CN111690748B - Probe set for detecting microsatellite instability by using high-throughput sequencing, kit and microsatellite instability detection method - Google Patents
Probe set for detecting microsatellite instability by using high-throughput sequencing, kit and microsatellite instability detection method Download PDFInfo
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Abstract
The invention provides a probe set for detecting microsatellite instability by using high-throughput sequencing, a kit and a method for detecting microsatellite instability, which relate to the technical field of gene detection, and the probe set provided by the invention can simultaneously capture 11 sites of BAT26, BAT25, BAT-34c4, BAT-40, D2S123, D5S346, MONO-27, NR21, NR24, NR27 and D17S250 by using high-throughput sequencing, is used for evaluating the microsatellite instability of an object to be detected, and provides theoretical basis and guidance for prognosis of tumor patients and formulation of clinical treatment schemes.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a probe set for detecting microsatellite instability by using high-throughput sequencing, a kit and a microsatellite instability detection method.
Background
Microsatellites refer to short tandem repeat fragments that are prevalent in the human genome, typically repeat units ranging from 1 to 5 bases, with repeat times varying from 10 to tens of times. Most microsatellite sequences are located in non-coding regions of the genome.
DNA repair systems are systems that are closely related to the occurrence of tumors. Studies have shown that multiple somatic mutations can result in the conversion of cells from normal cells to tumor cells. Mutations in DNA repair and replication genes can be detected in more than about 50% of tumors. When the DNA mismatch repair system functions properly, if a microsatellite repeat sequence is replicated incorrectly, the homolog of the mismatch repair protein MutS forms a heterodimer (hMutSα or hMutSβ) which can recognize the base mismatch on the newly synthesized DNA nucleotide chain together and bind to the mismatch site in an ATP-dependent manner. hMLHl is combined with hPMSL, hPMS2 and hMLH3 to form MutL dimer, and the MutL dimer is combined with DNA polymerase under the action of EX01 and PCNA proteins to delete mismatched bases, and then DNA ligase is used to link DNA chains together to complete the repair of mismatched base DNA nucleic acid chains. If errors are not repaired, these errors may result in permanent mutations, thereby promoting tumorigenesis. Mismatch repair systems typically increase replication accuracy by a factor of 1000 to 10000. Mismatch repair deficient cells cause genomic instability associated with microsatellite instability (MSI) and patient tumor susceptibility increases.
In 1997, for ease of comparison in the study, the national cancer institute (Nmional Cancer Institute, NCI) recommended a set of 5 microsatellite markers, called Bethesda (Bethesda) markers, including two single nucleotide repeats, N (BaT-25 and BAT-26), and three dinucleotide repeats, D5S346, D2S123 and D17S 250), 131, initially selected microsatellite sites were highly variable, and the definition criteria for MSI were established at the conference, the presence of two or more marker instability in the sample was defined as MSI-H, and those with one marker instability was defined as MSI-L, no marker instability was MSS, and when the marker point exceeded 5, the site instability was defined as MSI-H at > 30%. Subsequent Suraweera et al have found a set of 5 single nucleotide repeats (BAT-25, BAT-26, NR-21, NR-22 and NR-24).
Research shows that MSI detection plays an important guiding role in tumor immunotherapy. PD-1 antibody drug (Pembrolizumab (Keytruda)) was the first "broad-spectrum" antitumor drug in the 5 th 2017 to treat all solid tumor patients with microsatellite highly unstable (MSI-H) or mismatch repair deficiency (dMMR) in bulk.
A recent Clinical CancerResearch study has shown that detection of MSI status by liquid biopsy helps provide guidance for prognosis of tumor patients and formulation of clinical treatment regimens.
The existing MSI detection mainly uses a fluorescent PCR detection method, namely, a specific primer is used, a plurality of sites related to microsatellite markers are amplified simultaneously, and amplified products are analyzed through capillary electrophoresis and compared with normal tissues to check whether mobility is changed or not, so that the state of MSI is judged.
In view of this, the present invention has been made.
Disclosure of Invention
It is a first object of the present invention to provide a probe set for detecting microsatellite instability using high throughput sequencing to at least alleviate one of the technical problems of the prior art.
A second object of the present invention is to provide a kit for detecting microsatellite instability using high throughput sequencing.
A third object of the present invention is to provide a method for detecting microsatellite instability.
The present invention provides a probe set for detecting microsatellite instability using high throughput sequencing, the probe set comprising probes simultaneously capturing: BAT26, BAT25, BAT-34c4, BAT-40, D2S123, D5S346, MONO-27, NR21, NR24, NR27 and D17S250.
Further, the probes include sense strand probes;
preferably, sense strand probes that capture the positions of BAT-34c4, BAT-40, BAT25, MONO-27, NR21, BAT26, D5S346, D2S123, NR24, D17S250 and NR27 have nucleotide sequences as shown in SEQ ID No.1-10, SEQ ID No.11-20, SEQ ID No.21-32, SEQ ID No.33-42, SEQ ID No.43-53, SEQ ID No.54-62, SEQ ID No.63-72, SEQ ID No.73-84, SEQ ID No.85-93, SEQ ID No.94-105 and SEQ ID No.106-117, in that order.
Further, the probe is labeled with a label, preferably biotin.
Further, the probe is a DNA probe or an RNA probe.
The invention also provides a kit for detecting microsatellite instability by using high-throughput sequencing, which comprises the probe set.
In addition, the invention also provides a method for detecting microsatellite instability, which uses the probe set or the kit to enrich target genes through liquid phase hybridization, and performs high-throughput sequencing on the DNA sequences obtained through enrichment to obtain microsatellite instability results.
Further, constructing a target genome library, enriching target genes by liquid phase hybridization by using the probe set or the kit, and carrying out high-throughput sequencing on the DNA sequences obtained by enrichment to obtain a microsatellite instability result.
Further, after the whole genome of the target is fragmented, a target genome library is obtained through end repair.
Further, the target gene fragment is obtained by eluting the mixture of the probe set and the magnetic beads and adsorbing the mixture.
Further, the target gene fragment obtained by elution treatment is subjected to fragment amplification by utilizing PCR, and the amplified fragment with the length of 220-320bp is subjected to high-throughput sequencing and analysis, so that a microsatellite instability result is obtained.
The probe set provided by the invention can simultaneously capture 11 sites of BAT26, BAT25, BAT-34c4, BAT-40, D2S123, D5S346, MONO-27, NR21, NR24, NR27 and D17S250 through high-throughput sequencing for evaluating the unstable sites of the microsatellite, accurately evaluate the unstable state of the microsatellite of a to-be-tested object, and provide theoretical basis and guidance for prognosis of tumor patients and formulation of clinical treatment schemes. The probe set provided by the invention can be used by being mixed with a conventional probe, and the unstable state of a sample microsatellite can be detected without adding a special process while detecting conventional mutation, so that the detection cost is reduced.
According to the microsatellite instability detection method provided by the invention, the target gene is enriched by using the probe set provided by the invention through liquid phase hybridization, so that the capturing and enriching efficiency of fragments is greatly improved, and the DNA sequences obtained through enrichment are subjected to high-flux sequencing, so that the effects of low data repetition rate and high effective depth can be realized, and the efficiency of high-flux sequencing is improved. The probe set can effectively target and enrich relevant sites of microsatellite instability in tumor tissues and free DNA, and provides an efficient genotyping technology for research and application of microsatellite instability. Meanwhile, the target gene can be effectively enriched by using the liquid phase hybridization technology, the requirement on sample size is reduced, and meanwhile, the sequencing cost can be better controlled.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a conventional BAT26 probe design;
FIG. 2 is a schematic diagram of the design of probes for BAT26 sites according to the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In the present application, the use of the term "including" and other forms is non-limiting unless otherwise specified.
Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.
According to one aspect of the present invention, there is provided a probe set for detecting microsatellite instability using high throughput sequencing, the probe set comprising probes simultaneously capturing: BAT26, BAT25, BAT-34c4, BAT-40, D2S123, D5S346, MONO-27, NR21, NR24, NR27 and D17S250.
