CN106676637A - DNA library for detecting multiple osteochondromas (MO) pathogenic genes and application of DNA library - Google Patents
DNA library for detecting multiple osteochondromas (MO) pathogenic genes and application of DNA library Download PDFInfo
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Abstract
The invention discloses a DNA library for detecting MO pathogenic gene mutations through a targeted high-throughput semiconductor sequencing technique and application of the DNA library. Specifically, a primer pool is designed according to 15 MO pathogenic genes, sample genome DNA is subjected to super-multiplex PCR amplification, and an amplified product is sequenced by using the high-throughput semiconductor sequencing technique to find pathogenic mutations so as to provide a theoretical basis of genetics and molecular biology for clinical diagnosis. The DNA library for detecting the MO pathogenic genes disclosed by the invention has the characteristics of accuracy, rapidness, flexibility and low cost, and 15 gene detection regions related in the invention comprehensively cover all known pathogenic genes of MO, thereby having important significance and clinical values in diagnosis and differential diagnosis of MO.
Description
Technical field
The present invention relates to a kind of caused a disease by targeting high flux semiconductor sequencing technologies checkout and diagnosis osteochondroma multiple
The DNA library of gene and its application.Specifically according to osteochondroma multiple Disease-causing gene, design can be covered outside said gene
The super-multiplet PCR primer of aobvious son and contiguous zone, and the amplification of super-multiplet PCR is carried out to sample genomic dna, amplified production is utilized
High throughput sequencing technologies are sequenced, and find pathogenic mutation, specify the genetic factors of osteochondroma multiple, are clinical diagnosis
The theoretical foundation of science of heredity and molecular biology is provided, belongs to the genetic test skill in biomedical sector clinical detection technique
Art.
Background technology
Osteochondroma multiple (Multiple osteochondromas, MO) also known as multiple exostosis, are a kind of
Skeletal development exception, is characterized in that number can be formed on skeleton to differ, the bone protuberance for differing in size.When the pathology is serious also
Referred to as diaphyseal aclasis, being primarily referred to as disease, to cause the plastotype for entirely suffering from bone to occur abnormal, it might even be possible to makes all cartilage internalizations
There is different degrees of exception in the skeleton of bone.The morbidity of the disease generally be in bilateral symmetric property, be apt to occur in femur, shin bone, fibula,
Humerus, shoulder blade, ilium and rib, generally can occur at birth, and growth is just terminated after puberty.The conjunction of the disease
And disease includes：Skeleton deformity, fracture, blood vessel and neurotrosis and canceration are osteosarcoma (incidence is about 5%), and patient Jing is often needed
Carry out operative treatment.
At present, think that osteochondroma multiple is autosomal dominant disease in the world, most of patients have house
Race's Genetic history.Patient has the probability of half by disease genetic to the next generation, to family and society bring huge stress and
Financial burden.Clinically except relief of symptoms of performing the operation, still without effective immunotherapy targeted autoantibody method.If however, entered to patient
Row gene diagnosis, finds out pathogenic mutation, and carries out antenatal consulting and pre-natal diagnosis when patient prepares and gives birth to, then can effectively keep away
Exempt from the transmission of pathogenic mutation, allow patient to obtain normal offspring.Therefore, comprehensively, quickly and accurately examination Disease-causing gene is multiple
Property osteochondroma Precise Diagnosis and preventing and treating important prerequisite condition.But, traditional genetic test side being sequenced based on Sanger
It is expensive to there is flux low price in method, and the drawbacks of complex operation, primary first-order equation can only detect an amplification region, it is impossible to which satisfaction is caused a disease more
The ageing requirement that genopathy and multisample are detected simultaneously.
Therefore, it is necessary to seek a kind of method of new detection osteochondroma multiple Disease-causing gene mutation, diagnosis is improved
Accuracy rate, reduces cost and labour intensity simultaneously improve ageing.
