CN106282369A - A kind of probe groups for detecting congenital cataract related gene and test kit - Google Patents
A kind of probe groups for detecting congenital cataract related gene and test kit Download PDFInfo
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Abstract
The invention discloses a kind of probe groups for detecting congenital cataract related gene and test kit.The probe groups of the present invention includes the multiple probe sequences being respectively directed to the non-duplicate region of exon of 225 congenital cataract pathogenesis related genes, the present invention also provide for comprising this probe groups for areas captured and the test kit of secondary order-checking.225 related genes that congenital cataract can disposably be had been found that by the probe groups of the present invention and test kit carry out complete detection, improve coverage rate and the efficiency of molecular diagnosis, specificity is good, highly sensitive, significant to congenital cataract Prenatal Screening, early diagnosis, accurate prevention and control.
Description
Technical field
The invention belongs to gene detecting kit field, be specifically related to a kind of for detecting congenital cataract related gene
Probe groups and test kit.
Background technology
Congenital cataract be born i.e. exist or be born after the congenital heredity that just gradually forms or dysplasia cause
Lenticular opacity, is to cause child blind and the primary disease of inpairment of vision, accounts for child's blind (most preferably correcting defects of vision less than 0.05)
And the 50% of middle severe inpairment of vision (most preferably correcting defects of vision less than 0.3, be not less than 0.05) child sum, effectively preventing and treating is always for it
It is to receive much concern and an international difficult problem urgently to be resolved hurrily.
Congenital cataract about 30% infant has obvious Family inherited inclination, has three kinds of hereditary patterns, including often contaminating
Colour solid dominant inheritance, autosomal recessive inheritance, AR and X linkage inheritance.Wherein, autosomal dominant inheritance, AD is modal heredity
Form.Chain research based on isolated family and the order-checking of disease genetic correlation candidate gene, it was reported that the most identified elder generation
22 disease sites that it cataract is relevant and 17 Disease-causing genes, congenital cataract Disease-causing gene go deep into all-round exploration
It it is most important break-through point in its study of incident mechanism.
Recently as the development of secondary sequencing technologies, the congenital cataract Disease Gene reported grows with each passing day, extremely
Few 250 mutational sites having included 120 genes, but be correlated with still without more comprehensively congenital cataract in the market
The combination of gene region capture probe and test kit.
Summary of the invention
It is an object of the invention to for the most comprehensively congenital cataract related gene detection spy in prior art
Pin group and the deficiency of test kit, it is provided that a kind of probe groups for detecting congenital cataract related gene and test kit, utilize
It can detect congenital cataract related gene comprehensively, fast and accurately.
A kind of probe groups for detecting congenital cataract related gene, comprises energy specific recognition human congenital white
Multiple DNA sequence in the non-duplicate region of cataract related gene exon；
Described congenital cataract related gene is as follows:
PEX10,PEX14,EPHA2,HSPG2,POMGNT1,FOXE3,RPE65,CRYZ,DNASE2B,ABCD3,
COL11A1,GJA8,ADAMTSL4,GPR161,PROX1,RAB3GAP2,GNPAT,PXDN,PEX13,RAB3GAP1,LCT,
FIGN,LRP2,AGPS,CRYGD,CRYGC,CRYGB,CRYGA,CYP27A1,CRYBA2,KCNJ13,FYCO1,COL7A1,
LAMB2,FLNB,GPX1,PVRL3,CPOX,CASR,CNBP,BFSP2,SOX2,CRYGS,WFS1,HMX1,CC2D2A,
SRD5A3,CLOCK,AFF1,ANK2,ETFDH,CTNND2,ERCC8,VCAN,EFNA5,TGFBI,SIL1,SPARC,TFAP2A,
GCNT2,GCM2,NEU1,PEX6,LGSN,LCA5,GJA1,PEX7,IFNGR1,PEX3,EZR,FAM126A,GTF2IRD1,
HIP1,NRCAM,AGK,FDFT1,BIN3,DOCK5,ESCO2,WRN,ADAM9,EYA1,CNGB3,NBN,RECQL4,VLDLR,
CDKN2A,GALT,ALDH1A1,PTCH1,TDRD7,LMX1B,POMT1,VIM,ITGB1,ERCC6,ATOH7,PCBD1,PTEN,
SLC16A12,ALDH18A1,PITX3,OAT,HRAS,USH1C,LGR4,PAX6,CAT,PEX16,DHCR7,BEST1,LRP5,
MYO7A,FZD4,MMP1,ATM,CRYAB,APOA1,JAM3,PEX5,COL2A1,MIP,MVK,GJA3,GJB6,B3GALTL,
SLC25A15,ITM2B,COL4A1,SEC23A,OTX2,SIX6,VSX2,TGFB3,POMT2,DICER1,BUB1B,SORD,
FBN1,NR2E3,LOXL1,MIR184,POLG,GFER,TMEM114,NOD2,HSF4,ADAMTS18,MAF,YWHAE+,
GUCY2D,CRYBA1,UNC45B,PEX12,WNT3,NOG,GALK1,PYCR1,EPG5,CTDP1,ADAMTS10,SMARCA4,
MAN2B1,SIPA1L3,OPA3,SIX5,DMPK,FKRP,FTL,LIM2,TBC1D20,BFSP1,PHARC,CHMP4B,NCOA6,
SALL4,GNAS,CRYAA,COL18A1,LSS,PEX26,CRYBB3,CRYBB2,CRYBB1,CRYBA4,NF2,LARGE,
ARSE,HCCS,NHS,BCOR,NDP,RP2,PORCN,PQBP1,EBP,GLA,COL4A6,COL4A5,LAMP2,OCRL,ABCD1
+,IKBKG,AKR1E2,CYP51A1,FOXC1,MFSD6L,MYH9,PEX1,PEX2,PEX19,PITX2,RAB18,RNLS,
SOX2,TMEM70,TMEM114,CBS,ERCC2,ERCC3,FKTN,FOXD3,PEX5L,PEX11β,SLC2A1。
Described probe groups, a length of 78bp of each of which DNA sequence.The congenital cataract that each DNA sequence is mated
The section in the non-duplicate region of related gene exon is the most as shown in table 1.
The described probe groups for detecting congenital cataract related gene is at detection congenital cataract related gene
In application.
A kind of test kit for detecting congenital cataract related gene, including described for detect congenital white in
The probe groups of barrier related gene.
The described test kit for detecting congenital cataract related gene is at detection congenital cataract related gene
In application.
The beneficial effects of the present invention is: congenital cataract can disposably be sent out by the probe groups of the present invention and test kit
225 existing related genes detect, and improve coverage rate and the efficiency of molecular diagnosis, and specificity is good, highly sensitive, to elder generation
It cataract Prenatal Screening, early diagnosis, accurate prevention and control are significant.
Detailed description of the invention
Following example are to further illustrate the present invention rather than limitation of the present invention.
Embodiment 1: for detecting the probe groups of congenital cataract related gene
1, the screening of congenital cataract related gene determines
With " congenital cataract ", " inherited cataract ", " pediatric cataract ",
" infantile cataract ", " childhood cataract " etc. is term, at PubMed, Online Mendelian
Inheritance in Man (OMIM) and National Center for Biotechnology Information (NCBI)
Correlator website is searched for and has reported, by familial linkage analysis, crowd's association analysis, Sporadic cases gene test and animal
The congenital cataract related gene of the different Evidence grades that experiment finds.By further to clinical phenotypes and genetic background
Screening, finally includes 225 congenital cataract related genes in, carries out product design and detection.
The congenital cataract related gene of screening is as follows:
PEX10(5192),PEX14(5195),EPHA2(1969),HSPG2(3339),POMGNT1(55624),FOXE3
(2301),RPE65(6121),CRYZ(1429),DNASE2B(58511),ABCD3(5825),COL11A1(1301),GJA8
