CN106591441A - Probes, method and chip for detecting alpha and/or beta-thalassemia mutation based on whole-gene capture sequencing and application of such probes, such method and such chip - Google Patents
Probes, method and chip for detecting alpha and/or beta-thalassemia mutation based on whole-gene capture sequencing and application of such probes, such method and such chip Download PDFInfo
- Publication number
- CN106591441A CN106591441A CN201611095478.2A CN201611095478A CN106591441A CN 106591441 A CN106591441 A CN 106591441A CN 201611095478 A CN201611095478 A CN 201611095478A CN 106591441 A CN106591441 A CN 106591441A
- Authority
- CN
- China
- Prior art keywords
- detection
- kinds
- probe
- chip
- sequencing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000523 sample Substances 0.000 title claims abstract description 113
- 230000035772 mutation Effects 0.000 title claims abstract description 77
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 38
- 201000006288 alpha thalassemia Diseases 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 25
- 208000005980 beta thalassemia Diseases 0.000 title claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 120
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 63
- 238000012217 deletion Methods 0.000 claims abstract description 20
- 230000037430 deletion Effects 0.000 claims abstract description 18
- 238000005516 engineering process Methods 0.000 claims abstract description 10
- 239000012634 fragment Substances 0.000 claims description 21
- 206010043391 Thalassaemia beta Diseases 0.000 claims description 20
- 206010043390 Thalassaemia alpha Diseases 0.000 claims description 18
- 208000002903 Thalassemia Diseases 0.000 claims description 13
- 238000012937 correction Methods 0.000 claims description 11
- 238000013467 fragmentation Methods 0.000 claims description 9
- 238000006062 fragmentation reaction Methods 0.000 claims description 9
- 230000005856 abnormality Effects 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 4
- 238000007405 data analysis Methods 0.000 claims description 4
- 239000007791 liquid phase Substances 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000012165 high-throughput sequencing Methods 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 2
- 235000020958 biotin Nutrition 0.000 claims description 2
- 239000011616 biotin Substances 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 235000015170 shellfish Nutrition 0.000 claims 1
- 238000011144 upstream manufacturing Methods 0.000 abstract description 11
- 210000002593 Y chromosome Anatomy 0.000 abstract description 5
- 210000001766 X chromosome Anatomy 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 25
- 238000009396 hybridization Methods 0.000 description 14
- 239000011534 wash buffer Substances 0.000 description 14
- 239000011324 bead Substances 0.000 description 13
- 239000007788 liquid Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 8
- 238000005352 clarification Methods 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000008034 disappearance Effects 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 108010090804 Streptavidin Proteins 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 101150013707 HBB gene Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 208000007502 anemia Diseases 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 230000037433 frameshift Effects 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 210000005239 tubule Anatomy 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 206010064571 Gene mutation Diseases 0.000 description 3
- 102100027685 Hemoglobin subunit alpha Human genes 0.000 description 3
- 102100030378 Hemoglobin subunit theta-1 Human genes 0.000 description 3
- 101001009007 Homo sapiens Hemoglobin subunit alpha Proteins 0.000 description 3
- 101000843063 Homo sapiens Hemoglobin subunit theta-1 Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000000869 mutational effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 101100263837 Bovine ephemeral fever virus (strain BB7721) beta gene Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 101100316840 Enterobacteria phage P4 Beta gene Proteins 0.000 description 2
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 101150087698 alpha gene Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 231100000221 frame shift mutation induction Toxicity 0.000 description 2
- 206010019847 hepatosplenomegaly Diseases 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 108010009923 Hemoglobin Constant Spring Proteins 0.000 description 1
- 208000012925 Hemoglobin H disease Diseases 0.000 description 1
- 108010091256 Hemoglobin Westmead Proteins 0.000 description 1
- 101710128747 Hemoglobin subunit alpha-A Proteins 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 208000037062 Polyps Diseases 0.000 description 1
- 238000001190 Q-PCR Methods 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 101150059736 SRY gene Proteins 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108010051149 hemoglobin Bart's Proteins 0.000 description 1
- 108010073713 hemoglobin Q Thailand Proteins 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 108700010045 sry Genes Proteins 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000000216 zygoma Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides primers, a method and a chip for detecting alpha and/or beta-thalassemia point mutation and deletion mutation based on whole-gene capture sequencing and application of such primers, such method and such chip. The primers, the method, the chip and application thereof have the advantages that through designing of capture probes, relevant genes involved in alpha-thalassemia and beta-thalassemia are enriched and all mutation information including SNP and indel in full-length sequences of genes is detected; through addition of autosome, X-chromosome and Y-chromosome regions as well as upstream and downstream regions of coded genes as references, structure variations such as SNV and CNV are detected; compared with existing various hotspot mutation site detection technologies, the method is capable of detecting hotspot mutation information as well as some rare mutations and undiscovered new mutation types to detect and analyze full-length sequence specificity of target genes, fully covers the mutation types and makes up the defect that a conventional detection method easily causes missing detection of low-frequency mutations and rare mutations greatly.
Description
Technical field
The invention belongs to genomics and biology field, in particular to a kind of based on full genome capture sequencing
α and/or the detection probe of β-thalassaemia mutations, method, chip and application.
Background technology
Thalassemia is also known as thalassemia or Thalassemia.The mutation type of the lean related gene in ground
It is intricate with mutational site, up to thousands of or even thousand of kinds.As silent oscillation α lean (α α/α -) or (α α/α α T), light-duty α ground
Lean (α α/--) or (α -/α -) although patient is mostly without obvious clinical symptoms, but if Mr. and Mrs both sides are homotype carriers of mutation,
It is heavyly lean that then its children has 25% probability, and 50% probability is gene carrier；It is different from silent oscillation or lightly lean
Patient, Hemoglobin H disease (- α/--), (α -/--), (α α T/--) or (α α T/ α α T) patient can present it is slight to anemia and
Hepatosplenomegaly etc.；More serious HB Bart ' s schridde syndromes (--/--) more in third trimester of pregnancy (34～40 weeks) or postpartum
It is dead in a few hours.Similarly for the lean patient in β types ground, how asymptomatic light-duty patient clinical is or anemia, and heavy patient faces
It is general on bed anaemia occur within 3-6 month in birth, hepatosplenomegaly, the skeleton change such as cheekbone protuberance, eye distance be broadening.