High throughput sequencing technology (High-throughput sequencing), also known as "Next generation" sequencing technology ("Next-generation" sequencing technology), is marked by the ability to sequence hundreds of thousands to millions of DNA molecules in parallel at a time, and by the general short read lengths. It generally has high sensitivity. High throughput, low cost. Typical high-throughput sequencing platforms include, but are not limited to, large-scale parallel signature sequencing (Massively Parallel Signature Sequencing, MPSS), polymerase cloning (Polony Sequencing), 454pyrosequencing (454 pyrosequencing), illumina (Solexa) sequencing, ABI SOLiD sequencing, ion semiconductor sequencing (Ion semiconductor sequencing), DNA nanosphere sequencing (DNA nanoball sequencing), and the like.
The probe set provided by the invention can simultaneously capture 11 sites of BAT26, BAT25, BAT-34c4, BAT-40, D2S123, D5S346, MONO-27, NR21, NR24, NR27 and D17S250 through high-throughput sequencing, and can be used for evaluating the unstable states of microsatellites of an object to be tested. The probe set can effectively target and enrich microsatellite unstable related sites in tumor tissues and free DNA, and provides theoretical basis and guidance for prognosis of tumor patients and formulation of clinical treatment schemes. The probe set provided by the invention can be used by being mixed with a conventional probe, and the unstable state of a sample microsatellite can be detected without adding a special process while detecting conventional mutation, so that the detection cost is reduced.
In some embodiments, the sample used in the liquid biopsy is cfDNA, which has an average length of 160bp to 170bp and a lower total amount, and is more stringent for the probe. The probe set provided by the invention can efficiently and accurately detect the unstable condition of the microsatellite in the cfDNA sample, and has wider application fields.
In some preferred embodiments, the probe is a probe comprising a sense strand.
The mutation mode of the microsatellite sequence is mainly repeated insertion and deletion, and the conventional probe design mode only refers to the sense strand of the conventional genome to carry out probe design, for example, three probes are designed for each MSI microsatellite locus and are respectively located at the flank and the middle of the detection locus (shown in figure 1). The primary mutant form changes the number of repetitions of the repeat unit due to the sites associated with microsatellite instability. For example, BAT-26, which has the sequence TTCAGGT (A) 27GGGTT, when microsatellite instability occurs, the number of intermediate A repeats is randomly reduced, and the reduced number of repeats is generally greater than 10 repeats, resulting in mutant. One of the probes designed for this site in patent 201710647677.8 is: AGTATATGAAATTGGATATTGCAGCAGTCAGAGCCCTTAACCTTTTTCAGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTTAAAAATGTTGAATGGTTAAAAAATGTTTTCATTGAC, when the mutation occurs at the site, the repetition number is far lower than that of the actual probe, and the capture off-target of the probe is generated. In this embodiment, hybridization is performed on the sense strand design probes of the target, and all possible indels are designed, specifically, it is known in the art that MSI is mainly a reduction in repetitive sequence, and this embodiment designs, for example, AAAAAAA repetition, taking all the reduction possibilities into consideration, and in this embodiment, all probes with 1-7 reduction (AAAAAA, AAAAA, AAAA, AAA, AA, A, -) in repetitive sequence are designed specifically (as shown in fig. 2), so as to improve the specificity of the probes, improve the effective capturing information, and further improve the detectability of complex and difficult samples.
Preferably, sense strand probes that capture the positions of BAT-34c4, BAT-40, BAT25, MONO-27, NR21, BAT26, D5S346, D2S123, NR24, D17S250 and NR27 have nucleotide sequences as shown in SEQ ID No.1-10, SEQ ID No.11-20, SEQ ID No.21-32, SEQ ID No.33-42, SEQ ID No.43-53, SEQ ID No.54-62, SEQ ID No.63-72, SEQ ID No.73-84, SEQ ID No.85-93, SEQ ID No.94-105 and SEQ ID No.106-117, in that order.
In some preferred embodiments, the probe is labeled with a label, preferably biotin.
In some preferred embodiments, the probe is a DNA probe or an RNA probe.
Based on the beneficial effects of the probe set for detecting microsatellite instability by using high-throughput sequencing, the invention also provides a kit for detecting microsatellite instability by using high-throughput sequencing, which comprises the probe set provided by the invention. The kit uses the probe set provided by the invention as an effective detection substance, so that the kit has all the beneficial effects of the probe set provided by the invention. In addition, the kit may further include a conventional detection reagent, which is not limited in the present invention.
In addition, the invention also provides a method for detecting microsatellite instability, which uses the probe set or the kit to enrich target genes through liquid phase hybridization, and performs high-throughput sequencing on the DNA sequences obtained through enrichment to obtain microsatellite instability results.
According to the microsatellite instability detection method provided by the invention, the target gene is enriched by using the probe set provided by the invention through liquid phase hybridization, so that the capturing and enriching efficiency of fragments is greatly improved, and the DNA sequences obtained through enrichment are subjected to high-flux sequencing, so that the effects of low data repetition rate and high effective depth can be realized, and the efficiency of high-flux sequencing is improved. The probe set can effectively target and enrich relevant sites of microsatellite instability in tumor tissues and free DNA, and provides an efficient genotyping technology for research and application of microsatellite instability. Meanwhile, the target gene can be effectively enriched by using the liquid phase hybridization technology, the requirement on sample size is reduced, and meanwhile, the sequencing cost can be better controlled.
The method for detecting the microsatellite instability is only used for scientific research and is not a disease diagnosis and/or treatment method.
In some preferred embodiments, a target genomic library is first constructed, the target gene is enriched by liquid phase hybridization using the probe set or kit described above, and the DNA sequence obtained by enrichment is subjected to high throughput sequencing to obtain microsatellite instability results.
The target genomic library is a whole genomic library, specifically, genomic DNA may be extracted from a cell, body fluid or tissue sample of a human, for example, genomic DNA may be extracted from a peripheral blood sample, and processed to obtain the target genomic library. Preferably, the genomic library of interest is obtained by end repair after fragmenting the whole genome of interest.
In some preferred embodiments, the target gene fragments are obtained by elution treatment after mixing and adsorbing the probe set with magnetic beads. For example, each probe in the probe set provided by the invention can be labeled with biotin, then the hybridization product is adsorbed by streptavidin magnetic beads after hybridization, the hybridization product is adsorbed to the magnetic beads through the combination of biotin and streptavidin on the probe, and then the enriched microsatellite site fragments related to the instability of the microsatellite are released from the magnetic beads.
In some preferred embodiments, the target gene fragments obtained by elution treatment are amplified by PCR, and the amplified fragments with the length of 220-320bp are subjected to high-throughput sequencing and analysis, so that microsatellite instability results are obtained.
The invention is further illustrated by the following specific examples, which are not to be construed as limiting the scope of the invention in accordance with conventional or manufacturer's recommendations. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. The reaction system and the reaction conditions in the examples are only examples. The reaction system and reaction conditions in each step in the examples can be adjusted within an acceptable range to optimize the reaction conditions, and the invention is not limited thereto; the length of the probe will be different according to the length of the synthesized platform, and in this embodiment, the length of the probe is 121bp, and it can be understood that the design rule of the probe provided by the invention is only compounded.
Example 1 preparation of Capture probes
Probe sequences were synthesized by methods well known in the art, mixed homogeneously in a total volume of 1ml of TE, and 1 μl of the mixture was subjected to PCR amplification using universal PCR primers as shown in the following table, PCR amplification system: 1 μl of the probe solution; forward primer (20. Mu.M), 1. Mu.l; reverse primer (20. Mu.M), 1ul;47 mu L Platinum PCR Supermix, total volume 50. Mu.L. Amplification conditions: 95 ℃ for 2min; (94 ℃,30s,55 ℃,30s,72 ℃ 30 s) 20 cycles; 72℃for 5min.
| Sequence (5 '-3') |
| TAATACGACTCACTATAGGGATCGCACCAGCGTGT |
| AGAAGAGTCACATAC |
The PCR product was purified using a MinElute PCR Purification Kit purification kit and stored at-20 ℃. The PCR purified product was transcribed in vitro using an Ambion SP6 megascript kit at 500 ng. The in vitro transcribed RNA was purified using Qiagen RNeasy minikit and the purified product yielded a biotin-labeled probe of this example.