(2703),ADAMTSL4(54507),GPR161(23432),PROX1(5629),RAB3GAP2(25782),GNPAT(8443),
PXDN(7837),PEX13(5194),RAB3GAP1(22930),LCT(3938),FIGN(55137),LRP2(4036),AGPS
(8540),CRYGD(1421),CRYGC(1420),CRYGB(1419),CRYGA(1418),CYP27A1(1593),CRYBA2
(1412),KCNJ13(3769),FYCO1(79443),COL7A1(1294),LAMB2(3913),FLNB(2317),GPX1
(2876),PVRL3(25945),CPOX(1371),CASR(846),CNBP(7555),BFSP2(8419),SOX2(6657),
CRYGS(1427),WFS1(7466),HMX1(3166),CC2D2A(57545),SRD5A3(79644),CLOCK(9575),
AFF1(4299),ANK2(287),ETFDH(2110),CTNND2(1501),ERCC8(1161),VCAN(1462),EFNA5
(1946),TGFBI(7045),SIL1(64374),SPARC(6678),TFAP2A(7020),GCNT2(2651),GCM2
(9247),NEU1(4758),PEX6(5190),LGSN(51557),LCA5(167691),GJA1(2697),PEX7(5191),
IFNGR1(3459),PEX3(8504),EZR(7430),FAM126A(84668),GTF2IRD1(9569),HIP1(3092),
NRCAM(4897),AGK(55750),FDFT1(2222),BIN3(55909),DOCK5(80005),ESCO2(157570),WRN
(7486),ADAM9(8754),EYA1(2138),CNGB3(54714),NBN(4683),RECQL4(9401),VLDLR
(7436),CDKN2A(1029),GALT(2592),ALDH1A1(216),PTCH1(5727),TDRD7(23424),LMX1B
(4010),POMT1(10585),VIM(7431),ITGB1(3688),ERCC6(2074),ATOH7(220202),PCBD1
(5092),PTEN(5728),SLC16A12(387700),ALDH18A1(5832),PITX3(5309),OAT(4942),HRAS
(3265),USH1C(10083),LGR4(55366),PAX6(5080),CAT(12359),PEX16(9409),DHCR7
(1717),BEST1(7439),LRP5(4041),MYO7A(4647),FZD4(8322),MMP1(4312),ATM(472),
CRYAB(1410),APOA1(335),JAM3(83700),PEX5(5830),COL2A1(1280),MIP(4284),MVK
(4598),GJA3(2700),GJB6(10804),B3GALTL(145173),SLC25A15(10166),ITM2B(9445),
COL4A1(1282),SEC23A(10484),OTX2(5015),SIX6(4990),VSX2(338917),TGFB3(7043),
POMT2(29954),DICER1(23405),BUB1B(701),SORD(6652),FBN1(2200),NR2E3(10002),
LOXL1(4016),MIR184(406960),POLG(5428),GFER(2671),TMEM114(283953),NOD2(64127),
HSF4(3299),ADAMTS18(170692),MAF(4094),YWHAE+(7531),GUCY2D(3000),CRYBA1(1411),
UNC45B(146862),PEX12(5193),WNT3(7473),NOG(9241),GALK1(2584),PYCR1(5831),EPG5
(57724),CTDP1(9150),ADAMTS10(81794),SMARCA4(6597),MAN2B1(4125),SIPA1L3
(23094),OPA3(80207),SIX5(147912),DMPK(1760),FKRP(79147),FTL(2512),LIM2(3982),
TBC1D20(128637),BFSP1(631),PHARC(100272226),CHMP4B(128866),NCOA6(23054),SALL4
(57167),GNAS(2778),CRYAA(1409),COL18A1(80781),LSS(4047),PEX26(55670),CRYBB3
(1417),CRYBB2(1415),CRYBB1(1414),CRYBA4(1413),NF2(4771),LARGE(9215),ARSE
(415),HCCS(3052),NHS(4810),BCOR(54880),NDP(4693),RP2(6102),PORCN(64840),PQBP1
(10084),EBP(10682),GLA(2717),COL4A6(1288),COL4A5(1287),LAMP2(3920),OCRL
(4952),ABCD1+(215),IKBKG(8517),AKR1E2(83592),CYP51A1(1595),FOXC1(2296),MFSD6L
(162387),MYH9(4627),PEX1(5189),PEX2(5828),PEX19(5824),PITX2(5308),RAB18
(22931),RNLS(55328),SOX2(6657),TMEM70(54968),TMEM114(283953),CBS(875),ERCC2
(2068),ERCC3(2071),FKTN(2218),FOXD3(27022),PEX5L(51555),PEX11β(8799),SLC2A1
(6513)；Totally 225 genes, wherein, correspond to the Gene ID of this gene in bracket.
2, for detecting design and the preparation of the probe of congenital cataract related gene
The complete outer of above-mentioned congenital cataract related gene according to the human genome HG19 disclosed in Genebank shows
Subsequence, to the non-duplicate region of each exon according to probe sequence (the i.e. probe of base pair complementarity principle design 78bp
Sequence and corresponding congenital cataract related gene exon sequence are strictly complementary couplings), each probe sequence along
Design is moved in gene extron position, thus obtains and the spy of the congenital cataract non-duplicate Region Matching of related gene exon
Pin group.Utilize situ synthesis techniques, the probe of a large amount of compounding design, and it is substantial amounts of with biology to utilize the method for PCR to amplify
The probe groups of element labelling.