Up to now, China is heavy and the lean patient in osculant ground is 300,000 or so, and mutator carrier is more than 30,000,000
People, wherein more universal with Guangxi, Yunnan, Hainan, Guangdong, Guizhou and other places.The lean patient in ground there is no at present effective treatment method,
And medical expense is expensive, monthly cost is up to that 2000-3000 is first, and to patient and family huge financial burden and spirit are brought
Pain.
The seriousness endangered based on thalassemia and the diversity of associated gene mutation, exploitation is a kind of effectively, cover α
With the gene screening detection technique or corresponding detection kit of beta globin gene total length, for the population of Di Pin hotspots
Examination, to preventing or preventing generation of the disease in neonate, to improve the overall quality of newborns and have important meaning with the general level of the health
Justice.
At present, all of detection technique is mostly to carry out examination for specific mutational site, there is no specific detection α and β
The technology of globulin gene total length, its testing result can only ensure that detected site does not have mutation, but it cannot be guaranteed that all
Pathogenic mutation does not morph, the risk that there are a large amount of site missing inspections, it is difficult to which accurate reflection detection is individual and its offspring suffers from
The risk or probability of disease or carrying related mutation.
Presently relevant technology includes：Restriction fragment length polymorphism analysis linkage analysis technology, single-strand conformation polymorphism
Detection, mutant oligonucleotide extend amplification, specific mutation site (multiple) PCR amplification sequencing technologies, probe spot hybridization technique,
Breach PCR methods (Gap-PCR), fluorescent PCR, genetic chip etc..Its Patent (CN104789672A, 2015.07.22) is devised
A set of detection probe, but the probe remains and is designed on the basis of known mutations site, it is solid by color fluorescent detection
Fixed 3 α deletion forms mutation, 3 α Nondeletion mutations and 17 β Nondeletion mutations, rather than high flux all standing detection.
Similar patent also includes CN104372100,2015.02.25；CN104293937A, 2015.01.21；CN104204227A,
2014.12.10 and CN102277419A, 2011.12.14 etc., it is that probe and primer are designed by known mutations site, to phase
Answer mutational site to detect, it is impossible to detect all of abrupt information, can not be used to find new mutation type.Although patent
(CN102329876A, 2012.01.25 or CN102329876B, 2014.04.02) has invented a kind of by chip capture combination
The method of the nucleotide sequence of high-flux sequence detection disease associated nucleic acid molecules, but it is mainly for detection of familial
Sample polyp (catch position chr05:112073411), polycystic kindey syndrome (catch position chr06:51479999), section property brain is hard
Change syndrome (catch position chr09:135766620), PKU (catch position chr12:103231969), horse is all comprehensive
Close disease (catch position chr15:48700368) with Du Shi muscular dystrophy (catch position chrX:31137199) single-gene such as
Disease, remains in a sense the capture sequencing of specific site.Though patent is referred to can be used for thalassemic detection, so
And do not provide clear and definite ways and means.
Therefore, it is necessary to provide a kind of mutation type for being easy to detection new, the base of α and beta globin gene total length can be covered
Because of examination detection technique and detection kit.
The content of the invention
To solve the above problems, the invention provides a kind of α and/or β-thalassemia that sequencing is captured based on full genome
The detection probe of mutation, method, chip and application.
In a first aspect, a kind of the invention provides α and/or β-thalassaemia mutations that sequencing is captured based on full genome
Detection probe, the detection probe includes target-probe group and control probe group, wherein, the target-probe group includes the institute of table 1
Show at least 5 kinds, preferably at least 10 kinds, preferably at least 20 kinds, preferably at least 30 kinds in 1-96 probes, preferably at least 50
Kind, preferably at least 80 kinds, be preferably all 96 kinds；
The control probe include 97-579 probes shown in table 1 at least 10 kinds, preferably at least 20 kinds, preferably extremely
It is few 30 kinds, preferably at least 50 kinds, preferably at least 80 kinds, preferably at least 100 kinds, preferably at least 130 kinds, preferably at least 160 kinds, excellent
Choosing at least 190 kinds, preferably at least 220 kinds, preferably at least 250 kinds, preferably at least 280 kinds, preferably at least 310 kinds, preferably at least
340 kinds, preferably at least 370 kinds, preferably at least 400 kinds, preferably at least 430 kinds, be preferably all 483 kinds；
Wherein, for 1-579 detection probes shown in table 1 are defined as below：
Table 1 the 1st is classified as the species numbering of detection probe；
Table 1 the 2nd is classified as the optional gene loci region of detection probe sequence；
Table 1 the 3rd is classified as gene loci or the gene region that the need of detection probe sequence are covered；
Wherein, in 1-96 probes, the sequence of every probe takes the 2nd row optional gene loci region total length or the 2nd
Arrange the sequence that optional gene loci region arbitrarily selects continuous 75-105nt base as target-probe, the sequence of every probe
Row cover the gene loci or gene region shown in the row of probe correspondence the 3rd；
In 97-579 probes, the sequence of every probe arbitrarily selects continuous in the optional gene loci region of the 2nd row
Sequence of the 60-120nt base as control probe.
Preferably, the 97-579 probes of table 1 are replaceable region, and those skilled in the art can be according to existing mononucleotide
Pleomorphism site is replaced.
What deserves to be explained is, the detection probe that the present invention is provided can be the normal chain or minus strand of gene region shown in the row of table 1 the 2nd
Sequence.
In one embodiment of the invention, the normal chain of gene region is used as probe sequence shown in detection probe using the row of table 1 the 2nd.
The detection probe of table 1. is numbered and sequence information
Preferably, the detection probe covers the coding region of all coding for alpha gene clusters and β gene clusters.
It is further preferred that the detection probe also covers HBB gene upstream and downstream 3kb regions.
It is further preferred that the detection probe also covers HBZ upstream region of gene 22.8kb to HBQ1 downstream of gene 8.8kb
Region.
It is further preferred that the detection probe also covers X chromosome region.
It is further preferred that the detection probe also covers Y chromosome region.
It is further preferred that the detection probe is covered goes back autosome region.
Preferably, 5 ends of the detection probe are also associated with biotin and are marked, to capture target area.
The detection probe that first aspect present invention is provided has following advantage：
A, the coding region that can capture all coding for alpha gene clusters and β gene clusters, detect the various of all related gene total lengths
The information such as possible point mutation and little indel mutation；B, by the way that related gene is extended, such as HBB gene upstream and downstream 3kb,
HBZ upstream region of gene 22.8kb to HBQ1 downstream of gene 8.8kb is captured simultaneously；C, while the subregion for increasing X and Y chromosome is made
For internal reference, the detection of the big region disappearance of correlative coding gene is realized.