Example 2 specific Capture and sequencing
This example uses the capture probe set provided in example 1 and enriches the target sequence based on the sequence capture technique of liquid phase hybridization. The liquid phase hybridization technical route is as follows: 1) preparing a hybridization probe library, 2) enriching target genes by using probes, and 3) sequencing the enriched DNA sequences by using a high-throughput sequencer.
The library construction method comprises the following steps:
DNA extraction and disruption
DNA was extracted from 4 peripheral blood samples using QIAamp DNA Blood Mini Kit.
Using a Bioruptor Pico DNA breaking instrument, setting parameters of ON 30s and OFF 30s as 1 cycle after the temperature of the cold circulation instrument is reduced to 4 ℃, carrying out 3 rounds of breaking every 10cycles, placing the samples ON an oscillator after each group is finished, fully and uniformly mixing, and carrying out breaking ON the next round after short centrifugation.
1 μl of the sample was used for fragment detection using QSEP 100, and the main peak of the sample detection after normal disruption was about 150bp-200bp.
2. End repair
The 10×T4PNKbuffer and Natural dNTP Mix were removed from the kit stored at-20deg.C in advance, thawed on ice and thoroughly mixed with Vortex until no solid insoluble material was present in the Buffer, and the enzyme was removed from the-20deg.C refrigerator and placed on the-20deg.C ice bin.
The end repair reaction systems were prepared separately in 1.5ml centrifuge tubes as follows: the temperature bath is carried out for 30min at 20 ℃. The reaction product was purified using Ampure XP magnetic beads and dissolved in 20. Mu.l TE.
3. Terminal addition "A" (A-Tailing)
The end-to-end "A" reaction system was prepared separately in 1.5ml centrifuge tubes as follows: the temperature bath is carried out for 30min at 37 ℃. The reaction product was purified using Ampure XP magnetic beads and dissolved in 25. Mu.l TE.
| Reagent(s) | Volume of |
| Sample from 2 | 19.5μl |
| 10×Blue Buffer | 2.5μl |
| 1mM dATP | 2.5μl |
| Klenow(3’-5’exo-) | 0.5μl |
| Total | 25μl |
Adapter connection
The adapter ligation reaction system was prepared in a 1.5ml centrifuge tube. The temperature bath is carried out for 15min at 20 ℃. The post-reaction product was purified using Ampure XP magnetic beads and dissolved in 21. Mu.l TE:
| reagent(s) | Volume of |
| Sample from step 3 | 25μl |
| 2×Rapid ligation Buffer | 25μl |
| Adaptes | 1μl |
| T4 DNA ligase | 1μl |
| Total | 52μl |
PCR amplification of index
The PCR reaction system and the reaction conditions are as follows:
| reagent(s) | Volume of |
| Sample from step 4 | 21μl |
| HiFi Mix | 25μl |
| Index primers 1 | 2μl |
| Index primers 2 | 2μl |
| Total | 50μl |
The following procedure was run in a PCR instrument:
the reaction products were purified using Ampure XP magnetic beads. Dissolved in 50. Mu.l TE. The concentration of PCR products was detected using NanoDrop 1000. Library quantification was performed using Qubit 3.0, library concentrations >25 ng/. Mu.l reference as a qualified library. The main peak of the library is about 220-320bp, and the main peak is free from hetero peaks after and before the main peak by using QSEP 100 detection.
6. Hybridization
And taking out the Hyb Block and the Hyb Buffer from the refrigerator, melting the Hyb Block and the Hyb Buffer on ice, and placing the Hyb Buffer on a metal bath for preheating at 65 ℃.
The sample library was mixed with Hyb block, labeled B, according to the following system.
| Component | Volume for capture |
| Sample library | 600ng |
| Hyb Block | 5μl |
And (3) placing the Hyb Buffer in a new 200 mu l PCR tube, covering a tube cover, marking as A, and continuously placing in a 65 ℃ water bath for incubation for later use, wherein the Hyb Buffer is melted at room temperature, precipitation occurs before heating, the mixture is placed in the 65 ℃ water bath for preheating, and 20 mu l of the Hyb Buffer is placed in the new 200 mu l PCR tube after complete dissolution (without precipitation and turbidity).
Mu.l of RNase Block and 2. Mu.l of Probe were placed in a 200. Mu.l PCR tube, gently blotted and mixed, briefly centrifuged and placed on ice for use, labeled C.
Setting parameters of a PCR instrument, heat lid 100 ℃,95 ℃ for 5min;65 ℃, hold;
the PCR tube B was placed on a PCR instrument and the procedure was run.
When the temperature of the PCR instrument is reduced to 65 ℃, placing the PCR tube A on the PCR instrument for incubation, and covering a thermal cover of the PCR instrument; after 5min, placing the C on the PCR for incubation, and covering a thermal cover of the PCR instrument; placing the PCR tube C into a PCR instrument for 2min, adjusting the liquid transfer device to 13 μl, sucking 13 μl Hyb Buffer from the PCR tube A, transferring the samples in all the PCR tubes B into the PCR tube C, gently sucking 10 times, mixing thoroughly, avoiding generating a large amount of bubbles, sealing the tube cover, covering the thermal cover of the PCR instrument, and incubating overnight at 65 ℃ for 8-16 h.
7. Capturing
Wash Buffer2 (1.8 ml for each capture) was aliquoted in advance and placed on a thermo mixer for pre-heating at 65 ℃.
The hybridization product PCR tube C is kept on a PCR instrument, the product of the hybridized PCR tube C is added into 200 mu L of Biotin-bed, the mixture is sucked and beaten for 6 times by a pipette for uniform mixing, the mixture is placed on a rotary mixer (10 rpm/min) for 30min at room temperature, a centrifuge tube is placed on a magnetic rack for 2min, and then the supernatant is removed.
500. Mu.L of Wash Buffer 1 was added to the centrifuge tube, gently blotted 6 times to mix well, the beads resuspended, and the samples were mixed by shaking on a vortex mixer for 5s and incubated at room temperature for 15min.
Adding 500 μl of Wash Buffer2 preheated at 65deg.C, mixing by vortex for 5s, incubating on a thermo mixer at 65deg.C for 10min, and washing at 800 rpm.
The tube was placed on a magnetic rack for 2min and the supernatant removed. The washing was repeated 2 times for a total of 3 times. Wash Buffer2 was removed thoroughly last time (residue can be removed with a 10. Mu.l pipette).
Add 25. Mu.L of Nuclease-free water to the centrifuge tube, remove the tube from the magnetic rack, gently pipette 6 times to resuspend the beads for use.
8. Enrichment
Enrichment of the DNA library after capture was required and Mix was formulated according to the following table:
| Component | Volume for 4capture |
| samples from the previous step | 30μl |
| Post PCR Buffer | 18μl |
| Post PCR Primer(25μM) | 1μl |
| Post DNA Polymerase | 1μl |
| Total volume | 50μl |
After purification of the PCR products, the library was quantified using Qubit dsDNA HS Assay Kit and the library fragment length was determined using QSEP 100, the library length being approximately between 220-320 bp. Sequencing was performed using an Illumina sequencer.
The data was analyzed using xy_msifinder software and MSI typing results were obtained.
Example 3
20 colorectal cancer samples of known MSI type and plasma samples of corresponding patients were collected, and tested according to the procedure shown in example 2, with the test results shown below.
From the results, the probe of the invention is used for detecting microsatellite states of different samples, the accuracy of tissue samples reaches 100%, the specificity reaches 100%, the accuracy of plasma samples reaches 97.5%, and the specificity reaches 100%.