Specifically, the section of the congenital cataract related gene exon that single probe sequence is mated such as table 1 institute
Show.The order of the section of the congenital cataract related gene exon that probe sequence listed in table 1 is mated is that row 1 are by upper
Under to, from top to bottom, row 3 are from top to bottom for row 2.As in row 1, the 3rd row represents the chr10:103990252-of PITX3 gene
103990330 (calculate head and do not include tail, i.e. but do not comprise last alkali of this section from the beginning of the first of this section base position
Base position, lower with) exon region, altogether 78bp, to should the probe sequence in region be 5 '-GGATGATCTACGGGCGGGGCCG
CTCATACGGGCCTTTCCACGGCGTACTGGCACGGACTAAGGTTGGCTGCCGGGGGC-3’；In row 1, the 4th row represents
The exon region of the chr10:103990330-103990408 of PITX3 gene, altogether 78bp, to should the probe sequence in region
It is 5 '-GGCCCGTGCACAGCGGGGTAGCTGAAGGAGGCGTGCTGTTTGGCTTTGAGCCGCAG GCTGGCCAGGCTCGAG
TTACAC-3’；In row 2, the 2nd row represents the exon region of the chr19:47261713-47261791 of FKRP gene, altogether 78bp,
To should the probe sequence in region be 5 '-GTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAATATGAATAGGGTTT
TTTTTATGTTTCTTGCCTCATCCCAA-3’。
The section of the congenital cataract related gene exon that table 1 probe sequence is mated
Embodiment 2: for detecting the test kit of congenital cataract related gene
The test kit for detecting congenital cataract related gene of the present embodiment, is by detection congenital cataract
The sudden change of related gene exon carries out the test kit of Molecular genetic test.
The test kit being used for detecting congenital cataract related gene of the present embodiment includes: the probe that embodiment 1 obtains
Group, also include PCR ion amplification mix (PCR buffer, dNTPs, Taq enzyme etc.), 96 orifice plates and 96 orifice plate seal moulds and
QIAGEN Purification kit(Cat.No.159992)、Qiagen PCR kit(Cat.No.28104)、Qiagen
MinElute kit (Cat.No.28004) and Ampure beads XP (Cat.No.4471250) etc..
Embodiment 3: for detecting the application of the test kit of congenital cataract related gene
Use Covaris to crush instrument genomic DNA qualified for quality inspection and be broken into the sheet of a length of 180-280bp at random
Section, end reparation and tail end connect top connection respectively at fragment two ends after adding A-tailing and prepare DNA library.Will be with special
After DNA library polling of index and carry out solution hybridization with biotin labeled probe, re-use band Streptavidin
Magnetic bead the exon region of target gene (congenital cataract related gene) is captured, laggard through PCR linear amplification
Style of writing storehouse quality inspection, qualified after check order.
1. sample requirement
Genomic DNA sample (STb gene sample, OD260/280 value between 1.8-2.0, concentration 100ng/ μ L, always
Measure 50 μ L/ samples, and guarantee that DNA is pollution-free, without degraded)
2. genome dna library builds
2.1 genomic DNAs interrupt
2.1.1 the 80 μ L genomic DNAs being dissolved in TE buffer are joined Covaris microtube.
2.1.2 according to the program that interrupts arranged below:
2.1.3 sample QIAGEN Purification kit (Qiagen PCR kit or Qiagen interrupted
MinElute kit or Ampure beads XP) purification.
2.2 fragment ends reparations
2.2.1 1.5ml centrifuge tube or 200 μ L PCR pipe are taken, according to following preparation reaction system
DNA and H2O:37.5 μ L
End Repair Reaction Buffer (red): 7 μ L
End Repair Enzyme (orange): 5.5 μ L
Total volume 50 μ L, hatches 30 minutes for 20 DEG C
2.2.2 product Ampure beads is purified according to following steps
1) Ampure beads is taken out from refrigerator, turn upside down and make beads fully suspend.
2) the Ampure beads that 1.8 times of volumes of addition fully suspend in product (such as bulk product 50 μ L, then adds 90
The Ampure beads of μ L), with rifle head suction mixing at least ten times.
3) mixed liquor is put at room temperature 5 minutes, make DNA fully be attracted on beads.
4) on Magnet, room temperature places 2 minutes (not rotating tube).
5) 2 minutes or solution become limpid after, move to abandon by the liquid of clarification with pipettor and (note: rifle head keeps off
To magnetic bead).
6) Tube is placed on magnetic frame, and (ethanol is joined to add the ethanol of 750 μ L (adding 200 μ L in 200 μ L PCR pipe) 80%
Time of putting is within two weeks, and guarantees to close), close upper tube cap, room temperature is placed 30 seconds.
7) clear liquor is abandoned in shifting.
8) repeating step 6,7 once.
9) with the rifle head of 200 μ L, the residual liquid at the bottom of pipe is thoroughly blotted.
10) being removed from Magnet by tube, open lid, within 2 minutes, (this step is in order to vapor away residual in room temperature placement
Ethanol, in order to avoid affecting subsequent reactions).
11) adding the water without enzyme (RNase water) of 35 μ L, fully mix, room temperature is placed 2 minutes.
