried out;

(4) bioinformatics analysis of sequencing results

The sequencing result is a fastq file, and the sequencing result is analyzed by using an algorithm (published by Broad) for acquiring the gene mutation to obtain and annotate the gene mutation result;

the method mainly comprises the steps of quality control of fastq files, genome association, analysis of somatic mutation (somatic mutation) and germ cell mutation (germline mutation) and annotation;

the software used respectively had: the fastqc and fastx _ toolkit are used for quality control; bwa, and gatk and mutect2 obtain somatic mutation; obtaining embryo cell mutation by a HaplotpypeCaller method; ANNOVAR is annotated.

(5) Comparing the results to obtain mutation results

And comparing the sequencing result of the sample with the DNA screening lists in the tables 1 and 2 to obtain the gene mutation result of the patient with the sample.

In the clinic, the final diagnosis is obtained by combining clinical information.

Wherein, the steps (2) to (5) can be carried out by a sequencing company;

whole genome or whole exome sequencing may also be performed in step (3), such that the results of the analysis are compared to the multigenome screening lists listed in tables 1 and 2 in step (5) to obtain gene mutation results;

however, the cost can be reduced by applying the multi-gene screening probe for breast cancer.

The invention also provides a kit for screening the breast cancer genes, which comprises the breast cancer polygene screening probe.

The preparation principle of the breast cancer polygene screening probe provided by the invention is as follows: the method comprises the steps of selecting genes closely related to the occurrence, development and targeted therapy of breast cancer in a TCGA database, a METABRIC database, an MSKCC-IMPACT database, an 560-case U.S. breast cancer WGS database and a breast research institute database of a tumor hospital, enriching important exon regions and partial intron regions of 511 genes by using a biotin probe hybridization method based on a second-generation sequencing technology, and performing high-depth sequencing, so that the events such as gene mutation, copy number variation and the like having clear clinical relevance to the breast cancer can be accurately detected, the multi-gene screening list is made into a probe, and high-risk genes are detected by means of DNA sequencing in a targeted manner, so that the gene mutation having guiding significance for diagnosis and therapy can be more efficiently detected, and the breast cancer patients can be accurately typed. Has important guiding significance for clinical diagnosis and target point discovery of targeted therapy. The mutant gene can be detected in a targeted manner, and the cost of gene detection of a patient can also be reduced.

The method for screening the breast cancer gene can be applied in two aspects. Firstly, the method comprises the following steps: sequencing a sample by a whole genome or a whole exon, analyzing to obtain mutation conditions of all genes, finding the genes listed by the invention, and carrying out next analysis by combining clinic; secondly, the gene probes listed in the invention are constructed, only the genes listed in the table 1 and the table 2 of the sample are specifically sequenced, and the mutation condition is analyzed and then combined with clinic to carry out the next diagnosis.

According to the invention, the multigene probe is provided, accurate sequencing is carried out, the gene mutation result is obtained, and then information such as clinic and the like of a patient sample is combined, so that powerful reference can be provided for clinical diagnosis of the patient, the cancer development condition of the patient can be further understood, and a treatment scheme can be conveniently and accurately formulated.

Detailed Description

The specific implementation process of the technical scheme of the invention (the process of preparing and using the product, including steps, conditions, parameters, results, and the like, which is described in detail in the specification) is described in detail by combining the examples, so that the implementation can be performed by a person skilled in the art.

Wherein:

1. from step (2) to step (5), the sequencing work is completed by the precision center of the tumor hospital;

2. primer and probe synthesis: suzhou Hongxn Biotechnology GmbH

3. The data analysis methods and software used in the examples are algorithms issued by Broad for obtaining gene mutations:

the original file for analysis is data in a fastq format; bwa is used for matching, and a haplotypeCaller method is used for obtaining germline mutation; somatic mutation analysis of tissue samples, using muttec 2, was then annotated using ANNOVAR. The concrete contents are as follows:

the first embodiment is as follows:

1. a clinically confirmed cancer tissue and Blood sample of a breast cancer patient (subsidiary tumor hospital of the university of Compound Dane) was taken and DNA was extracted using a Kit of Tiangen Biochemical technology (Beijing) Co., Ltd., TGuide Cells/tissue genomic DNA Kit and Tiangen Biochemical technology (Beijing) Co., Ltd., TGuide Large Volume Blood genomic DNA Kit (1-3ml), respectively.