From the above, it has been shown that the invention has the following advantages: 1. the method has high accuracy, high sensitivity and high flux, and can effectively and accurately detect the microsatellite instability of tumor patients. 2. The microsatellite instability detection method based on the second-generation sequencing data can eliminate the need of independent and special detection of MSI states, and can detect the clinically important tumor phenotype of microsatellite instability while detecting sensitive mutations on other genes. 3. The method can detect more microsatellite loci than the conventional detection method.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
SEQUENCE LISTING
<110> Hangzhou mascot medical laboratory Co., ltd
Zhang Guangmou
<120> detection of microsatellite instability Using high throughput sequencing Probe set, kit and detection of microsatellite instability
Method of
<160> 117
<170> PatentIn version 3.5
<210> 1
<211> 130
<212> DNA
<213> artificial sequence
<400> 1
gaagcgagga tcaacttcct ttttttgttg ttgcttgtgt ggttttcttt tttttttttt 60
tttttttttt tttttttttt tttgagacaa ggtggtcttg ctctaccacc caggccattg 120
cgtgaaccga 130
<210> 2
<211> 130
<212> DNA
<213> artificial sequence
<400> 2
gaagcgagga tcaacttcct ttttttgttg ttgcttgtgt ggttttcttt tttttttttt 60
tttttttttt tttttttttt ttttgagaca aggtggtctt gctctaccac ccaggcattg 120
cgtgaaccga 130
<210> 3
<211> 130
<212> DNA
<213> artificial sequence
<400> 3
gaagcgagga tcaactcctt tttttgttgt tgcttgtgtg gttttctttt tttttttttt 60
tttttttttt tttttttttt ttttgagaca aggtggtctt gctctaccac ccaggcattg 120
cgtgaaccga 130
<210> 4
<211> 130
<212> DNA
<213> artificial sequence
<400> 4
gaagcgagga tcaactcctt tttttgttgt tgcttgtgtg gttttctttt tttttttttt 60
tttttttttt tttttttttt tttttgagac aaggtggtct tgctctacca cccagcattg 120
cgtgaaccga 130
<210> 5
<211> 130
<212> DNA
<213> artificial sequence
<400> 5
gaagcgagga tcaactcttt ttttgttgtt gcttgtgtgg ttttcttttt tttttttttt 60
tttttttttt tttttttttt tttttgagac aaggtggtct tgctctacca cccagcattg 120
cgtgaaccga 130
<210> 6
<211> 130
<212> DNA
<213> artificial sequence
<400> 6
gaagcgagga tcaactcttt ttttgttgtt gcttgtgtgg ttttcttttt tttttttttt 60
tttttttttt tttttttttt ttttttgaga caaggtggtc ttgctctacc acccacattg 120
cgtgaaccga 130
<210> 7
<211> 130
<212> DNA
<213> artificial sequence
<400> 7
gaagcgagga tcaacttttt tttgttgttg cttgtgtggt tttctttttt tttttttttt 60
tttttttttt tttttttttt ttttttgaga caaggtggtc ttgctctacc acccacattg 120
cgtgaaccga 130
<210> 8
<211> 130
<212> DNA
<213> artificial sequence
<400> 8
gaagcgagga tcaacttttt tttgttgttg cttgtgtggt tttctttttt tttttttttt 60
tttttttttt tttttttttt tttttttgag acaaggtggt cttgctctac caccccattg 120
cgtgaaccga 130
<210> 9
<211> 130
<212> DNA
<213> artificial sequence
<400> 9
gaagcgagga tcaactctca agccaagatt aacttcctac accacaaccc tgcttttgtt 60
cctttttttg ttgttgcttg tgtggttttc tttttttttt tttttttttt tttttcattg 120
cgtgaaccga 130
<210> 10
<211> 130
<212> DNA
<213> artificial sequence
<400> 10
gaagcgagga tcaacttttt tttttttttt tttttttttt tgagacaagg tggtcttgct 60
ctaccaccca ggctagagtg cagaggtgtg atcttggctc actgccagga accagcattg 120
cgtgaaccga 130
<210> 11
<211> 130
<212> DNA
<213> artificial sequence
<400> 11
gaagcgagga tcaactttgg ccaactaaaa aagaaaaaaa agtaaaacca ggattttttt 60
tttttttttt tttttttgag gcagagtctt gctctgtctc ccacgctgga gtgcacattg 120
cgtgaaccga 130
<210> 12
<211> 130
<212> DNA
<213> artificial sequence
<400> 12
gaagcgagga tcaactttgg ccaactaaaa aagaaaaaaa agtaaaacca ggattttttt 60
tttttttttt ttttttttga ggcagagtct tgctctgtct cccacgctgg agtgccattg 120
cgtgaaccga 130
<210> 13
<211> 130
<212> DNA
<213> artificial sequence
<400> 13
gaagcgagga tcaacttggc caactaaaaa agaaaaaaaa gtaaaaccag gatttttttt 60
tttttttttt ttttttttga ggcagagtct tgctctgtct cccacgctgg agtgccattg 120
cgtgaaccga 130
<210> 14
<211> 130
<212> DNA
<213> artificial sequence
<400> 14
gaagcgagga tcaacttggc caactaaaaa agaaaaaaaa gtaaaaccag gatttttttt 60
tttttttttt tttttttttg aggcagagtc ttgctctgtc tcccacgctg gagtgcattg 120
cgtgaaccga 130
<210> 15
<211> 130
<212> DNA
<213> artificial sequence
<400> 15
gaagcgagga tcaactggcc aactaaaaaa gaaaaaaaag taaaaccagg attttttttt 60
tttttttttt tttttttttg aggcagagtc ttgctctgtc tcccacgctg gagtgcattg 120
cgtgaaccga 130
<210> 16
<211> 130
<212> DNA
<213> artificial sequence
<400> 16
gaagcgagga tcaactggcc aactaaaaaa gaaaaaaaag taaaaccagg attttttttt 60
tttttttttt tttttttttt gaggcagagt cttgctctgt ctcccacgct ggagtcattg 120
cgtgaaccga 130
<210> 17
<211> 130
<212> DNA
<213> artificial sequence
<400> 17
gaagcgagga tcaactgcca actaaaaaag aaaaaaaagt aaaaccagga tttttttttt 60
tttttttttt tttttttttt gaggcagagt cttgctctgt ctcccacgct ggagtcattg 120
cgtgaaccga 130
<210> 18
<211> 130
<212> DNA
<213> artificial sequence
<400> 18
gaagcgagga tcaactgcca actaaaaaag aaaaaaaagt aaaaccagga tttttttttt 60
tttttttttt tttttttttt tgaggcagag tcttgctctg tctcccacgc tggagcattg 120
cgtgaaccga 130
<210> 19
<211> 130
<212> DNA
<213> artificial sequence
<400> 19
gaagcgagga tcaactgatg tggttctgtc tcctttctta agggtggatc aaatttcact 60
tggccaacta aaaaagaaaa aaaagtaaaa ccaggatttt tttttttttt tttttcattg 120
cgtgaaccga 130
<210> 20
<211> 130
<212> DNA
<213> artificial sequence
<400> 20
gaagcgagga tcaacttttt tttttttttt tttttgaggc agagtcttgc tctgtctccc 60
acgctggagt gcagtggcgc aatctcagct cactgcaatc tccaccccct gggttcattg 120
cgtgaaccga 130
<210> 21
<211> 130
<212> DNA
<213> artificial sequence
<400> 21
gaagcgagga tcaactgata ttgcagcagt cagagccctt aacctttttc aggtaaaaaa 60
aaaaaaaaaa aaaaaaaggg ttaaaaatgt tgaatggtta aaaaatgttt tcattcattg 120
cgtgaaccga 130
<210> 22
<211> 130
<212> DNA
<213> artificial sequence
<400> 22
gaagcgagga tcaactatat tgcagcagtc agagccctta acctttttca ggtaaaaaaa 60
aaaaaaaaaa aaaaaaaggg ttaaaaatgt tgaatggtta aaaaatgttt tcattcattg 120
cgtgaaccga 130
<210> 23
<211> 130
<212> DNA
<213> artificial sequence
<400> 23
gaagcgagga tcaactatat tgcagcagtc agagccctta acctttttca ggtaaaaaaa 60
aaaaaaaaaa aaaaaaaagg gttaaaaatg ttgaatggtt aaaaaatgtt ttcatcattg 120
cgtgaaccga 130
<210> 24
<211> 130
<212> DNA
<213> artificial sequence
<400> 24
gaagcgagga tcaacttatt gcagcagtca gagcccttaa cctttttcag gtaaaaaaaa 60