The content of the invention
For achieving the above object, the present invention provides a kind of DNA library of the Disease-causing gene of osteochondroma multiple, wherein,
The library includes at least one gene of serial number 1-8.
Table 1：Osteochondroma multiple Disease-causing gene 1
| Sequence number | Gene name | OMIM is numbered |
| 1 | COL2A1 | 120140 |
| 2 | CTTNBP2NL | 615100 |
| 3 | EXTL1 | 601738 |
| 4 | EXTL2 | 602411 |
| 5 | IDH1 | 147700 |
| 6 | IDH2 | 147650 |
| 7 | PTPN11 | 176876 |
| 8 | SDC2 | 142460 |
Table 2：Osteochondroma multiple Disease-causing gene 2
| Sequence number | Gene name | OMIM is numbered |
| 9 | EXT1 | 608177 |
| 10 | EXT2 | 608210 |
| 11 | EXTL3 | 605744 |
| 12 | FGFR3 | 134934 |
| 13 | GRPR | 305670 |
| 14 | PTH1R | 168468 |
| 15 | PTHLH | 168470 |
Preferably, the DNA library includes the gene of serial number 1-8
Preferably, the DNA library is also including at least one gene of serial number 9-15.
It is furthermore preferred that the DNA library includes the gene of serial number 1-15.
The present invention also provides a kind of detection reagent of above-mentioned DNA library.
Preferably, the detection reagent is the primer that can expand above-mentioned DNA library
It is furthermore preferred that the primer is, according to above-mentioned DNA library, design can cover said gene extron and adjoin tune
The primer pond suitable for super-multiplet PCR in control region, so as to carry out the amplification of super-multiplet PCR to sample genomic dna.
Preferably, it is described to adjoin regulatory region for each extron edge at least 5bp regions.
Preferably, the primer pond carries out the amplification design of super-multiplet PCR by software so that in each reaction tube once
Synchronous parallel expands thousands of amplicons by amplification, so as to carry out high efficiency amplification easy to operate to target area.
The present invention also provides a kind of above-mentioned DNA library or the detection reagent of above-mentioned DNA library is preparing diagnostic kit
In application, the kit be used for diagnose osteochondroma multiple.
The present invention also provides the detection reagent of a kind of above-mentioned DNA library or above-mentioned DNA library in diagnostic device is prepared
Application, the diagnostic device be used for diagnose osteochondroma multiple.Preferably, the diagnostic device is genetic chip.
Preferably, the detection reagent is the primer that can expand above-mentioned DNA library.
It is furthermore preferred that the primer is, according to above-mentioned DNA library, design can cover said gene extron and adjoin tune
The primer pond suitable for super-multiplet PCR in control region, so as to carry out the amplification of super-multiplet PCR to sample genomic dna.
Preferably, it is described to adjoin regulatory region for each extron edge at least 5bp regions.
Preferably, the primer pond carries out the amplification design of super-multiplet PCR by software so that in each reaction tube once
Synchronous parallel expands thousands of amplicons by amplification, so as to carry out high efficiency amplification easy to operate to target area.
It is furthermore preferred that the application comprises the following steps：
(1) genomic DNA of experimenter's sample is provided；
(2) from Ion TorrentTMHigh-flux sequence platform is automatically synthesized the amplimer pond for covering above-mentioned Disease-causing gene,
The amplification of super-multiplet PCR is carried out to target area；
(3) digestion is carried out to the amplified production that step (2) is obtained；
(4) digestion products for obtaining to step (3) plus Barcode joints, the Barcode joints come from high pass measurement
Sequence builds storehouse kit；
(5) connection product that step (4) is obtained is purified；
(6) secondary amplification is carried out with universal primer to the purified product that step (5) is obtained, the universal primer comes from high pass
Measure sequence and build storehouse kit；
(7) Piece Selection and concentration mensuration are carried out to the secondary amplified production that step (6) is obtained, that is, builds up patients target area
Domain expands library；
(8) to the library obtained by step (7) after Water-In-Oil pcr amplification reaction, by sequence purpose fragment to be measured connection
In ISP pearls, reactant liquor is obtained；
(9) reactant liquor comprising ISP pearls that step (8) is obtained is clicked and entered into chip, upper Ion TorrentTMHigh pass is measured
Sequence instrument is sequenced；
(10) base sequence information that step (9) is obtained is carried out into bioinformatics comparison process, obtains related to disease
Mutational site；
(11) Sanger PCR sequencing PCR checkings are carried out to the mutational site that step (10) is obtained.