TABLE 3 tissue sample quality control

The table 3 mainly includes quality evaluation of DNA sample extraction and quality evaluation of sequencing, which are the most basic parts of the whole experiment, and the failure of data quality will affect the accuracy and reliability of subsequent results.

2. Preparation of DNA sample library:

DNA was randomly disrupted using a Bioruptor UCD-300 non-contact fully automated sonicator.

DNA400ng was diluted to 50. mu.l with nuclease-free water, transferred to a 0.5ml pipehead, mixed well, centrifuged briefly and placed on ice until needed.

Placing a sample: and symmetrically placing centrifuge tubes (if single tubes exist, adding water into the empty tubes for balancing), screwing the rotating head, placing the centrifuge tubes on an ice box, and precooling for 1-2 min. And (3) carrying out ultrasonic disruption of 150-200 bp. The preceding ultrasonication step was repeated for 9 cycles, ending about 90 min.

Library Preparation was then performed using the KAPA Library construction Kit (KAPA LTP Library Preparation Kit, Roche sequencing).

3. Preparation of RNA probe:

for all the high-risk mutant genes with high breast cancer incidence in the table 1 and the table 2, downloading exon coding sequences of each gene, and adding 100bp flanking sequences at the 3 'end and the 5' end respectively (the genome version in the ucsc database selects hg19/GRCH 37);

processing the gene sequence by using Oligowiz2.0 software to obtain an output.owz file;

opening the output. owz file under a windows system by utilizing OligoWiz-2.1.0_ OFFLINE. jar software, selecting the minimum probe spacing to be 40bp, selecting the maximum probe number to be unlimited, using default values for other parameters, and deriving a probe list;

adding a primer sequence of SEQ NO.1 at the 5 'end of the probe, adding a primer sequence of SEQ NO.2 at the 3' end, and synthesizing the probe.

The oligo pool of the target genes in Table 1 and Table 2 was obtained from the company, and diluted to 1 ng/. mu.l with 1 XTE (pH 8.0). The oligo sequence was amplified with the Herculase II Fusion DNA Polymerase kit (Agilent). RNA transcription was performed using Ambion SP6megascript kit (thermo Fisher scientific) and probe preparation was completed. The RNA probe was stored at-80 ℃ and a portion of the RNA probe library was diluted to 100 ng/. mu.l.

The probe prepared by the method can be applied to 200-300 samples, and the cost of sequencing is greatly reduced.

4. Hybrid capture and sample presentation sequencing

1. Mixing 95 μ l hybridization blocking reagent (complementary primer sequences with SEQ NO.1 and SEQ NO.2, respectively) and DNA library with total amount of no less than 500ng and volume of about 5 μ l, centrifuging, marking as "DNA-block", placing on PCR instrument, covering with hot cover, and keeping at 95 deg.C for 5 min; hold at 65 ℃.

2. And matching a 'bait' Mix reaction system in the PCR reaction tube, marking the RNA probe as 'bait', placing the 'bait' tube on the PCR instrument for incubation when the temperature of the PCR instrument is reduced to 65 ℃ for 2.5min, and covering a hot cover.

TABLE 4 composition of "Bait" Mix reaction system

3. Placing the PCR tube 'bait' into a PCR instrument for 2.5min, sucking 13 mu l of Hyb Buffer from the hyb Buffer hybridization Buffer solution, transferring the Hyb Buffer to a DNA-block sample, sucking 6 mu l of the Hyb Buffer from a 'bait' hole, transferring the DNA-block sample to the 'bait' sample, sucking and beating the mixture for 10 times by light, fully mixing the mixture to avoid generating a large amount of bubbles, pasting a membrane, covering a hot cover of the PCR instrument, and incubating the mixture overnight for 24h at 65 ℃.