aaaaaaaaaa aaaaaaaagg gttaaaaatg ttgaatggtt aaaaaatgtt ttcatcattg 120
cgtgaaccga 130
<210> 25
<211> 130
<212> DNA
<213> artificial sequence
<400> 25
gaagcgagga tcaacttatt gcagcagtca gagcccttaa cctttttcag gtaaaaaaaa 60
aaaaaaaaaa aaaaaaaaag ggttaaaaat gttgaatggt taaaaaatgt tttcacattg 120
cgtgaaccga 130
<210> 26
<211> 130
<212> DNA
<213> artificial sequence
<400> 26
gaagcgagga tcaactattg cagcagtcag agcccttaac ctttttcagg taaaaaaaaa 60
aaaaaaaaaa aaaaaaaaag ggttaaaaat gttgaatggt taaaaaatgt tttcacattg 120
cgtgaaccga 130
<210> 27
<211> 130
<212> DNA
<213> artificial sequence
<400> 27
gaagcgagga tcaactattg cagcagtcag agcccttaac ctttttcagg taaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa gggttaaaaa tgttgaatgg ttaaaaaatg ttttccattg 120
cgtgaaccga 130
<210> 28
<211> 130
<212> DNA
<213> artificial sequence
<400> 28
gaagcgagga tcaactttgc agcagtcaga gcccttaacc tttttcaggt aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa gggttaaaaa tgttgaatgg ttaaaaaatg ttttccattg 120
cgtgaaccga 130
<210> 29
<211> 130
<212> DNA
<213> artificial sequence
<400> 29
gaagcgagga tcaactttgc agcagtcaga gcccttaacc tttttcaggt aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa agggttaaaa atgttgaatg gttaaaaaat gttttcattg 120
cgtgaaccga 130
<210> 30
<211> 130
<212> DNA
<213> artificial sequence
<400> 30
gaagcgagga tcaactagtt tgaactgact acttttgact tcagccagta tatgaaattg 60
gatattgcag cagtcagagc ccttaacctt tttcaggtaa aaaaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 31
<211> 130
<212> DNA
<213> artificial sequence
<400> 31
gaagcgagga tcaacttttg aactgactac ttttgacttc agccagtata tgaaattgga 60
tattgcagca gtcagagccc ttaacctttt tcaggtaaaa aaaaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 32
<211> 130
<212> DNA
<213> artificial sequence
<400> 32
gaagcgagga tcaactaaaa aaaaaaaaaa aaaaagggtt aaaaatgttg aatggttaaa 60
aaatgttttc attgacatat actgaagaag cttataaagg agctaaaata ttttgcattg 120
cgtgaaccga 130
<210> 33
<211> 130
<212> DNA
<213> artificial sequence
<400> 33
gaagcgagga tcaactacat tgctggaagt tctggccaga gaaattagaa cacacacaca 60
cacacacaca cacacacaca ctattttata gatagataga tggtatccaa gtcagcattg 120
cgtgaaccga 130
<210> 34
<211> 130
<212> DNA
<213> artificial sequence
<400> 34
gaagcgagga tcaactgctg gaagttctgg ccagagaaat tagaacacac acacacacac 60
acacacacac acacacacac acacactatt ttatagatag atagatggta tccaacattg 120
cgtgaaccga 130
<210> 35
<211> 130
<212> DNA
<213> artificial sequence
<400> 35
gaagcgagga tcaactctgg aagttctggc cagagaaatt agaacacaca cacacacaca 60
cacacacaca cacacacaca cacacactat tttatagata gatagatggt atccacattg 120
cgtgaaccga 130
<210> 36
<211> 130
<212> DNA
<213> artificial sequence
<400> 36
gaagcgagga tcaacttgga agttctggcc agagaaatta gaacacacac acacacacac 60
acacacacac acacacacac acacacacta ttttatagat agatagatgg tatcccattg 120
cgtgaaccga 130
<210> 37
<211> 130
<212> DNA
<213> artificial sequence
<400> 37
gaagcgagga tcaactggaa gttctggcca gagaaattag aacacacaca cacacacaca 60
cacacacaca cacacacaca cacacacact attttataga tagatagatg gtatccattg 120
cgtgaaccga 130
<210> 38
<211> 130
<212> DNA
<213> artificial sequence
<400> 38
gaagcgagga tcaactgaag ttctggccag agaaattaga acacacacac acacacacac 60
acacacacac acacacacac acacacacac tattttatag atagatagat ggtatcattg 120
cgtgaaccga 130
<210> 39
<211> 130
<212> DNA
<213> artificial sequence
<400> 39
gaagcgagga tcaactaagt tctggccaga gaaattagaa cacacacaca cacacacaca 60
cacacacaca cacacacaca cacacacaca ctattttata gatagataga tggtacattg 120
cgtgaaccga 130
<210> 40
<211> 130
<212> DNA
<213> artificial sequence
<400> 40
gaagcgagga tcaactaaaa caggatgcct gcctttaaca ctgctattca acattgctgg 60
aagttctggc cagagaaatt agacacagtg atacacacac acacacacac acacacattg 120
cgtgaaccga 130
<210> 41
<211> 130
<212> DNA
<213> artificial sequence
<400> 41
gaagcgagga tcaactcaca cacacacaca cacacacaca cacatatttt atagatagat 60
agatggtatc caagtcagaa agggagaagt aaaactatcc ctattgtaga tgacacattg 120
cgtgaaccga 130
<210> 42
<211> 130
<212> DNA
<213> artificial sequence
<400> 42
gaagcgagga tcaactcaca cacacacaca cacacacaca cacacacata ttttatagat 60
agatagatgg tatccaagtc agaaagggag aagtaaaact atccctattg tagatcattg 120
cgtgaaccga 130
<210> 43
<211> 130
<212> DNA
<213> artificial sequence
<400> 43
gaagcgagga tcaactctga ctccaaaaac tcttctcttc cctgggccca gtcctatttt 60
tttttttttt ttttgtgaga cagagtctca ctctgtcacc caggttggaa tgcaacattg 120
cgtgaaccga 130
<210> 44
<211> 130
<212> DNA
<213> artificial sequence
<400> 44
gaagcgagga tcaactctga ctccaaaaac tcttctcttc cctgggccca gtcctatttt 60
tttttttttt tttttgtgag acagagtctc actctgtcac ccaggttgga atgcacattg 120
cgtgaaccga 130
<210> 45
<211> 130
<212> DNA
<213> artificial sequence
<400> 45
gaagcgagga tcaacttgac tccaaaaact cttctcttcc ctgggcccag tcctattttt 60
tttttttttt tttttgtgag acagagtctc actctgtcac ccaggttgga atgcacattg 120
cgtgaaccga 130
<210> 46
<211> 130
<212> DNA
<213> artificial sequence
<400> 46
gaagcgagga tcaacttgac tccaaaaact cttctcttcc ctgggcccag tcctattttt 60
tttttttttt ttttttgtga gacagagtct cactctgtca cccaggttgg aatgccattg 120
cgtgaaccga 130
<210> 47
<211> 130
<212> DNA
<213> artificial sequence
<400> 47
gaagcgagga tcaactgact ccaaaaactc ttctcttccc tgggcccagt cctatttttt 60
tttttttttt ttttttgtga gacagagtct cactctgtca cccaggttgg aatgccattg 120
cgtgaaccga 130
<210> 48
<211> 130
<212> DNA
<213> artificial sequence
<400> 48
gaagcgagga tcaactgact ccaaaaactc ttctcttccc tgggcccagt cctatttttt 60
tttttttttt tttttttgtg agacagagtc tcactctgtc acccaggttg gaatgcattg 120
cgtgaaccga 130
<210> 49
<211> 130
<212> DNA
<213> artificial sequence
<400> 49
gaagcgagga tcaactactc caaaaactct tctcttccct gggcccagtc ctattttttt 60
tttttttttt tttttttgtg agacagagtc tcactctgtc acccaggttg gaatgcattg 120
cgtgaaccga 130
<210> 50
<211> 130
<212> DNA
<213> artificial sequence
<400> 50
gaagcgagga tcaactactc caaaaactct tctcttccct gggcccagtc ctattttttt 60
tttttttttt ttttttttgt gagacagagt ctcactctgt cacccaggtt ggaatcattg 120
cgtgaaccga 130
<210> 51
<211> 130
<212> DNA
<213> artificial sequence
<400> 51
gaagcgagga tcaactctcc aaaaactctt ctcttccctg ggcccagtcc tatttttttt 60
tttttttttt ttttttttgt gagacagagt ctcactctgt cacccaggtt ggaatcattg 120
cgtgaaccga 130
<210> 52
<211> 130
<212> DNA
<213> artificial sequence
<400> 52