Preferably, peripheral blood, body fluid, histoorgan sample of the sample from experimenter.
It is furthermore preferred that the application is further used for instructing selective fertility, for example：Specify that by the method multiple
The pathogenic mutation of osteochondroma patient, carries out fine hair puncture under the premise of the pregnant early stage informed consent of patient or amniocentesis obtains tire
Youngster's correlation DNA sample, and detected, to determine whether fetus carries the Disease-causing gene, decide whether to continue gestation then, from
And prevent pathogenic mutation from passing to the next generation.
The present invention also provides a kind of diagnostic kit, wherein, the kit includes the cause of above-mentioned osteochondroma multiple
The detection reagent of the DNA library of ospc gene or above-mentioned DNA library.
The present invention also provides a kind of diagnostic device, wherein, described device includes the pathogenic base of above-mentioned osteochondroma multiple
The detection reagent of the DNA library of cause or above-mentioned DNA library.Preferably, described device is genetic chip.
Preferably, the detection reagent is the primer that can expand above-mentioned DNA library.
It is furthermore preferred that the primer is, according to above-mentioned DNA library, design can cover said gene extron and adjoin tune
The primer pond suitable for super-multiplet PCR in control region, so as to carry out the amplification of super-multiplet PCR to sample genomic dna.
Preferably, it is described to adjoin regulatory region for each extron edge at least 5bp regions.
Preferably, the primer pond carries out the amplification design of super-multiplet PCR by software so that in each reaction tube once
Synchronous parallel expands thousands of amplicons by amplification, so as to carry out high efficiency amplification easy to operate to target area.
The present invention provides a kind of using above-mentioned DNA library, the detection reagent of DNA library, diagnostic kit or diagnostic device
The method detected to patient, methods described comprises the following steps：
(1) genomic DNA of experimenter's sample is provided；
(2) from Ion TorrentTMHigh-flux sequence platform is automatically synthesized the amplimer pond of above-mentioned Disease-causing gene, to mesh
Mark region carries out the amplification of super-multiplet PCR；
(3) digestion is carried out to the amplified production that step (2) is obtained；
(4) digestion products for obtaining to step (3) plus Barcode joints, the Barcode joints come from high pass measurement
Sequence builds storehouse kit；
(5) connection product that step (4) is obtained is purified；
(6) secondary amplification is carried out with universal primer to the purified product that step (5) is obtained, the universal primer comes from high pass
Measure sequence and build storehouse kit；
(7) Piece Selection and concentration mensuration are carried out to the secondary amplified production that step (6) is obtained, that is, builds up patients target area
Domain expands library；
(8) to the library obtained by step (7) after Water-In-Oil pcr amplification reaction, by sequence purpose fragment to be measured connection
In ISP pearls, reactant liquor is obtained；
(9) reactant liquor comprising ISP pearls that step (8) is obtained is clicked and entered into chip, upper Ion TorrentTM high passes are measured
Sequence instrument is sequenced；
(10) base sequence information that step (9) is obtained is carried out into bioinformatics comparison process, obtains related to disease
Mutational site；
(11) Sanger PCR sequencing PCR checkings are carried out to the mutational site that step (10) is obtained.
(12) if there is the mutant of above-mentioned Disease-causing gene in experimenter's sample, experimenter can be diagnosed as multiple bone
Chondroma.