Capture magnetic beads (Dynabeads M-270Streptavidin, Thermo FisherScientific) were then prepared (capture magnetic beads were used to capture DNA fragments). According to the requirement of 3 multiplied by 165 mu l of high-stringency buffer for each sample, the high-stringency buffer is subpackaged in a 96-hole plate, a drying instrument is opened, the temperature of the drying instrument is adjusted to 65 ℃, and the drying instrument is placed in the drying instrument for preheating when the temperature of the drying instrument is stabilized at 65 ℃. Then, capture of the target region DNA library is performed, and enrichment of the DNA library is completed (Post-PCR reaction). The library was then mixed and sample-fed sequenced.

5. Then comparing the detection result with the DNA screening list to obtain the gene mutation result of the patient, which is shown in the following table 5-8,

TABLE 5 detection results of leukocyte gene mutation of blood cells

Table 5 shows details of gene variations of mutated blood leukocyte (germline mutation) related to the genes listed in the foregoing Table 1 in the results of bioinformatics analysis described previously after sequencing of DNA samples of patients.

TABLE 6 detection results of multiple gene mutations (pathological mutations) in tumor tissues

TABLE 7 tissue polygene copy number variation detection results

TABLE 8 summary of test results and guidelines for drug administration

Table 8 shows the drug resistance and other conditions of the patients according to germline mutation, physiological mutation and copy number variation of the key genes, and the medication guidance scheme shown in Table 8 is established.

Sequence listing

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gaagcgagga tcaact 16

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<213> Artificial Sequence (Artificial Sequence)

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cattgcgtga accga 15

Claims (5)

1. A multi-gene screening probe for breast cancer is characterized by being prepared by the following method:

aiming at high-risk mutant genes with high incidence of breast cancer, downloading coding sequences of exons of each gene, and adding 100bp flanking sequences at the 3 'end and the 5' end respectively;

processing the gene sequence by using Oligowiz2.0 software to obtain an output.owz file;

opening the output. owz file under a windows system by utilizing OligoWiz-2.1.0_ OFFLINE. jar software, selecting the minimum probe spacing to be 40bp, selecting the maximum probe number to be unlimited, using default values for other parameters, and deriving a probe list;

adding a primer sequence of SEQ NO.1 at the 5 'end of the probe, adding a primer sequence of SEQ NO.2 at the 3' end of the probe, and then synthesizing the probe.

2. The breast cancer polygene screening probe as claimed in claim 1, wherein the specific parameters for processing gene sequences using Oligowiz2.0 software are set as follows:

-length: the target Oligo is preferably 80 bases in length;

-lmax and-lmin: the length of the probe is strictly limited and must be 80 bp;

-minlh: the software default value is 15;

the result of the program run is redirected to owz suffix named files.

3. The multi-gene screening probe for breast cancer according to claim 1, wherein the high-risk mutant genes for high incidence of breast cancer include 401 mutant genes and 110 copy number variant genes, which are specifically shown in tables 1 and 2.

4. A method for screening breast cancer genes is characterized by comprising the following steps:

(1) DNA sample preparation

Extracting lesion tissues and paracancer or blood tissues of a patient to perform DNA extraction, and optionally embedding tissues with paraffin;

(2) DNA sample library construction

Randomly fragmenting the DNA into small fragments of hundreds of bases or shorter, and then adopting a KAPA library construction kit to prepare the library;

(3) hybrid Capture and sequencing

The multi-gene screening probe for breast cancer, which is applied to the DNA library, is hybridized and captured, and is sent for sequencing;

(4) bioinformatics analysis of sequencing results

Analyzing the sequencing result by using an algorithm for acquiring the gene mutation issued by Broad to obtain the gene mutation result and annotating;

(5) comparing the results to obtain mutation results

And comparing the sequencing result of the sample with the DNA screening lists in the tables 1 and 2 to obtain the gene mutation result of the patient with the sample.

5. A kit for screening breast cancer genes, which comprises the breast cancer polygene screening probe of claim 1.