gaagcgagga tcaacttaag gtctgcctta acgtgatccc cattgctgaa ttttacctcc 60
tgactccaaa aactcttctc ttccctgggc ccagtcctat tttttttttt tttttcattg 120
cgtgaaccga 130
<210> 53
<211> 130
<212> DNA
<213> artificial sequence
<400> 53
gaagcgagga tcaacttttt tttttttttt ttgtgagaca gagtctcact ctgtcaccca 60
ggttggaatg caatggcaca atctccgctc actgcaagct ccgcctcccg ggttccattg 120
cgtgaaccga 130
<210> 54
<211> 130
<212> DNA
<213> artificial sequence
<400> 54
gaagcgagga tcaactgtgg gagtgattct ctaaagagtt ttgtgttttg tttttttttt 60
tttttttttt tttttttttt tgagaacaga gcattttaga gccatagtta aaatgcattg 120
cgtgaaccga 130
<210> 55
<211> 130
<212> DNA
<213> artificial sequence
<400> 55
gaagcgagga tcaacttggg agtgattctc taaagagttt tgtgttttgt tttttttttt 60
tttttttttt tttttttttt tgagaacaga gcattttaga gccatagtta aaatgcattg 120
cgtgaaccga 130
<210> 56
<211> 130
<212> DNA
<213> artificial sequence
<400> 56
gaagcgagga tcaacttggg agtgattctc taaagagttt tgtgttttgt tttttttttt 60
tttttttttt tttttttttt ttgagaacag agcattttag agccatagtt aaaatcattg 120
cgtgaaccga 130
<210> 57
<211> 130
<212> DNA
<213> artificial sequence
<400> 57
gaagcgagga tcaactggga gtgattctct aaagagtttt gtgttttgtt tttttttttt 60
tttttttttt tttttttttt ttgagaacag agcattttag agccatagtt aaaatcattg 120
cgtgaaccga 130
<210> 58
<211> 130
<212> DNA
<213> artificial sequence
<400> 58
gaagcgagga tcaactggga gtgattctct aaagagtttt gtgttttgtt tttttttttt 60
tttttttttt tttttttttt tttgagaaca gagcatttta gagccatagt taaaacattg 120
cgtgaaccga 130
<210> 59
<211> 130
<212> DNA
<213> artificial sequence
<400> 59
gaagcgagga tcaactggag tgattctcta aagagttttg tgttttgttt tttttttttt 60
tttttttttt tttttttttt tttgagaaca gagcatttta gagccatagt taaaacattg 120
cgtgaaccga 130
<210> 60
<211> 130
<212> DNA
<213> artificial sequence
<400> 60
gaagcgagga tcaactggag tgattctcta aagagttttg tgttttgttt tttttttttt 60
tttttttttt tttttttttt ttttgagaac agagcatttt agagccatag ttaaacattg 120
cgtgaaccga 130
<210> 61
<211> 130
<212> DNA
<213> artificial sequence
<400> 61
gaagcgagga tcaactcagg tggcaaaggg catggctttc ctcgcctcca agaatgtaag 60
tgggagtgat tctctaaaga gttttgtgtt ttgttttttt gatttttttt tttttcattg 120
cgtgaaccga 130
<210> 62
<211> 130
<212> DNA
<213> artificial sequence
<400> 62
gaagcgagga tcaacttttt tttttttttt ttttttttga gaacagagca ttttagagcc 60
atagttaaaa tgcagaatgt cattttgaag tgtggtaacc aaaagcagag gaaatcattg 120
cgtgaaccga 130
<210> 63
<211> 130
<212> DNA
<213> artificial sequence
<400> 63
gaagcgagga tcaacttttt tcagggaatt gagagttaca ggttactctg tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg taaatttccc gatttatcac tagagtgagt aactacattg 120
cgtgaaccga 130
<210> 64
<211> 130
<212> DNA
<213> artificial sequence
<400> 64
gaagcgagga tcaacttttt cagggaattg agagttacag gttactctgt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtaaatttcc cgatttatca ctagagtgag taactcattg 120
cgtgaaccga 130
<210> 65
<211> 130
<212> DNA
<213> artificial sequence
<400> 65
gaagcgagga tcaacttttc agggaattga gagttacagg ttactctgtg tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg tgtaaatttc ccgatttatc actagagtga gtaaccattg 120
cgtgaaccga 130
<210> 66
<211> 130
<212> DNA
<213> artificial sequence
<400> 66
gaagcgagga tcaactttca gggaattgag agttacaggt tactctgtgt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtgtaaattt cccgatttat cactagagtg agtaacattg 120
cgtgaaccga 130
<210> 67
<211> 130
<212> DNA
<213> artificial sequence
<400> 67
gaagcgagga tcaacttcag ggaattgaga gttacaggtt actctgtgtg tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg tgtgtaaatt tcccgattta tcactagagt gagtacattg 120
cgtgaaccga 130
<210> 68
<211> 130
<212> DNA
<213> artificial sequence
<400> 68
gaagcgagga tcaactcagg gaattgagag ttacaggtta ctctgtgtgt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtgtgtaaat ttcccgattt atcactagag tgagtcattg 120
cgtgaaccga 130
<210> 69
<211> 130
<212> DNA
<213> artificial sequence
<400> 69
gaagcgagga tcaactaggg aattgagagt tacaggttac tctgtgtgtg tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg tgtgtgtaaa tttcccgatt tatcactaga gtgagcattg 120
cgtgaaccga 130
<210> 70
<211> 130
<212> DNA
<213> artificial sequence
<400> 70
gaagcgagga tcaactggga attgagagtt acaggttact ctgtgtgtgt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtgtgtgtaa atttcccgat ttatcactag agtgacattg 120
cgtgaaccga 130
<210> 71
<211> 130
<212> DNA
<213> artificial sequence
<400> 71
gaagcgagga tcaactgtgt gtgtgtgtgt gtgtgtgtgt aaatttcccg atttatcact 60
agagtgagta actaactaac taactgcttt ataaagctat cctggtattc atatgcattg 120
cgtgaaccga 130
<210> 72
<211> 130
<212> DNA
<213> artificial sequence
<400> 72
gaagcgagga tcaactgtgt gtgtgtgtgt gtgtgtgtgt gtgtaaattt cccgatttat 60
cactagagtg agtaactaac taactaactg ctttataaag ctatcctggt attcacattg 120
cgtgaaccga 130
<210> 73
<211> 130
<212> DNA
<213> artificial sequence
<400> 73
gaagcgagga tcaactctgt gagatccagg aaaccatgct tgcaaaccac tggtaaaaaa 60
aaaaaaaaaa aaaaaaagcc acagtgactt gcttattggt cattgctagt attatcattg 120
cgtgaaccga 130
<210> 74
<211> 130
<212> DNA
<213> artificial sequence
<400> 74
gaagcgagga tcaacttgtg agatccagga aaccatgctt gcaaaccact ggtaaaaaaa 60
aaaaaaaaaa aaaaaaagcc acagtgactt gcttattggt cattgctagt attatcattg 120
cgtgaaccga 130
<210> 75
<211> 130
<212> DNA
<213> artificial sequence
<400> 75
gaagcgagga tcaacttgtg agatccagga aaccatgctt gcaaaccact ggtaaaaaaa 60
aaaaaaaaaa aaaaaaaagc cacagtgact tgcttattgg tcattgctag tattacattg 120
cgtgaaccga 130
<210> 76
<211> 130
<212> DNA
<213> artificial sequence
<400> 76
gaagcgagga tcaactgtga gatccaggaa accatgcttg caaaccactg gtaaaaaaaa 60
aaaaaaaaaa aaaaaaaagc cacagtgact tgcttattgg tcattgctag tattacattg 120
cgtgaaccga 130
<210> 77
<211> 130
<212> DNA
<213> artificial sequence
<400> 77
gaagcgagga tcaactgtga gatccaggaa accatgcttg caaaccactg gtaaaaaaaa 60
aaaaaaaaaa aaaaaaaaag ccacagtgac ttgcttattg gtcattgcta gtattcattg 120
cgtgaaccga 130
<210> 78
<211> 130
<212> DNA
<213> artificial sequence
<400> 78
gaagcgagga tcaacttgag atccaggaaa ccatgcttgc aaaccactgg taaaaaaaaa 60
aaaaaaaaaa aaaaaaaaag ccacagtgac ttgcttattg gtcattgcta gtattcattg 120
cgtgaaccga 130
<210> 79
<211> 130
<212> DNA
<213> artificial sequence
<400> 79