Preferably, peripheral blood, body fluid, histoorgan sample of the sample from experimenter.
It is furthermore preferred that methods described is further used for instructing selective fertility, for example：Specify that by the method multiple
The pathogenic mutation of osteochondroma patient, carries out fine hair puncture under the premise of the pregnant early stage informed consent of patient or amniocentesis obtains tire
Youngster's correlation DNA sample, and detected, to determine whether fetus carries the Disease-causing gene, decide whether to continue gestation then, from
And prevent pathogenic mutation from passing to the next generation.
Using the present invention DNA library osteochondroma multiple patient is carried out gene diagnosis have following significance and
Effect：
1. the DNA library of the application is 15 bases that applicant selects from numerous osteochondroma multiple Disease-causing genes
Cause, these genes are especially suitable for the detection including the yellow race patient including Chinese；Lack in the world at present large-scale
Chinese yellow race's osteochondroma multiple gene diagnosis relevant information, most known osteochondroma multiple Disease-causing genes
All essentially from the report of American-European crowd, they are to Chinese pathogenic and feature currently without play-by-play.Inventor enters
Long-term clinical observation and the gene diagnosis research of the yellow race of extensive China osteochondroma multiple are gone, have summarized and excavate
A series of Disease-causing gene of Chinese yellow race's osteochondroma multiple patients, and in the world it has been reported that Disease-causing gene
Frequency of disease development in Chinese patients and pathogenic observed and verified.It is exhausted big in 15 genes selected by the present invention
Most genes are the Disease-causing gene that inventor verifies in yellow race crowd and finds.
2. using based on Ion Torrent in the present inventionTMHigh throughput sequencing technologies are carried out to the amplified production of target area
Detection, can simultaneously detect in once sequencing reaction 15 related genes that be related in the present invention whole extrons and
Contiguous zone, and detection sample size can be adjusted according to the size of different chip data amounts, ensureing average sequencing depth
500 × on the premise of, detect the sample size that 1 to 96 people do not wait, whole sequencing reaction and data analysis interpretation can two days it
Inside complete, greatly reduce the cost and labour intensity of amplified reaction, improve it is ageing, meanwhile, the detection zone have covers
Cover degree is wide, and overall coverage reaches 99.5%；.By the sequencing to target amplification region and data interpretation, can accurately identify
The mutation related to disease, judges kinds of Diseases and the cause of disease, and for clinic reliable examining report in time is provided.Present invention design
Detection method verifies through Sanger PCR sequencing PCRs, in two generations, were sequenced depth reach 100 × point mutation, the degree of accuracy of this method reaches
To 100%；
3. gene diagnosis contributes to pre-natal diagnosis and neonatal screening etc..Because of osteochondroma multiple, patient will face one in future
Serial severe complication, and effective radical cure method is there is no, bring heavy medical burden to patient home and society.Patient
Household pays special attention to the health of patient offspring.Osteochondroma multiple is often dyeing dominant hereditary disease, and patient offspring has 50%
Initiation potential.Making a definite diagnosis for patient's Disease-causing gene mutation, can provide clear and definite genetic counselling clothes for the next generation for breeding health
Business.
4. it is furthermore preferred that the present invention is provided cover the cause of disease known to osteochondroma multiple comprehensively, comprising 15 correlations of causing a disease
The DNA library of Disease-causing gene.This 15 gene selects are based on the clinical Disease-causing gene database that the world has been reported and generally acknowledged
(omim database, HGMD databases, ClinVar databases) and the subject study content of inventor oneself.This 15 genes
On pathogenic mutation both can individually cause a disease, it is also possible to mutually Combination pathogenicity causes more serious and complicated clinical phenotypes, this
Invention is accomplished disposably comprehensively to be sequenced them first, is to be currently known to be caused a disease base for osteochondroma multiple
Because covering a kind of most comprehensively targeting sequencing detection.