Second aspect, the invention provides a kind of α and/or β-thalassaemia mutations based on full genome capture sequencing
Detection chip, the chip is liquid-phase chip or solid phase chip, the detection probe being combined with thereon described in any one of first aspect.
Preferably, the liquid-phase chip is the liquid-phase chip with DNA oligo as hybridization probe.
The third aspect, the invention provides a kind of α and/or β-thalassaemia mutations based on full genome capture sequencing
Detection method, including the detection chip using the detection probe described in any one of first aspect or described in any one of second aspect with
The step of DNA sample to be captured is hybridized.
Fourth aspect, the invention provides a kind of α and/or β-thalassaemia mutations based on full genome capture sequencing
Detection method, including the detection chip using the detection probe described in any one of first aspect or described in any one of second aspect with
The step of DNA sample to be captured is hybridized；Carry out to capturing the target dna for obtaining with using second generation high throughput sequencing technologies
The step of sequencing.
Preferably, the detection method comprises the steps：
1) genomic DNA sample to be detected is broken into into fragment, is preferably broken into the fragment that length is 200-500bp,
More have and elect the fragment for being broken into that length is 250-350bp as；
2) to step 1) fragment that interrupts is purified, end is repaired, plus A, plus joint；
3) by step 2) product that obtains and the detection probe described in any one of first aspect or any one of second aspect institute
The detection chip stated is hybridized, and captures the DNA fragmentation in target acquistion region；After the DNA fragmentation in wash-out target acquistion region
Carry out library amplification；
4) to step 3) sequencing library that obtains carries out high-flux sequence；
5) by step 4) sequencing gained sequence compare, obtain sequencing result.
It is further preferred that the high-flux sequence is second generation high-flux sequence, and ensure each described sequencing library
Original data volume reach more than 0.6Gb, the sequencing depth of target area reaches 400 × more than, target area coverage reaches
More than 99%.
Preferably, the step 7) specifically include：
A. basic statistics：Target area is divided into into multiple minizones, and counts the sequencing piece included by each minizone
Section number accounts for the percentage reads% and average G/C content of non-autosome interval to be measured fragment number；
B. skewed popularity amendment is expanded：The reads% for falling into each statistics minizone is grouped by average G/C content, each point is taken
The reads% medians of group obtain correction factor divided by all interval medians；And by each original interval reads%
It is multiplied by correction factor to obtain that correction value reads% of fragment percentage is sequenced in each interval '；
C. number variation detection is copied：In data analysis process, a normal population is introduced as check sample, take control
The copy number of the thalassemia related gene of sample defines human genome thalassemia related gene as control collection
Copy number baseline, and calculate interval correction value reads% of thalassemia related gene in each sample to be tested ' sum,
And in control this interval mean value of centralized calculation and variance；And then each interval degree of peeling off in check sample is calculated, with z values
Represent, if z is less than -3 or more than 3, the interval there occurs numerical abnormality.
Preferably, described in any one of first aspect detection chip described in detection probe, any one of second aspect, the 3rd
The gene mutation that detection method described in aspect or any one of fourth aspect is detected, the gene mutation is in following mutation
One or more：The nucleotides G of HBB gene upstream the 519th sport A, 578 T sport A, 591 G sport T, 390
Position G sports A, 591 deleted nucleotides G, 601 A and sports G, 753 A and sport G；The 672nd core of HBA1 upstream region of gene
Thuja acid A sports G, 384 G and sports A, 276 sports T for C；The 1031st nucleotides T of HBA2 upstream region of gene sport C,
771 nucleotides T sport C；HBA2 downstream of gene 3 end non-coding region, 795 nucleotides A sport G.
5th aspect, the present invention provides a kind of inspection of the α and/or β-thalassaemia mutations that sequencing is captured based on full genome
Test agent box, including the detection probe described in any one of first aspect or the detection chip described in any one of second aspect, also wrap
Include detection reagent.
6th aspect, the present invention provides a kind of detection probe, any one of second aspect institute as described in any one of one side
Detection reagent described in the detection method described in detection chip, the third aspect or any one of fourth aspect stated or the 5th aspect
Application of the box in detection α and/or β-thalassaemia mutations.
As described in the present invention, in arbitrary first aspect to α and/or β-thalassaemia mutations described in the 6th aspect
Form includes that α and/or β-thalassemia point mutation and/or deletion form are mutated；Preferably base replace, insertion or lack and
Fragment deletion or amplification.It is specifically including but not limited to nonsynonymous mutation, frameshift mutation, frameshift deletion, translation termination increase, translation
Terminate disappearance.
The beneficial effect of the technical scheme that the present invention is provided：
The technical scheme that the present invention is provided not only is enriched with the lean phase in all participation α and β ground by designing synthetic capture probe
The abrupt informations such as correlation gene, all SNP, the indel in each full length gene sequence of detection, by increasing such as X and Y chromosome area
Domain, while increasing the upstream and downstream region of each encoding gene as reference, can detect the structure variations such as various SNV, CNV.It is real
The detection of the big region disappearance of existing correlative coding gene.
Relative to the technology of various detection hot mutant sites at present, the present invention not only can detect the information of hot spot mutation,
And some rare mutation and undiscovered new mutation type can be detected, realize genes of interest full length sequence specific detection
Analysis, mutation type all standing greatly makes up common detection methods to low frequency mutation and the missing inspection of rare mutation.
Experimentally, by joint of the design with difference index, Optimal Experimental condition, realize that once capture can be simultaneously
Complete 32 samples builds storehouse and sequencing, significantly reduces cost, improves the flux of detection.
Description of the drawings
Fig. 1 is the detection stream of the α of full genome capture sequencing provided in an embodiment of the present invention and/or β-thalassaemia mutations
Cheng Tu；
Fig. 2 is the fragment distribution results that the high-throughput sequencing library 2100 that the embodiment of the present invention builds detects library；
Fig. 3-4 is the interval copy number statistics of the lean related gene in bioinformatics ground provided in an embodiment of the present invention.
Specific embodiment
Described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as
Protection scope of the present invention.
In the embodiment of the present invention without special instruction outward, agents useful for same and consumptive material are commercial goods.