gaagcgagga tcaacttgag atccaggaaa ccatgcttgc aaaccactgg taaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa gccacagtga cttgcttatt ggtcattgct agtatcattg 120
cgtgaaccga 130
<210> 80
<211> 130
<212> DNA
<213> artificial sequence
<400> 80
gaagcgagga tcaactgaga tccaggaaac catgcttgca aaccactggt aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa gccacagtga cttgcttatt ggtcattgct agtatcattg 120
cgtgaaccga 130
<210> 81
<211> 130
<212> DNA
<213> artificial sequence
<400> 81
gaagcgagga tcaactcctg gaaacaaagc attgaagtct gcagttgaaa agcccaacgt 60
ctgtgagatc caggaaacca tgcttgcaaa ccactggtaa aaaaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 82
<211> 130
<212> DNA
<213> artificial sequence
<400> 82
gaagcgagga tcaacttgga aacaaagcat tgaagtctgc agttgaaaag cccaacgtct 60
gtgagatcca ggaaaccatg cttgcaaacc actggtaaaa aaaaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 83
<211> 130
<212> DNA
<213> artificial sequence
<400> 83
gaagcgagga tcaactaaaa aaaaaaaaaa aaagccacag tgacttgctt attggtcatt 60
gctagtatta tcgactcaga acctctttac taatggctag taaatcataa ttgagcattg 120
cgtgaaccga 130
<210> 84
<211> 130
<212> DNA
<213> artificial sequence
<400> 84
gaagcgagga tcaactaaaa aaaaaaaaaa aaaaagccac agtgacttgc ttattggtca 60
ttgctagtat tatcgactca gaacctcttt actaatggct agtaaatcat aattgcattg 120
cgtgaaccga 130
<210> 85
<211> 130
<212> DNA
<213> artificial sequence
<400> 85
gaagcgagga tcaactttct ggtcactcgc gtttacaaac aagaaaagtg ttgctaaaaa 60
aaaaaaaaaa aaaaaggcca ggggagacat acatttaaat ataaaaatag aactgcattg 120
cgtgaaccga 130
<210> 86
<211> 130
<212> DNA
<213> artificial sequence
<400> 86
gaagcgagga tcaactttct ggtcactcgc gtttacaaac aagaaaagtg ttgctaaaaa 60
aaaaaaaaaa aaaaaaggcc aggggagaca tacatttaaa tataaaaata gaactcattg 120
cgtgaaccga 130
<210> 87
<211> 130
<212> DNA
<213> artificial sequence
<400> 87
gaagcgagga tcaacttctg gtcactcgcg tttacaaaca agaaaagtgt tgctaaaaaa 60
aaaaaaaaaa aaaaaaggcc aggggagaca tacatttaaa tataaaaata gaactcattg 120
cgtgaaccga 130
<210> 88
<211> 130
<212> DNA
<213> artificial sequence
<400> 88
gaagcgagga tcaacttctg gtcactcgcg tttacaaaca agaaaagtgt tgctaaaaaa 60
aaaaaaaaaa aaaaaaaggc caggggagac atacatttaa atataaaaat agaaccattg 120
cgtgaaccga 130
<210> 89
<211> 130
<212> DNA
<213> artificial sequence
<400> 89
gaagcgagga tcaactctgg tcactcgcgt ttacaaacaa gaaaagtgtt gctaaaaaaa 60
aaaaaaaaaa aaaaaaaggc caggggagac atacatttaa atataaaaat agaaccattg 120
cgtgaaccga 130
<210> 90
<211> 130
<212> DNA
<213> artificial sequence
<400> 90
gaagcgagga tcaactagca gataaaagag aacacgaaaa atattcctac tccgcattca 60
cactttctgg tcactcgcgt ttacaaacaa gaaaagtgtt gctaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 91
<211> 130
<212> DNA
<213> artificial sequence
<400> 91
gaagcgagga tcaactcaga taaaagagaa cacgaaaaat attcctactc cgcattcaca 60
ctttctggtc actcgcgttt acaaacaaga aaagtgttgc taaaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 92
<211> 130
<212> DNA
<213> artificial sequence
<400> 92
gaagcgagga tcaactaaaa aaaaaaaagg ccaggggaga catacattta aatataaaaa 60
tagaactgtg ccagcgactc cggctggaat tctgctgaaa gggatgtgtc ttcagcattg 120
cgtgaaccga 130
<210> 93
<211> 130
<212> DNA
<213> artificial sequence
<400> 93
gaagcgagga tcaactaaaa aaaaaaaaaa ggccagggga gacatacatt taaatataaa 60
aatagaactg tgccagcgac tccggctgga attctgctga aagggatgtg tcttccattg 120
cgtgaaccga 130
<210> 94
<211> 130
<212> DNA
<213> artificial sequence
<400> 94
gaagcgagga tcaactagct ggccatatat atatttaaac catttgaaag tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg tttgaaacca tttgaaagtt tatgtatgtg tatatcattg 120
cgtgaaccga 130
<210> 95
<211> 130
<212> DNA
<213> artificial sequence
<400> 95
gaagcgagga tcaactgctg gccatatata tatttaaacc atttgaaagt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtttgaaacc atttgaaagt ttatgtatgt gtatacattg 120
cgtgaaccga 130
<210> 96
<211> 130
<212> DNA
<213> artificial sequence
<400> 96
gaagcgagga tcaactctgg ccatatatat atttaaacca tttgaaagtg tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg tgtttgaaac catttgaaag tttatgtatg tgtatcattg 120
cgtgaaccga 130
<210> 97
<211> 130
<212> DNA
<213> artificial sequence
<400> 97
gaagcgagga tcaacttggc catatatata tttaaaccat ttgaaagtgt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtgtttgaaa ccatttgaaa gtttatgtat gtgtacattg 120
cgtgaaccga 130
<210> 98
<211> 130
<212> DNA
<213> artificial sequence
<400> 98
gaagcgagga tcaactggcc atatatatat ttaaaccatt tgaaagtgtg tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg tgtgtttgaa accatttgaa agtttatgta tgtgtcattg 120
cgtgaaccga 130
<210> 99
<211> 130
<212> DNA
<213> artificial sequence
<400> 99
gaagcgagga tcaactgcca tatatatatt taaaccattt gaaagtgtgt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtgtgtttga aaccatttga aagtttatgt atgtgcattg 120
cgtgaaccga 130
<210> 100
<211> 130
<212> DNA
<213> artificial sequence
<400> 100
gaagcgagga tcaactccat atatatattt aaaccatttg aaagtgtgtg tgtgtgtgtg 60
tgtgtgtgtg tgtgtgtgtg tgtgtgtttg aaaccatttg aaagtttatg tatgtcattg 120
cgtgaaccga 130
<210> 101
<211> 130
<212> DNA
<213> artificial sequence
<400> 101
gaagcgagga tcaactcata tatatattta aaccatttga aagtgtgtgt gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtgtgtgttt gaaaccattt gaaagtttat gtatgcattg 120
cgtgaaccga 130
<210> 102
<211> 130
<212> DNA
<213> artificial sequence
<400> 102
gaagcgagga tcaactctca gcctccgaaa gtgctgggat tacaggcatg agccactcag 60
ctggccatat atatatttaa accatttgaa agtgtgtgtg tgtgtgtgtg tgtgtcattg 120
cgtgaaccga 130
<210> 103
<211> 130
<212> DNA
<213> artificial sequence
<400> 103
gaagcgagga tcaactgcct ccgaaagtgc tgggattaca ggcatgagcc actcagctgg 60
ccatatatat atttaaacca tttgaaagtg tgtgtgtgtg tgtgtgtgtg tgtgtcattg 120
cgtgaaccga 130
<210> 104
<211> 130
<212> DNA
<213> artificial sequence
<400> 104
gaagcgagga tcaactgtgt gtgtgtgtgt gtgtgtgtgt ttgaaaccat ttgaaagttt 60
atgtatgtgt atatatatat ataaacacac acatattttt attgtctatt tgattcattg 120
cgtgaaccga 130
<210> 105
<211> 130
<212> DNA
<213> artificial sequence
<400> 105
gaagcgagga tcaactgtgt gtgtgtgtgt gtgtgtgtgt gtgtttgaaa ccatttgaaa 60
gtttatgtat gtgtatatat atatataaac acacacatat ttttattgtc tatttcattg 120
cgtgaaccga 130
<210> 106
<211> 130
<212> DNA
<213> artificial sequence
<400> 106
gaagcgagga tcaactccag cctgggcaac aaagcgagac ccagtctcaa agaaaaaaaa 60