12) being put on Magnet by tube, room temperature places two minutes (or treating that solution is the limpidest), shifts the clear of 33.5 μ L
Clear liquid (DNA is in clear liquor) is in a new centrifuge tube or PCR pipe, in case lower step uses.
The tail end of 2.3 fragments adds A (A Tailing)
2.3.1 reaction system is prepared according to following components
DNA in H2O: 33.5μL
A-Tailing Reaction Buffer (yellow): 15 μ L
A-Tailing Enzyme (green): 1.5 μ L
Total volume 50 μ L, hatches 30min for 37 DEG C
2.3.2 product Ampure beads is purified according to following steps
1) Ampure beads is taken out from refrigerator, turn upside down and make beads fully suspend.
2) the Ampure beads that 1.8 times of volumes of addition fully suspend in product (such as bulk product 50 μ L, then adds 90
The Ampure beads of μ L), with rifle head suction mixing at least ten times.
3) mixed liquor is put at room temperature 5 minutes, make DNA fully be attracted on beads.
4) on Magnet, room temperature places 2 minutes (not rotating tube).
5) 2 minutes or solution become limpid after, move to abandon by the liquid of clarification with pipettor and (note: rifle head keeps off
To magnetic bead).
6) Tube is placed on magnetic frame, and (ethanol is joined to add the ethanol of 750 μ L (adding 200 μ L in 200 μ L PCR pipe) 80%
Time of putting is within two weeks, and guarantees to close), close upper tube cap, room temperature is placed 30 seconds.
7) clear liquor is abandoned in shifting.
8) repeating step 6,7 once.
9) with the rifle head of 200 μ L, the residual liquid at the bottom of pipe is thoroughly blotted.
10) being removed from Magnet by tube, open lid, within 2 minutes, (this step is in order to vapor away residual in room temperature placement
Ethanol, in order to avoid affecting subsequent reactions).
11) adding the water without enzyme (RNase water) of 30 μ L, fully mix, room temperature is placed 2 minutes.
12) being put on Magnet by tube, room temperature places two minutes (or treating that solution is the limpidest), the clarification of transferase 12 7 μ L
Liquid (DNA is in clear liquor) is in a new centrifuge tube or PCR pipe, in case lower step uses.
2.4 connect illumina adaptor
2.4.1 reaction system is prepared according to following components
DNA in EB:27 μ L (from A-tailing)
Ligation Reaction Buffer (white): 30 μ L
Ligation Enzyme (purple) 3 μ L:3 μ L
Total volume 70 μ L, hatches 10min for 25 DEG C.
2.4.2 product Ampure beads is purified according to following steps
1) Ampure beads is taken out from refrigerator, turn upside down and make beads fully suspend.
2) the Ampure beads that 1.5 times of volumes of addition fully suspend in product (such as bulk product 70 μ L, then adds
The Ampure beads of 105 μ L), with rifle head suction mixing at least ten times.
3) mixed liquor is put at room temperature 5 minutes, make DNA fully be attracted on beads.
4) on Magnet, room temperature places 2 minutes (not rotating tube).
5) 2 minutes or solution become limpid after, move to abandon by the liquid of clarification with pipettor and (note: rifle head keeps off
To magnetic bead).
6) Tube is placed on magnetic frame, and (ethanol is joined to add the ethanol of 750 μ L (adding 200 μ L in 200 μ L PCR pipe) 80%
Time of putting is within two weeks, and guarantees to close), close upper tube cap, room temperature is placed 30 seconds.
7) clear liquor is abandoned in shifting.
8) repeating step 6,7 once.
9) with the rifle head of 200 μ L, the residual liquid at the bottom of pipe is thoroughly blotted.
10) being removed from Magnet by tube, open lid, within 2 minutes, (this step is in order to vapor away residual in room temperature placement
Ethanol, in order to avoid affecting subsequent reactions).
11) adding the water without enzyme (RNase water) of 32 μ L, fully mix, room temperature is placed 2 minutes.
12) being put on Magnet by tube, room temperature places two minutes (or treating that solution is the limpidest), shifts the clarification of 30 μ L
Liquid (DNA is in clear liquor), in a new PCR pipe, carries out following PCR reaction.
2.5PCR
2.5.1 PCR reaction system is prepared according to following components
2.5.2PCR program is provided that
Stage1:98 DEG C of 2min
Stage2:9x (98 DEG C, 30sec；65℃,30sec；72℃,30sec)
Stage3:72 DEG C of 5min, 4 DEG C of 10s
2.5.3PCR product with Ampure beads according to following steps purify (PCR primer of first purification half, if
Yield poorly, require second half product adds expansion respective cycle number according to it)
1) Ampure beads is taken out from refrigerator, turn upside down and make beads fully suspend.Take out from product
In 50 μ L to new PCR pipe (drawing again after first mixing for several times up and down with suction nozzle during absorption).