In general, DNA library, the detection reagent of DNA library and its application being related in the present invention has accurate, spirit
Living, quick, inexpensive the characteristics of；Through clinical assessment, the invention has good auxiliary diagnosis valency to osteochondroma multiple
Value.
Description of the drawings
Ion Torrent of Fig. 1TMThe data acquisition synoptic diagram of high-flux sequence reaction
Fig. 2 is once sequenced to the target area of 15 genes, the data amount information that different specimens are obtained
Fig. 3 after primary data analysis, the mutating alkali yl quantity of the different specimens for obtaining
Fig. 4 Sanger PCR sequencing PCRs are verified to the mutational site that detection method is obtained
Specific embodiment
With reference to specific embodiment, the invention will be further described, not the restriction to inventing, according to this area
Known prior art, embodiments of the present invention are not limited to this, therefore all this areas according to done by present disclosure
Equivalent, belong to protection scope of the present invention.
Embodiment 1
1. reagent used in the method：
Ion AmpliSeqTMLibrary Kit 2.0, Ion PGMTMTemplate OT2 200Kit v3, IonSequencing 200Kit v2, Ion Xpress Barcode Adaptors 1-16Kit, Ion 318TM Chip
Kit v2
2. collection of specimens and preservation
(1) collection of specimens：Sample is peripheral blood in patients.Blood is conventional extracting vein blood 5ml, and EDTA anti-freezings are processed.
(2) preserve：Can detect immediately, 4 DEG C preserve one week, -80 DEG C of preservations more than a week.
3. detecting step and interpretation of result：
(1) extraction of sample genomic DNA：The extraction of specimen dna is according to TIANGEN Biotech (Beijing) Co., Ltd.
Blood DNA extracts kit operating instruction is carried out.
(2) super-multiplet PCR of object detection area expands and builds storehouse：With the full extron of 15 genes being related in the present invention
For detection zone, based on Ion AmpliSeqTMDesigner is automatically synthesized super-multiplet PCR primer, the target area to specimen dna
Domain is expanded and is built storehouse, is embodied as follows:
A. the amplification of target area：
Reaction condition：
B. amplified production is two-in-one, digestion：
Reaction condition：
C. jointing：
Reaction condition：
22℃ 30min
72℃ 10min
10℃ Up to 1h
D. the secondary amplification of purifying and purified product：Purification step is according to Ion Ampliseq Library
Preparation operation manuals are carried out, and final product is dissolved in 52 μ l reaction systems, and the reaction system is consisted of：
Reaction condition：
E. Piece Selection：Piece Selection step is entered according to Ion Ampliseq Library Preparation operation manuals
OK, obtain finally build storehouse product Qubit 2.0Fluorometer it is quantitative after build storehouse and complete.
(3) high-flux sequence：Sequencing and early-stage preparations step are according to Ion PGMTMTemplate OT2 200Kit v3 and
IonSequencing 200Kit v2 operation manuals are carried out：
A. Water-In-Oil PCR reactions：
Above-mentioned reaction system is added in Ion OneTouch 2 carries out Water-In-Oil PCR reactions.
B. after the completion of Water-In-Oil PCR reactions, the Ion PGM Template OT2 200Ion of sequencing template are connected with
Sphere Particles are purified through Ion Onetouch ES, add sequencing primer and archaeal dna polymerase：
After room temperature 5min, the reactant liquor comprising ISP pearls for obtaining is clicked and entered into chip, upper Ion TorrentTM high fluxs
Sequenator is sequenced.
(4) data analysis：Sequencing data is analyzed through coverage analysis and variant caller, obtains alkali
Basic sequence and mutational site, after Ion Reporter are in line annotation, obtaining diagnosing genetic cardiomyopathies has in mutational site
The mutational site of meaning.
(5) Sange methods checking：For the mutational site for obtaining, verified using Sanger PCR sequencing PCRs.