With reference to Fig. 1, the α and/or β-thalassaemia mutations of a kind of full genome capture sequencing are embodiments provided
Detection method, specifically include：
1. target gene and reference sequences design and synthesis
In the present embodiment, target area includes：The region and HBZ upstream region of gene of HBB gene and its upstream and downstream 3kb
The region of 22.8kb to HBQ1 downstream of gene 8.8kb.Meanwhile, the sry gene region on Y chromosome is have chosen as experimentation
In Quality Control site；Have chosen and amount on other chromosomes in genome range 270 sites as copy number type detection
With reference to site.Finally, have selected ROCHE NimbleGen platforms carries out design and the building-up process of probe, final chip region
Domain size is 125kb.
2. the fragmentation of genomic DNA
Choose each 2ug of Whole Blood Genomic DNA of the lean patient in ground of 32 known mutations types, using Bioruptor or
Covaris interrupts instrument and interrupts, (DNA fragmentation master tape concentrates on 300bp, pollutes without albumen, RNA), Jing after electrophoresis detection is qualified
Further fragment screening is purified Ampure XP Beads (Agencourt), by the DNA fragmentation back dissolving of about 300bp to 32 μ l's
In Elution Buffer (Qiagen).
3. the end of fragmentation DNA is repaired：
1) DNA accordings to the form below obtained in the previous step are prepared into end in the centrifuge tube of 1.5ml and repairs reaction system：
2) centrifuge tube is put into and is adjusted to react 30min on 20 DEG C of Thermomixer (Eppendrf).Use after having reacted
QIAquick PCR Purification Kit (Qiagen) is purified, and finally sample is dissolved in into 34 μ l Elution
Buffer(EB)。
4.DNA fragments 3' end adds " A " base：
1) DNA accordings to the form below obtained in the previous step are prepared plus " A " reaction system in the centrifuge tube of 1.5ml：
2) and then by pipe it is put into and is adjusted to react 30min on 37 DEG C of Thermomixer (Eppendrf).Use after having reacted
MiniElute PCR Purification Kit (Qiagen) is purified, and finally sample is dissolved in into 20 μ L Elution
Buffer(EB)。
The connection of 5.index Adapter：
1) DNA accordings to the form below obtained in the previous step are prepared into Adapter coupled reaction systems, each sample connection is different
Index joints：
1) and then it is put into and is adjusted to react 15min on 20 DEG C of Thermomixer (Eppendrf).Use after having reacted
MiniElute PCR Purification Kit (Qiagen) is purified, and finally sample is dissolved in into 22 μ L Elution
Buffer(EB)。
Index Adapter:
TACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNT(SEQ NO.1)
NNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC(SEQ NO.2)
N is any base
6. the hybridization of target area and probe
1) with Qubit fluorometer and corresponding Quant-iT dsDNA HS Assay Kit (Invitrogen)
Product to connecting difference index joints is quantitative, and each sample takes the EP that the connection product of 32ng is added to a new 1.5ml
Guan Zhong, and add 10 μ L 1mg/ml Cot1DNA (Invitrogen) and each 1nmol tab closure sequence B lock1 and
Block2, mixture is placed in SpeedVac and is evaporated, and temperature setting is 60 DEG C.
2) 2 × SC Hybridiation Buffer and SC Hybridiation are separately added in the EP pipes being evaporated
Component A prepare following hybridization reaction system：
Block1：GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN(SEQ NO.3)
Block2：ANNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA(SEQ NO.4)
N be any base, and respectively with the N base complementrities of index adapter；
It is placed on centrifuge after sample concussion is mixed and is centrifuged 10 seconds at full speed.Sample is transferred to 95 DEG C of heat after centrifugation
It is incubated 10 minutes in block, makes DNA denaturation.
3) take out sample, concussion mix after room temperature at full speed centrifugation 10 seconds, transfer them in the PCR pipe of a 0.2mL or
In 96 hole PCR plates, and add the Probe Library of the design of 4.5 μ L.Concussion is placed on centrifuge after mixing and is centrifuged at full speed 10 seconds, will
Reaction system is put in PCR instrument, 47 DEG C of hybridization 16h-72h, and the hot lid temperature setting of PCR instrument is 57 DEG C.
7. the capture of hybridization sequences and washing is eluted
1) washing lotion is prepared
A) dilute following four Wash buffer (10 × SC Wash Buffer I, 10 × SC Wash Buffer II,
10 × SC Wash Buffer III and 2 × Stringent Wash Buffer, NimbleGen) store to after 1 × solution, storage
The time is deposited no more than 2 week.
B) 47 DEG C preheat 1mL Stringent Wash Buffer (NimbleGen) and 1mL SC Wash for preparing
Two kinds of solution of Buffer I (NimbleGen)
2) strepavidin magnetic beads are prepared
A) Dynabeads M-280Streptavidin (Invitrogen) magnetic bead is taken out from refrigerator, after fully mixing
Take 100 μ L to be added in the EP pipes of a new 1.5ml；
B) EP pipes are placed in into magnetic frame up to clarify, with the removal supernatant that pipettor is careful, add 200 μ L's
Streptavidin Dynabead Binding and Wash Buffer(NimbleGen)；
C) Vortex is mixed after 10s, EP pipes is placed back in into magnetic frame and is clarified to liquid, with the careful removal of pipettor
Clearly；
D) repeated washing once, is altogether washed twice；
E) with Streptavidin Dynabead Binding and Wash Buffer (NimbleGen) weights of 100 μ L
Outstanding magnetic bead, and proceeded in the tubule of 0.2mL；
F) magnetic bead (tubule being come on magnetic frame) is combined with magnetic frame, until liquid clarification, is gone with pipettor is careful
Except supernatant.
3) DNA fragmentation of strepavidin magnetic beads capture hybridization
Hybridization mixture is sucked out (record residual volume after hybridization) in being added to ready magnetic bead, piping and druming mixes 10
Tubule is placed in PCR instrument after secondary, (the hot lid temperature of PCR instrument should be set to 57 DEG C to 47 DEG C of incubation 45min, take out every 15min
Vortex 3s are precipitated with preventing magnetic bead).