2) the Ampure beads that 1.5 times of volumes of addition fully suspend in product (such as bulk product 50 μ L, then adds 75
The Ampure beads of μ L), with rifle head suction mixing at least ten times.
3) mixed liquor is put at room temperature 5 minutes, make DNA fully be attracted on beads.
4) on Magnet, room temperature places 2 minutes (not rotating tube).
5) 2 minutes or solution become limpid after, move to abandon by the liquid of clarification with pipettor and (note: rifle head keeps off
To magnetic bead).
6) Tube is placed on magnetic frame, and (ethanol is joined to add the ethanol of 750 μ L (adding 200 μ L in 200 μ L PCR pipe) 80%
Time of putting is within two weeks, and guarantees to close), close upper tube cap, room temperature is placed 30 seconds.
7) clear liquor is abandoned in shifting.
8) repeating step 6,7 once.
9) with the rifle head of 200 μ L, the residual liquid at the bottom of pipe is thoroughly blotted.
10) being removed from Magnet by tube, open lid, within 2 minutes, (this step is in order to vapor away residual in room temperature placement
Ethanol, in order to avoid affecting subsequent reactions).
11) adding the water without enzyme (RNase water) of 35 μ L, fully mix, room temperature is placed 2 minutes.
12) being put on Magnet by tube, room temperature places two minutes (or treating that solution is the limpidest), shifts the clarification of 33 μ L
Liquid (DNA is in clear liquor), in a new centrifuge tube performing labelling, remains 3 μ L samples for gel electrophoresis in PCR pipe.
The quality testing in 2.6 libraries
1) take the library sample that 1 μ L prepares, test its OD value with Nanodrop 2000, take the library sample that 3 μ L prepare
This, do the agarose gel electrophoresis experiment of 1%, and electrophoresis result clip size is between 300bp to 500bp.
2) the library sample short time prepared can leave 4 DEG C in, and long-time preservation is then saved in-20 DEG C.
3. target area capture and order-checking
Take prepared by the genome dna library for preparing and embodiment 1 with biotin labeled congenital for detecting
The probe groups of cataract related gene carries out solution hybridization, and the magnetic bead re-using band Streptavidin is congenital by be combined with probe
The exon sequence of cataract related gene captures, through the laggard style of writing storehouse quality inspection of PCR linear amplification, qualified after carry out two
Generation order-checking.
Embodiment 4: use the detection probe of the present invention and test kit to carry out clinical congenital cataract Samples detection
Choose three people in Congenital Cataract Pedigree (father is the most ill, and son is ill with mother) to detect, according to real
The idiographic flow executing example 3 carries out sample preparation and Sequence Detection.
As a example by mother's sample in above-mentioned Congenital Cataract Pedigree, order-checking original data volume is 1022Mb, target area
The order-checking coverage in territory reaches 99.91%, and the average order-checking degree of depth of target area reaches 1401.99, the target area base order-checking degree of depth
Ratio more than 20x reaches 99.31%.The sequencing data (clean data) removing joint sequence and low quality base is used
Burrows-Wheeler Aligner (BWA) comparison is on human genome；With GATK software, SNP and InDel is made a variation
Detection, associates multiple data base (such as dbsnp, 1000g, ESP etc.) with ANNOVAR software simultaneously and annotates variation result.
The analysis of congenital cataract related gene is carried out again after the variation result obtained is carried out the filtration of necessity.
According to the mode of inheritance of general diseases, according to dominant inheritance (AD:Autosomal Dominant), recessive inheritance
(AR:Autosomal Recessive) is analyzed.1) dominant inheritance: patient and mother have an identical heterozygous mutant, and father
Not this mutational site；2) recessive inheritance: patient and mother have identical homozygous mutation, and father is that heterozygosis is dashed forward in this site simultaneously
Become.Congenital cataract dominant inheritance's mode related mutation genetic results such as table 2 of obtaining of screening, lists sudden change and is and does not reports
The mutational site crossed.Result above proves that the cataract gene test product before the probe groups of the present invention and test kit are relatively has
Gene mutation recall rate the most extensive, efficient.
Table 2 congenital cataract dominant inheritance and recessive inheritance's mode related mutation gene
Note: Gene: Gene Name；Chr: chromosome；Begin: original position；End: final position；MutPosition:
The amino acid sites of sudden change.