As a result illustrate and analyze
Disposably the target area of 3 samples is detected (such as by the high throughput sequencing technologies being related in the present invention
Fig. 1), the total amount of data for obtaining is 797M (Total Base), and the fragment that can be always obtained 5,214,030 reads long data
(Total Reads), the middle position of each fragment reads a length of 140bp.3 samples averagely can be tested to 571,129 (510,003-
634,564) the long Reads (such as Fig. 2) of individual fragment reading, above sequencing result provides the data of abundance for the sequence analysis of next step
Amount.Sequencing result is analyzed through variant Caller, and each sample averagely has 55 variations (Variants) to be read (as schemed
3).By Ion Reporter after line annotation, the mutational site related to disease is surveyed through Sanger for the variation of detection zone
C.79C the checking of sequence method as Fig. 4 is enumerated, EXT1 genes is shown at Fig. 4 arrow indications (such as Fig. 4)>T
(p.Gln27Ter) heterozygosis nonsense mutation, the genetic defect type of final clear and definite patient, for clinic diagnosis basis are provided.Simultaneously should
Gene genetic pattern is autosomal dominant inheritance, and the siblings and its children of patient have 50% probability and patient one
Sample carries the pathogenic mutation, also faces onset risk, should carry out the examination of the pathogenic sites, is following selective fertility and product
Front diagnosis provides foundation.
The clinical practice illustration of the present invention
Using method provided by the present invention, 50 are carried out with the true patient of osteochondroma multiple but etiology unknown
Genetic test.The patient 48 of pathogenic mutation is found altogether.The method reaches to the total positives rate that osteochondroma multiple is detected
96%, with reference to clinical manifestation, imaging examination, and doctor's last diagnostic result, the false positive rate of this method is 0, i.e., all Jing
The carrying pathogenic mutation patient that this method confirms meets the Clinical symptoms of correlated inheritance disease, and can give clear and definite clinic
Diagnosis.Detection method according to the present invention is to be mutated quick screening implement, with Sanger sequencings using high throughput sequencing technologies
Method is the final goldstandard for determining mutation, therefore with quick, accurate feature.With high-flux sequence mean depth 100 × be
Example, the target area of 94.99% for being covered can be sequenced, and the point mutation that screening is obtained can be by Sanger PCR sequencing PCRs
Checking, therefore be 100% to the degree of accuracy that area above is detected.According to the clinic diagnosis interpretation of result of 50 tested patients, receive
Survey patient and false negative rate do not occur.The diagnosis report be given based on detection method according to the present invention obtains the accreditation of clinician
With adopt.It is accurate that detection method according to the present invention has, quickly, diagnosis the characteristics of inexpensive, to osteochondroma multiple
Important in inhibiting and practical value.In 50 patients, complex mutation is found that in 2 patients, two patient clinical tables
Type and age of onset have increased.It would therefore be desirable to comprehensive Disease-causing gene scanning is carried out to patient, with more accurately right
Its pathogenesis is analyzed, so as to being that clinical treatment from now on and genetic counselling lay the first stone.
Embodiment 2：
The newfound osteochondroma multiple Disease-causing gene of inventor 8 is included in the present invention in primer pond.Such as above-mentioned table 1
It is shown.Newfound 8 osteochondroma multiple Disease-causing genes come from inventor's research accumulation for many years and family line investigation.
Inventor is retrieved first in known osteochondroma multiple Disease-causing gene by KEGG signal paths, and homologous gene storehouse is sought
The key gene looked on the homologous gene and identical pathogenic path of known Disease-causing gene.Then in the Chinese yellow people for accumulating for many years
Plant in patient's large sample amount case Sample Storehouse carries out targeting high-flux sequence to the Disease-causing gene of candidate, and carries out bioinformatics
Analysis, screening and searching pathogenic mutation.Case to screening pathogenic mutation carries out follow-up and further pedigree analysis, to suffering from
The whole family of person carries out pathogenic sites sequence verification, and calculate pathogenic mutation isolates coefficient.When the cause for finding candidate gene
When disease mutation is isolated completely in two and the uncorrelated family of the above, you can tentatively judge that the candidate disease causing genes are new
Disease-causing gene.It is a discovery of the invention that the pathogenic mutation of above-mentioned 8 genes occurs completely in two and the uncorrelated family of the above
Isolate.