4) washing of the strepavidin magnetic beads of capture dna is combined
A) it is incubated after 45min, in the EP pipes that mixture is proceeded to into 1.5mL from the tubule of 0.2mL, EP pipes is placed in into magnetic frame
Up to liquid clarification, careful removal supernatant；
B) 1 × Wash Buffer I for adding 100 μ L to be preheating to 47 DEG C, mix 10s on vortex, EP pipes are placed in into magnetic
The up to liquid clarification of power frame, careful removal supernatant；
C) EP pipes are removed from magnetic frame, the 1 × Stringent Wash Buffer for adding 200 μ L to be preheating to 47 DEG C are moved
The piping and druming of liquid device is mixed 10 times (operation should be rapid so that the liquid in pipe is not less than 47 DEG C)；
D) after 47 DEG C of incubation 5min, EP pipes are placed in into magnetic frame up to liquid clarification, careful removal supernatant；
E) repeat step c)-d), share 1 × Stringent Wash Buffer and wash twice；
F) 1 × Wash Buffer I for adding 200 μ L room temperatures to place, vortex mixes 2min, if liquid splashes lid
On, with finger flick EP pipes make its focus on pipe it is low, by EP pipes be placed in magnetic frame up to liquid clarification, careful removal supernatant；
G) 1 × Wash Buffer II for adding 200 μ L room temperatures to place, vortex mix 1min, EP pipes are placed in into magnetic frame
Up to liquid clarification, careful removal supernatant；
H) 1 × Wash Buffer III for adding 200 μ L room temperatures to place, vortex mix 30s, EP pipes are placed in into magnetic frame
Up to liquid clarification, careful removal supernatant；
I) the EB buffer of 60ul are added in the magnetic bead of capture, is well mixed.
8. the PCR amplifications and library for capturing magnetic bead template selects purifying：
1) DNA obtained in the previous step is prepared into PCR reaction systems by following system：
PCR reaction conditions：
2) PCR primer is returned Jing after 2% agarose electrophoresis with QIAquick gel extraction kit (Qiagen)
The library of purifying 300-450bp fragments is received, or directly uses Agencourt AMPure Beads (Beckman Coulter) magnetic bead
Purifying reclaims PCR primer.
9. library detection：
Bioanalyzer analysis system (Agilent, Santa Clara, USA) detect library inserts
Size and content, the testing result of library 2100 is shown in Fig. 2；The concentration in Q-PCR accurate quantifications library.
10. sequencing and data analysis：
1) sequence alignment：
The DNA sequence dna (reads) that each sample sequencing is obtained is carried out with standard human's genome reference sequences (hg19)
Compare, obtain the information that surveyed DNA sequence dna is positioned at genome relevant position.Comparison process can from bwa, soap2,
Bowtie2 etc. routinely compares software, and the present embodiment is compared using bwa softwares.Meanwhile, in order to avoid repetitive sequence is to follow-up
The interference of analysis, only choosing the sequence fragment uniquely compared with human genome reference sequences carries out follow-up statistics.
2) point mutation and little insertion/deletion are detected：
A. comparison data pretreatment：Comparison data result stores into BAM file formats, subsequent addition sequence unpaired message,
The repeating label that sequence group information and pcr amplification primer rise；
B. point mutation and little insertion/deletion are identified using GATKUnifiedGenotyper softwares；
C. point mutation and little insertion/deletion are annotated using ANNOVAR softwares, judge each position point mutation and
Whether little insertion/deletion causes the nonsynonymous mutation of gene, frameshift mutation, frameshift deletion, translation termination increases, translation termination lacks
Lose etc.；
D. compare with known lean database, it is determined that known pathogenic mutation.
Final 32 samples are visible with little insertion/deletion type mutation result in the point mutation of thalassemia related gene
Table 2, wherein, 12 samples show no obvious abnormalities, the detection point mutation of 20 samples and little insertion/deletion, wherein, 4 17 heterozygosis,
2 17 heterozygosis and β E heterozygosis, 1-28 miscellaneous and 41-42 heterozygosis, 2 41-42 heterozygosis, 1 654 heterozygosis, 1 Hb CS heterozygosis, 1
Example Hb Q-Thailand heterozygosis, 1 HbQuong homozygosis simultaneously -28 heterozygosis, 2 HbWestmead homozygosis, 5 β E heterozygosis.
3) number variation detection is copied：
A. basic statistics：Target area is divided into into multiple zonules in order to data statistics.Count each minizone institute
The sequencing fragment number for falling accounts for the percentage reads% and average G/C content of non-autosome region segments number to be measured.
B. skewed popularity amendment is expanded：The interval reads% of each statistics will be fallen into be grouped by average GC, in taking each packet
Digit obtains correction factor except the median in all regions.Each original interval reads% is multiplied by into correction factor to obtain
Correction value reads% of fragment percentage is sequenced in each interval '.
C. number variation detection is copied：In data analysis process, the control collection of a normal population is introduced defining people
The copy number baseline of Mediterranean anaemia related gene in genoid group.Calculate thalassemia related gene in each sample to be tested
Reads% ' the sums in region, in the mean value and variance in this region of control centralized calculations.And then each region is calculated right
In the same old way degree of peeling off in this, is represented with z values.If less than -3 or more than 3, the region there occurs numerical abnormality to z.
Finally, 32 samples are mutated the visible table 2 of result in the deletion type of thalassemia related gene, wherein, 18
Sample shows no obvious abnormalities, 10 sample detection SEA heterozygous disappearances, and 3.7 type heterozygous deletions, 4.2 type heterozygous deletions, SEA are multiple
Close the compound 3.7 types disappearance of 4.2 types disappearance, SEA a case each.
Fig. 3-4 is the interval copy number statistics of the lean related gene in bioinformatics ground provided in an embodiment of the present invention
(ordinate is copy number ratio copyratio, and abscissa is gene loci Locus).
The lean patient whole blood's genome testing result in 2. 32 ground of table
To further illustrate the beneficial effect of the embodiment of the present invention, the embodiment of the present invention additionally provides check experiment, including：
To each testing sample in the table 2 of the embodiment of the present invention, simultaneously using the detection (contrast of following 3 kinds of kits
Method is conventional method in industry, is also official's detection method that current Shenzhen family planning is adopted；Specifically, this detection method
In, deletion form is detected using gap-PCR；PCR amplifications combine reverse dot blot hybridization and detect saltant type)：
Gene of alpha thalassemia detection kit (gap-PCR methods) is sub- can be biological；
β-thalassemia gene detecting kit (PCR- revert dot blot hybridizations) reagent I, sub- energy is biological；
β-thalassemia gene detecting kit (PCR- revert dot blot hybridizations) reagent II, sub- energy is biological；
Non-deletion type gene of alpha thalassemia mutation detection kit (PCR- revert dot blot hybridizations) reagent I, Asia can give birth to
Thing；
Non-deletion type gene of alpha thalassemia mutation detection kit (PCR- revert dot blot hybridizations) reagent II, Asia can give birth to
Thing；
The analysis found that, be analyzed for being mutated in Hbvar databases：
Using control methods, No. 12 samples are only able to detect a kind of pathogenic mutation of SEA heterozygosis (and adopt the present invention enforcement
The detection probe of example offer, detection method can be detected simultaneously by Hb Westmead homozygosis pathological forms)；
Result and detection probe provided in an embodiment of the present invention, detection that control methods is detected to table 2 other samples
The testing result of method is completely the same.