Wherein, probe (the corresponding exon section chr21:46925269-of the A1203V sudden change of COL18A1 is detected
46925347) sequence is: 5 '-CCTGCAGCTATCAGCGTTCCCGGCCCTCCGGGCCCCCCTGGGCCCCCTGGGCCCCC TG
GAACCATGGGCGCCTCCTCA-3’；Detect the L994M sudden change of HIP1 probe (corresponding exon section chr7:
75168697-75168775) sequence is: 5 '-GCTCTCCCAGTTTTTGACGCTCCTTCTGCAATTCATTTTCTAGCTCTA
GCACCCTAACCTGTAAGGGAAAGTAAGTGG-3’；Probe (the corresponding exon 1 of the G196fs sudden change of MIP detected
Section chr12:56846818-56846896) sequence is: 5 '-TCCAGAAGGGCTTCAGGATCTTGCCCCTCTCCCTTCACTCA
CCCAGTGGTTAGTGAAGTTCCCAGTGAGAATGGCAGG-3’；Detect that the probe of P96S sudden change of SORD is (outside Dui Ying
Aobvious sub-segments chr15:45353227-45353305) sequence is: 5 '-TCCCACCCTCGGACATGGTGCCATCTTTGTTTTCC
TCTCCAGGTGATCGTGTTGCCATCGAGCCTGGTGCTCCCCGAG-3’；(the correspondence of the R3092P sudden change of VCAN detected
Exon section chr5:82841334-82841412) probe sequence is: 5 '-TTTCTTACTTTCCTGAATATGGTAGGAC
CTGATCGCTGCAAAATGAACCCGTGCCTTAACGGAGGCACCTGTTATCCT-3’；The P1872L sudden change of VCAN detected
Probe (corresponding exon section chr5:82834364-82834442) sequence be: 5 '-ACCACTGTTTCTTCATTTTCA
TTAAACGTAGAGTATGCAATTCAAGCCGAAAAGGAAGTAGCTGGCACTTTGTCTCCG-3’。
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (5)
1. the probe groups being used for detecting congenital cataract related gene, it is characterised in that comprising can specific recognition people
Multiple DNA sequence in the non-duplicate region of class congenital cataract related gene exon；
Described congenital cataract related gene is as follows:
PEX10,PEX14,EPHA2,HSPG2,POMGNT1,FOXE3,RPE65,CRYZ,DNASE2B,ABCD3,COL11A1,
GJA8,ADAMTSL4,GPR161,PROX1,RAB3GAP2,GNPAT,PXDN,PEX13,RAB3GAP1,LCT,FIGN,LRP2,
AGPS,CRYGD,CRYGC,CRYGB,CRYGA,CYP27A1,CRYBA2,KCNJ13,FYCO1,COL7A1,LAMB2,FLNB,
GPX1,PVRL3,CPOX,CASR,CNBP,BFSP2,SOX2,CRYGS,WFS1,HMX1,CC2D2A,SRD5A3,CLOCK,
AFF1,ANK2,ETFDH,CTNND2,ERCC8,VCAN,EFNA5,TGFBI,SIL1,SPARC,TFAP2A,GCNT2,GCM2,
NEU1,PEX6,LGSN,LCA5,GJA1,PEX7,IFNGR1,PEX3,EZR,FAM126A,GTF2IRD1,HIP1,NRCAM,
AGK,FDFT1,BIN3,DOCK5,ESCO2,WRN,ADAM9,EYA1,CNGB3,NBN,RECQL4,VLDLR,CDKN2A,GALT,
ALDH1A1,PTCH1,TDRD7,LMX1B,POMT1,VIM,ITGB1,ERCC6,ATOH7,PCBD1,PTEN,SLC16A12,
ALDH18A1,PITX3,OAT,HRAS,USH1C,LGR4,PAX6,CAT,PEX16,DHCR7,BEST1,LRP5,MYO7A,
FZD4,MMP1,ATM,CRYAB,APOA1,JAM3,PEX5,COL2A1,MIP,MVK,GJA3,GJB6,B3GALTL,
SLC25A15,ITM2B,COL4A1,SEC23A,OTX2,SIX6,VSX2,TGFB3,POMT2,DICER1,BUB1B,SORD,
FBN1,NR2E3,LOXL1,MIR184,POLG,GFER,TMEM114,NOD2,HSF4,ADAMTS18,MAF,YWHAE+,
GUCY2D,CRYBA1,UNC45B,PEX12,WNT3,NOG,GALK1,PYCR1,EPG5,CTDP1,ADAMTS10,SMARCA4,
MAN2B1,SIPA1L3,OPA3,SIX5,DMPK,FKRP,FTL,LIM2,TBC1D20,BFSP1,PHARC,CHMP4B,NCOA6,
SALL4,GNAS,CRYAA,COL18A1,LSS,PEX26,CRYBB3,CRYBB2,CRYBB1,CRYBA4,NF2,LARGE,
ARSE,HCCS,NHS,BCOR,NDP,RP2,PORCN,PQBP1,EBP,GLA,COL4A6,COL4A5,LAMP2,OCRL,ABCD1
+,IKBKG,AKR1E2,CYP51A1,FOXC1,MFSD6L,MYH9,PEX1,PEX2,PEX19,PITX2,RAB18,RNLS,
SOX2,TMEM70,TMEM114,CBS,ERCC2,ERCC3,FKTN,FOXD3,PEX5L,PEX11β,SLC2A1。
Probe groups for detecting congenital cataract related gene the most according to claim 1, it is characterised in that it is every
The a length of 78bp of individual DNA sequence, the congenital cataract related gene exon of each DNA sequence institute specific recognition is non-duplicate
The section in region is the most as follows:
3. the probe groups for detecting congenital cataract related gene described in claim 1 is in detection congenital cataract phase
Application in correlation gene.