aaaaaaaaaa aaaaaaaaga aattgaccct gagcataaaa caagtcttgg tggatcattg 120
cgtgaaccga 130
<210> 107
<211> 130
<212> DNA
<213> artificial sequence
<400> 107
gaagcgagga tcaactccag cctgggcaac aaagcgagac ccagtctcaa agaaaaaaaa 60
aaaaaaaaaa aaaaaaaaag aaattgaccc tgagcataaa acaagtcttg gtggacattg 120
cgtgaaccga 130
<210> 108
<211> 130
<212> DNA
<213> artificial sequence
<400> 108
gaagcgagga tcaactcagc ctgggcaaca aagcgagacc cagtctcaaa gaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaag aaattgaccc tgagcataaa acaagtcttg gtggacattg 120
cgtgaaccga 130
<210> 109
<211> 130
<212> DNA
<213> artificial sequence
<400> 109
gaagcgagga tcaactcagc ctgggcaaca aagcgagacc cagtctcaaa gaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa gaaattgacc ctgagcataa aacaagtctt ggtggcattg 120
cgtgaaccga 130
<210> 110
<211> 130
<212> DNA
<213> artificial sequence
<400> 110
gaagcgagga tcaactagcc tgggcaacaa agcgagaccc agtctcaaag aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa gaaattgacc ctgagcataa aacaagtctt ggtggcattg 120
cgtgaaccga 130
<210> 111
<211> 130
<212> DNA
<213> artificial sequence
<400> 111
gaagcgagga tcaactagcc tgggcaacaa agcgagaccc agtctcaaag aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa agaaattgac cctgagcata aaacaagtct tggtgcattg 120
cgtgaaccga 130
<210> 112
<211> 130
<212> DNA
<213> artificial sequence
<400> 112
gaagcgagga tcaactgcct gggcaacaaa gcgagaccca gtctcaaaga aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa agaaattgac cctgagcata aaacaagtct tggtgcattg 120
cgtgaaccga 130
<210> 113
<211> 130
<212> DNA
<213> artificial sequence
<400> 113
gaagcgagga tcaactgcct gggcaacaaa gcgagaccca gtctcaaaga aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa aagaaattga ccctgagcat aaaacaagtc ttggtcattg 120
cgtgaaccga 130
<210> 114
<211> 130
<212> DNA
<213> artificial sequence
<400> 114
gaagcgagga tcaactgggg aggcaaaggc tgcagtaagc caagatcacg ccactccact 60
ccagcctggg caacaaagcg agacccagtc tcaaagaaaa agaaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 115
<211> 130
<212> DNA
<213> artificial sequence
<400> 115
gaagcgagga tcaactggag gcaaaggctg cagtaagcca agatcacgcc actccactcc 60
agcctgggca acaaagcgag acccagtctc aaagaaaaag aaaaaaaaaa aaaaacattg 120
cgtgaaccga 130
<210> 116
<211> 130
<212> DNA
<213> artificial sequence
<400> 116
gaagcgagga tcaactaaaa aaaaaaaaga aaaaagaaat tgaccctgag cataaaacaa 60
gtcttggtgg atccagatca tcatatacaa gagatgaaat cctccagggt gtgggcattg 120
cgtgaaccga 130
<210> 117
<211> 130
<212> DNA
<213> artificial sequence
<400> 117
gaagcgagga tcaactaaaa aaaaaaaaaa gaaaaaagaa attgaccctg agcataaaac 60
aagtcttggt ggatccagat catcatatac aagagatgaa atcctccagg gtgtgcattg 120
cgtgaaccga 130
Claims (10)
1. A probe set for detecting microsatellite instability using high throughput sequencing, said probe set comprising probes simultaneously capturing: BAT26, BAT25, BAT-34c4, BAT-40, D2S123, D5S346, MONO-27, NR21, NR24, NR27 and D17S250;
the probes include sense strand probes;
sense strand probes that capture the positions of BAT-34c4, BAT-40, BAT25, MONO-27, NR21, BAT26, D5S346, D2S123, NR24, D17S250 and NR27 have nucleotide sequences shown as SEQ ID NO.1-10, SEQ ID NO.11-20, SEQ ID NO.21-32, SEQ ID NO.33-42, SEQ ID NO.43-53, SEQ ID NO.54-62, SEQ ID NO.63-72, SEQ ID NO.73-84, SEQ ID NO.85-93, SEQ ID NO.94-105 and SEQ ID NO.106-117, in that order.
2. The probe set of claim 1, wherein the probes are labeled with a label.
3. The set of probes of claim 2, wherein the probes are biotin-labeled.
4.A set of probes according to any one of claims 1 to 3, wherein the probes are DNA probes or RNA probes.
5. A kit for detecting microsatellite instability using high throughput sequencing, wherein the kit comprises the probe set of any one of claims 1-4.
6. A method for detecting microsatellite instability for non-diagnostic purposes is characterized in that a probe set according to any one of claims 1 to 4 or a kit according to claim 5 is used for enriching target genes through liquid phase hybridization, and DNA sequences obtained through enrichment are subjected to high-throughput sequencing to obtain microsatellite instability results.
7. The method according to claim 6, wherein a genomic library of interest is constructed, and then the target gene is enriched by liquid phase hybridization using the probe set according to any one of claims 1 to 4 or the kit according to claim 5, and the DNA sequence obtained by the enrichment is subjected to high throughput sequencing to obtain a microsatellite instability result.
8. The method according to claim 7, wherein the whole genome of the target is fragmented and then subjected to end repair to obtain a genomic library of the target.
9. The method according to claim 7, wherein the target gene fragment is obtained by elution treatment after mixing and adsorbing the probe set with magnetic beads.
10. The method according to claim 9, wherein the target gene fragment obtained by elution treatment is subjected to fragment amplification by PCR, and the amplified fragment with the length of 220-320bp is subjected to high-throughput sequencing and analysis, thereby obtaining a microsatellite instability result.
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| CN107475375A (en) * | 2017-08-01 | 2017-12-15 | 南京世和基因生物技术有限公司 | A kind of DNA probe storehouse, detection method and kit hybridized for microsatellite locus related to microsatellite instability |
| WO2019011971A1 (en) * | 2017-07-12 | 2019-01-17 | Institut Curie | Method for detecting a mutation in a microsatellite sequence |
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| CN110352252A (en) * | 2016-09-22 | 2019-10-18 | 威廉马歇莱思大学 | The molecular hybridization probe for capturing and analyzing for complex sequence |
| WO2019011971A1 (en) * | 2017-07-12 | 2019-01-17 | Institut Curie | Method for detecting a mutation in a microsatellite sequence |
| CN107475375A (en) * | 2017-08-01 | 2017-12-15 | 南京世和基因生物技术有限公司 | A kind of DNA probe storehouse, detection method and kit hybridized for microsatellite locus related to microsatellite instability |
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