Additionally, can also find not remembering in Hbvar databases using detection probe provided in an embodiment of the present invention, detection method
The mutation of load, these mutation are as shown in table 3 (and control methods does not detect these mutation in table 3)：
The mutation do not recorded in table 3.Hbvar databases
Illustrate that detection probe provided in an embodiment of the present invention, detection method are not only simple and efficient.Routine can also be obtained
The variation type that commercial test kit can not be detected.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
SEQUENCE LISTING
<110>Mutable gene Science and Technology Ltd. of Shenzhen
<120>A kind of inspection of the α and/or β-thalassemia point mutation and deletion form mutation that sequencing is captured based on full genome
Survey primer, method, chip and application
<130> 2016
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 41
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (35)..(40)
<223> n is a, c, g, or t
<400> 1
tacactcttt ccctacacga cgctcttccg atctnnnnnn t 41
<210> 2
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(6)
<223> n is a, c, g, or t
<400> 2
nnnnnnagat cggaagagca cacgtctgaa ctccagtcac 40
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (35)..(40)
<223> n is a, c, g, or t
<400> 3
gtgactggag ttcagacgtg tgctcttccg atctnnnnnn 40
<210> 4
<211> 41
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (2)..(7)
<223> n is a, c, g, or t
<400> 4
annnnnnaga tcggaagagc gtcgtgtagg gaaagagtgt a 41
Claims (10)
1. it is a kind of based on full genome capture sequencing α and/or β-thalassaemia mutations detection probe, it is characterised in that institute
Detection probe is stated including target-probe group and control probe group, wherein, the target-probe group includes No. 1-96 spy shown in table 1
At least 5 kinds, preferably at least 10 kinds, preferably at least 20 kinds, preferably at least 30 kinds, preferably at least 50 kinds in pin, preferably at least 80
Plant, be preferably all 96 kinds；
The control probe include 97-579 probes shown in table 1 at least 10 kinds, preferably at least 20 kinds, preferably at least 30
Kind, preferably at least 50 kinds, preferably at least 80 kinds, preferably at least 100 kinds, preferably at least 130 kinds, preferably at least 160 kinds, preferably extremely
Few 190 kinds, preferably at least 220 kinds, preferably at least 250 kinds, preferably at least 280 kinds, preferably at least 310 kinds, preferably at least 340
Kind, preferably at least 370 kinds, preferably at least 400 kinds, preferably at least 430 kinds, be preferably all 483 kinds；
Wherein, for 1-579 detection probes shown in table 1 are defined as below：
Table 1 the 1st is classified as the species numbering of detection probe；
Table 1 the 2nd is classified as the optional gene loci region of detection probe sequence；
Table 1 the 3rd is classified as gene loci or the gene region that detection probe sequence need to be covered；
Wherein, in 1-96 probes, the sequence of every probe takes the 2nd row optional gene loci region total length or can in the 2nd row
The sequence for selecting gene loci region arbitrarily to select continuous 75-105nt base as target-probe, the sequence of every probe is covered
Cover the gene loci or gene region shown in the row of probe correspondence the 3rd；
In 97-579 probes, the sequence of every probe arbitrarily selects continuous 60- in the optional gene loci region of the 2nd row
Sequence of the 120nt base as control probe.
It is 2. as claimed in claim 1 to be based on the detection probe that full genome captures the α and/or β-thalassaemia mutations of sequencing,
Characterized in that, 5 ends of the detection probe have also been coupled biotin being marked.
3. it is a kind of based on full genome capture sequencing α and/or β-thalassaemia mutations detection chip, it is characterised in that institute
Chip is stated for liquid-phase chip or solid phase chip, thereon the detection probe with reference to described in just like any one of claim 1.
4. it is a kind of based on full genome capture sequencing α and/or β-thalassaemia mutations detection method, it is characterised in that bag
Include the detection probe or the detection chip described in any one of claim 3 and DNA to be captured described in usage right 1 any one of requirement
The step of sample is hybridized.
5. it is a kind of based on full genome capture sequencing α and/or β-thalassaemia mutations detection method, it is characterised in that bag
Include the detection probe or the detection chip described in any one of claim 3 and DNA to be captured described in usage right 1 any one of requirement
The step of sample is hybridized；With the step being sequenced to the target dna that capture is obtained using second generation high throughput sequencing technologies
Suddenly.
It is 6. as claimed in claim 5 to be based on the detection method that full genome captures the α and/or β-thalassaemia mutations of sequencing,
Characterized in that, the detection method comprises the steps：
1) genomic DNA sample to be detected is broken into into fragment, is preferably broken into the fragment that length is 200-500bp, more had
Elect the fragment for being broken into that length is 250-350bp as；
2) to step 1) fragment that interrupts is purified, end is repaired, plus A, plus joint；
3) by step 2) product that obtains with described in the detection probe or any one of second aspect described in any one of first aspect
Detection chip is hybridized, and captures the DNA fragmentation in target acquistion region；Carry out after the DNA fragmentation in wash-out target acquistion region
Amplify in library；
4) to step 3) sequencing library that obtains carries out high-flux sequence；
5) by step 4) sequencing gained sequence compare, obtain sequencing result.
It is 7. as claimed in claim 6 to be based on the detection method that full genome captures the α and/or β-thalassaemia mutations of sequencing,
Characterized in that, the step 5) specifically include：
A. basic statistics：Target area is divided into into multiple minizones, and counts the sequencing fragment included by each minizone
Number accounts for the percentage reads% and average G/C content of non-autosome interval to be measured fragment number；
B. skewed popularity amendment is expanded：The reads% for falling into each statistics minizone is grouped by average G/C content, each packet is taken
Reads% medians obtain correction factor divided by all interval medians；And be multiplied by each original interval reads%
Correction factor obtains that correction value reads% of fragment percentage is sequenced in each interval '；
C. number variation detection is copied：In data analysis process, a normal population is introduced as check sample, take check sample
Thalassemia related gene copy number as control collection defining copying for human genome thalassemia related gene
Shellfish base line, and calculate interval correction value reads% of thalassemia related gene in each sample to be tested ' sum, and
Control this interval mean value of centralized calculation and variance；And then each interval degree of peeling off in check sample is calculated, with z value tables
Show, if z is less than -3 or more than 3, the interval there occurs numerical abnormality.