Claims (10)
1. it is a kind of diagnosis osteochondroma multiple DNA library, it is characterised in that the library include serial number 1-8 at least one
Individual gene.
2. DNA library as claimed in claim 1, it is characterised in that the DNA library includes the gene of serial number 1-8.
3. DNA library as described in claim 1 or 2, it is characterised in that the DNA library is also including serial number 9-15
At least one gene, it is preferred that the DNA library includes the gene of serial number 1-15.
4. the detection reagent of the arbitrarily described DNA library of a kind of claim 1-3.
5. detection reagent as claimed in claim 4, it is characterised in that the detection reagent is to expand above-mentioned DNA library
Primer, it is furthermore preferred that the primer is, according to above-mentioned DNA library, design can cover said gene extron and adjoin control region
The primer pond suitable for super-multiplet PCR in domain, so as to carry out the amplification of super-multiplet PCR to sample genomic dna.
6. the arbitrarily described detection reagent of the arbitrarily described DNA library of claim 1-3 or claim 4-5 is preparing diagnosis
Application in kit, it is characterised in that the kit is used to diagnose osteochondroma multiple.
7. the arbitrarily described detection reagent of the arbitrarily described DNA library of claim 1-3 or claim 4-5 is preparing diagnosis
Application in device, it is characterised in that the diagnostic device is used to diagnose osteochondroma multiple, it is preferred that the diagnosis dress
It is genetic chip to put.
8. the application as described in any claim of claim 6-7, it is characterised in that the application comprises the following steps：
(1) genomic DNA of experimenter's sample is provided；
(2) from Ion TorrentTMHigh-flux sequence platform is automatically synthesized the amplimer pond for covering above-mentioned Disease-causing gene, to mesh
Mark region carries out the amplification of super-multiplet PCR；
(3) digestion is carried out to the amplified production that step (2) is obtained；
(4) digestion products for step (3) being obtained plus Barcode joints；
(5) connection product that step (4) is obtained is purified；
(6) secondary amplification is carried out with universal primer to the purified product that step (5) is obtained；
(7) Piece Selection and concentration mensuration are carried out to the secondary amplified production that step (6) is obtained, that is, builds up the expansion of patients target region
Increase library；
(8) to the library obtained by step (7) after Water-In-Oil pcr amplification reaction, sequence purpose fragment to be measured is connected into ISP
Pearl, obtains reactant liquor；
(9) reactant liquor comprising ISP pearls that step (8) is obtained is clicked and entered into chip, upper Ion TorrentTMHigh-flux sequence instrument
It is sequenced；
(10) base sequence information that step (9) is obtained is carried out into bioinformatics comparison process, obtains related to disease dashing forward
Become site；
(11) Sanger PCR sequencing PCR checkings are carried out to the mutational site that step (10) is obtained.
9. a kind of diagnostic kit, it is characterised in that the kit include the arbitrarily described DNA library of claim 1-3 or
The arbitrarily described detection reagent of person's claim 4-5.
10. a kind of diagnostic device, it is characterised in that described device includes the arbitrarily described DNA library of claim 1-3 or power
Profit requires the arbitrarily described detection reagents of 4-5, it is preferred that the diagnostic device is genetic chip.
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| WO2023272672A1 (en) * | 2021-07-01 | 2023-01-05 | 北京市肿瘤防治研究所 | Rapid screening apparatus for germline mutations of tumor susceptibility genes in mixed samples and application |
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| WO2023272672A1 (en) * | 2021-07-01 | 2023-01-05 | 北京市肿瘤防治研究所 | Rapid screening apparatus for germline mutations of tumor susceptibility genes in mixed samples and application |
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