4. the test kit being used for detecting congenital cataract related gene, it is characterised in that include described in claim 1
For detecting the probe groups of congenital cataract related gene.
5. the test kit for detecting congenital cataract related gene described in claim 4 is in detection congenital cataract phase
Application in correlation gene.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342467A (en) * | 2017-01-23 | 2018-07-31 | 黄秀峰 | A kind of diagnostic method, gene probe and its application of MAC Disease-causing genes |
| CN109112134A (en) * | 2018-09-28 | 2019-01-01 | 中国人民解放军陆军军医大学第附属医院 | A kind of the BEST1 new mutation Disease-causing gene and its kit of retinal degenerative disease |
| CN111607641A (en) * | 2019-02-26 | 2020-09-01 | 中国人民解放军第四军医大学 | Novel gene mutation site causing congenital membrane cataract, detection method and application |
| CN113774128A (en) * | 2021-09-24 | 2021-12-10 | 中国人民解放军东部战区总医院 | Gja8 application of gene mutation site in preparation of product for diagnosing cataract disease |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168775A (en) * | 2007-10-29 | 2008-04-30 | 哈尔滨医科大学 | Cataract gene detecting kit |
| CN102978284A (en) * | 2012-11-19 | 2013-03-20 | 金子兵 | Gene chip for screening various ophthalmological hereditary diseases as well as preparation and usage method of gene chip |
| CN104561016A (en) * | 2014-12-29 | 2015-04-29 | 深圳华大基因科技有限公司 | CC (congenital cataract) PITX3 gene novel mutation |
-
2016
- 2016-09-18 CN CN201610831906.7A patent/CN106282369A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168775A (en) * | 2007-10-29 | 2008-04-30 | 哈尔滨医科大学 | Cataract gene detecting kit |
| CN102978284A (en) * | 2012-11-19 | 2013-03-20 | 金子兵 | Gene chip for screening various ophthalmological hereditary diseases as well as preparation and usage method of gene chip |
| CN104561016A (en) * | 2014-12-29 | 2015-04-29 | 深圳华大基因科技有限公司 | CC (congenital cataract) PITX3 gene novel mutation |
Non-Patent Citations (1)
| Title |
|---|
| 刘晖等: "先天性白内障致病基因的研究进展", 《中国儿童保健杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342467A (en) * | 2017-01-23 | 2018-07-31 | 黄秀峰 | A kind of diagnostic method, gene probe and its application of MAC Disease-causing genes |
| CN109112134A (en) * | 2018-09-28 | 2019-01-01 | 中国人民解放军陆军军医大学第附属医院 | A kind of the BEST1 new mutation Disease-causing gene and its kit of retinal degenerative disease |
| CN109112134B (en) * | 2018-09-28 | 2020-06-02 | 中国人民解放军陆军军医大学第一附属医院 | BEST1 new mutation pathogenic gene of retinal degeneration disease and kit thereof |
| CN111607641A (en) * | 2019-02-26 | 2020-09-01 | 中国人民解放军第四军医大学 | Novel gene mutation site causing congenital membrane cataract, detection method and application |
| CN111607641B (en) * | 2019-02-26 | 2022-08-09 | 中国人民解放军第四军医大学 | Molecular marker of new gene mutation site causing congenital membrane cataract and kit thereof |
| CN113774128A (en) * | 2021-09-24 | 2021-12-10 | 中国人民解放军东部战区总医院 | Gja8 application of gene mutation site in preparation of product for diagnosing cataract disease |
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