8. it is a kind of based on full genome capture sequencing α and/or β-thalassaemia mutations detection kit, it is characterised in that
Including the detection chip described in the detection probe described in any one of claim 1 or any one of claim 3, also including detection examination
Agent.
9. detection chip, the claim as described in detection probe as described in any one of claim 1 or any one of claim 3
The detection kit described in detection method or claim 8 described in any one of 4-7 is prominent in detection α and/or β-thalassemia
Application in change.
10. α as claimed in claim 9 and/or β-thalassaemia mutations include α and/or β-thalassemia point mutation and/
Or deletion form mutation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611095478.2A CN106591441B (en) | 2016-12-02 | 2016-12-02 | Alpha and/or beta-thalassemia mutation detection probe, method and chip based on whole gene capture sequencing and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611095478.2A CN106591441B (en) | 2016-12-02 | 2016-12-02 | Alpha and/or beta-thalassemia mutation detection probe, method and chip based on whole gene capture sequencing and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106591441A true CN106591441A (en) | 2017-04-26 |
| CN106591441B CN106591441B (en) | 2021-07-09 |
Family
ID=58596938
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611095478.2A Active CN106591441B (en) | 2016-12-02 | 2016-12-02 | Alpha and/or beta-thalassemia mutation detection probe, method and chip based on whole gene capture sequencing and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106591441B (en) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227368A (en) * | 2017-07-19 | 2017-10-03 | 臻悦生物科技江苏有限公司 | Intestinal cancer clinical application mutator detection kit |
| CN107236818A (en) * | 2017-07-19 | 2017-10-10 | 臻悦生物科技江苏有限公司 | Lung cancer clinical medication mutator detection kit |
| CN107688726A (en) * | 2017-09-21 | 2018-02-13 | 深圳市易基因科技有限公司 | The method of monogenic disease correlation copy number missing is judged based on liquid phase capture technique |
| CN107841545A (en) * | 2017-12-07 | 2018-03-27 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | The method of poor genotype based on high throughput sequencing technologies detection α, β |
| CN109777858A (en) * | 2018-12-20 | 2019-05-21 | 天津诺禾医学检验所有限公司 | The probe and method of hybrid capture are carried out to Duplication region |
| CN110184337A (en) * | 2019-05-16 | 2019-08-30 | 南京诺禾致源生物科技有限公司 | Probe compositions, the reagent comprising it, kit, detection method and application |
| CN110373458A (en) * | 2019-06-27 | 2019-10-25 | 东莞博奥木华基因科技有限公司 | A kind of kit and analysis system of thalassemia detection |
| CN110863041A (en) * | 2018-08-27 | 2020-03-06 | 深圳华大生命科学研究院 | Mutant gene related to thalassemia and detection reagent and application thereof |
| CN110938685A (en) * | 2019-12-11 | 2020-03-31 | 福建福君基因生物科技有限公司 | Gene detection probe set for neonatal hereditary metabolic disease and hemoglobinopathy and application thereof |
| CN111009288A (en) * | 2019-11-28 | 2020-04-14 | 苏州元德基因生物科技有限公司 | Probe design method of CEBPA gene and application thereof |
| CN111326211A (en) * | 2020-01-07 | 2020-06-23 | 深圳市早知道科技有限公司 | Method and device for detecting thalassemia genetic variation |
| CN112359109A (en) * | 2020-11-26 | 2021-02-12 | 北京迈基诺基因科技股份有限公司 | Probe set and kit for detecting alpha thalassemia and beta thalassemia related pathogenic genes |
| CN112634987A (en) * | 2020-12-25 | 2021-04-09 | 北京吉因加医学检验实验室有限公司 | Method and device for detecting copy number variation of single-sample tumor DNA |
| CN114774515A (en) * | 2022-03-24 | 2022-07-22 | 北京安智因生物技术有限公司 | Capture probe, kit and detection method for detecting polycystic kidney disease gene mutation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014023076A1 (en) * | 2012-08-10 | 2014-02-13 | 深圳华大基因科技有限公司 | Thalassemia typing method and use thereof |
| CN105886617A (en) * | 2016-04-16 | 2016-08-24 | 广州市达瑞生物技术股份有限公司 | Thalassemia gene detection method based on high-throughput sequencing technology |
-
2016
- 2016-12-02 CN CN201611095478.2A patent/CN106591441B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014023076A1 (en) * | 2012-08-10 | 2014-02-13 | 深圳华大基因科技有限公司 | Thalassemia typing method and use thereof |
| CN105886617A (en) * | 2016-04-16 | 2016-08-24 | 广州市达瑞生物技术股份有限公司 | Thalassemia gene detection method based on high-throughput sequencing technology |
Non-Patent Citations (2)
| Title |
|---|
| ARIANE BLATTNER等: "Detection of germline rearrangements in patients with α- and β-thalassemia using high resolution array CGH", 《BLOOD CELLS, MOLECULES AND DISEASES》 * |
| DANIEL E. SABATH等: "Characterization of Deletions of the HBA and HBB Loci by Array Comparative Genomic Hybridization", 《THE JOURNAL OF MOLECULAR DIAGNOSTICS》 * |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107236818A (en) * | 2017-07-19 | 2017-10-10 | 臻悦生物科技江苏有限公司 | Lung cancer clinical medication mutator detection kit |
| CN107227368A (en) * | 2017-07-19 | 2017-10-03 | 臻悦生物科技江苏有限公司 | Intestinal cancer clinical application mutator detection kit |
| CN107227368B (en) * | 2017-07-19 | 2021-11-05 | 臻悦生物科技江苏有限公司 | Kit for detecting clinical medication mutant gene of intestinal cancer |
| CN107688726B (en) * | 2017-09-21 | 2021-09-07 | 深圳市易基因科技有限公司 | Method for judging single-gene-disease-related copy number deficiency based on liquid phase capture technology |
| CN107688726A (en) * | 2017-09-21 | 2018-02-13 | 深圳市易基因科技有限公司 | The method of monogenic disease correlation copy number missing is judged based on liquid phase capture technique |
| CN107841545A (en) * | 2017-12-07 | 2018-03-27 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | The method of poor genotype based on high throughput sequencing technologies detection α, β |
| CN110863041A (en) * | 2018-08-27 | 2020-03-06 | 深圳华大生命科学研究院 | Mutant gene related to thalassemia and detection reagent and application thereof |
| CN109777858A (en) * | 2018-12-20 | 2019-05-21 | 天津诺禾医学检验所有限公司 | The probe and method of hybrid capture are carried out to Duplication region |
| CN110184337A (en) * | 2019-05-16 | 2019-08-30 | 南京诺禾致源生物科技有限公司 | Probe compositions, the reagent comprising it, kit, detection method and application |
| CN110373458A (en) * | 2019-06-27 | 2019-10-25 | 东莞博奥木华基因科技有限公司 | A kind of kit and analysis system of thalassemia detection |
| CN111009288A (en) * | 2019-11-28 | 2020-04-14 | 苏州元德基因生物科技有限公司 | Probe design method of CEBPA gene and application thereof |
| CN111009288B (en) * | 2019-11-28 | 2023-08-29 | 苏州元德友勤医学检验所有限公司 | Probe design method of CEBPA gene and application thereof |
| CN110938685A (en) * | 2019-12-11 | 2020-03-31 | 福建福君基因生物科技有限公司 | Gene detection probe set for neonatal hereditary metabolic disease and hemoglobinopathy and application thereof |
| CN111326211A (en) * | 2020-01-07 | 2020-06-23 | 深圳市早知道科技有限公司 | Method and device for detecting thalassemia genetic variation |
| CN111326211B (en) * | 2020-01-07 | 2023-12-19 | 深圳市早知道科技有限公司 | Method and device for detecting thalassemia gene variation |
| CN112359109A (en) * | 2020-11-26 | 2021-02-12 | 北京迈基诺基因科技股份有限公司 | Probe set and kit for detecting alpha thalassemia and beta thalassemia related pathogenic genes |
| CN112634987B (en) * | 2020-12-25 | 2021-07-27 | 北京吉因加医学检验实验室有限公司 | Method and device for detecting copy number variation of single-sample tumor DNA |
| CN112634987A (en) * | 2020-12-25 | 2021-04-09 | 北京吉因加医学检验实验室有限公司 | Method and device for detecting copy number variation of single-sample tumor DNA |
| CN114774515A (en) * | 2022-03-24 | 2022-07-22 | 北京安智因生物技术有限公司 | Capture probe, kit and detection method for detecting polycystic kidney disease gene mutation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106591441B (en) | 2021-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106591441A (en) | Probes, method and chip for detecting alpha and/or beta-thalassemia mutation based on whole-gene capture sequencing and application of such probes, such method and such chip | |
| EP3329010B1 (en) | Nucleic acids and methods for detecting chromosomal abnormalities | |
| JP2020054401A (en) | High degree multiple pcr method and composition | |
| CN105243295B (en) | With the relevant heredity of cancer or the detection of molecular distortion | |
| WO2020214547A1 (en) | Improved liquid biopsy using size selection | |
| US9206417B2 (en) | Multiplexed sequential ligation-based detection of genetic variants | |
| CN105441432B (en) | Composition and its purposes in sequencing and variation detection | |
| Tobias et al. | Essential medical genetics | |
| CN108485940A (en) | Copy the detection and classification of number variation | |
| HUE034854T2 (en) | Fetal genomic analysis from a maternal biological sample | |
| AU2011255641A1 (en) | Methods for non-invasive prenatal ploidy calling | |
| CN113308562B (en) | Cotton whole genome 40K single nucleotide site and application thereof in cotton genotyping | |
| CN110628891B (en) | Method for screening embryo genetic abnormality | |
| WO2018121468A1 (en) | Method, device and kit for detecting fetal genetic mutation | |
| AU2022237538A1 (en) | Methods for determination of transplant rejection | |
| JP2022541387A (en) | Methods and compositions for proximity ligation | |
| CN110734974B (en) | SNP locus combination and detection primer for cancer chemotherapy drugs | |
| Keren et al. | Oligonucleotide microarrays in constitutional genetic diagnosis | |
| Spanakis et al. | The molecular basis of genetic variation: mutation detection methodologies and limitations | |
| EP2847347B1 (en) | Highly multiplex pcr methods and compositions | |
| EP3696279A1 (en) | Methods for noninvasive prenatal testing of fetal abnormalities | |
| Trotta | Genetics of Primary Immunodeficiency in Finland | |
| CN110423808A (en) | Hepatic injury SNP site caused by the fleece-flower root and the application in disease forecasting and diagnosis | |
| Patterson et al. | Three assays for in-solution enrichment of ancient human DNA at more than a million SNPs | |
| CN117778588A (en) | Preparation method and application of sheep 1K liquid phase chip based on targeted capturing sequencing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200610 Address after: 518000 comprehensive building, Beishan Road, Beishan Industrial Zone, Yantian District, Guangdong, Shenzhen Applicant after: Wang Junwen Applicant after: Gao Fei Address before: Pingshan Pingshan street Shenzhen City, Guangdong Province Jinniu Road 518000 No. 16 Huahan science and Technology Industrial Park, No. 2 building room 323 Applicant before: SHENZHEN E-GENE TECHNOLOGY Co.,Ltd. |
|
| CB03 | Change of inventor or designer information |
Inventor after: Wang Junwen Inventor after: Gao Fei Inventor before: Wang Junwen Inventor before: Li Xuchao Inventor before: Gao Fei |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210914 Address after: 518000 Shenzhen City Investment creative factory life science park b3301, xiuxin community, Kengzi street, Pingshan District, Shenzhen City, Guangdong Province Patentee after: SHENZHEN E-GENE TECHNOLOGY Co.,Ltd. Address before: 518000 Beishan industrial complex building, Beishan Road, Yantian District, Shenzhen City, Guangdong Province Patentee before: Wang Junwen Patentee before: Gao Fei |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230911 Address after: Building 27, Huanbi Road 216, Yantian District, Shenzhen City, Guangdong Province, 518000, Building 211, Daguanju 505, Wanke East Coast Zone 3 Patentee after: Gao Fei Patentee after: Wang Junwen Address before: 518000 Shenzhen City Investment creative factory life science park b3301, xiuxin community, Kengzi street, Pingshan District, Shenzhen City, Guangdong Province Patentee before: SHENZHEN E-GENE TECHNOLOGY Co.,Ltd. |