CN112626193A - Gene chip, kit comprising gene chip and application of gene chip - Google Patents

Gene chip, kit comprising gene chip and application of gene chip Download PDF

Info

Publication number
CN112626193A
CN112626193A CN202011485199.3A CN202011485199A CN112626193A CN 112626193 A CN112626193 A CN 112626193A CN 202011485199 A CN202011485199 A CN 202011485199A CN 112626193 A CN112626193 A CN 112626193A
Authority
CN
China
Prior art keywords
seq
gene
gene chip
hereditary
ocular
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.)
Pending
Application number
CN202011485199.3A
Other languages
Chinese (zh)
Inventor
雷博
郝冰涛
李亚
游雅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to CN202011485199.3A priority Critical patent/CN112626193A/en
Publication of CN112626193A publication Critical patent/CN112626193A/en
Pending legal-status Critical Current

Links

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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention provides a gene chip for detecting hereditary anterior ocular segment disease and hereditary ocular syndrome, which comprises a gene probe specifically directed against the genes shown in table 1. The invention diagnoses and screens the molecular genes of the hereditary anterior ocular segment diseases and the hereditary ocular syndromes by designing the known pathogenic genes and the mutated probes aiming at the hereditary anterior ocular segment diseases and the hereditary ocular syndromes, and detects the known pathogenic genes by the target sequencing of the target region, thereby having the advantages of strong pertinence, high correctness, deep sequencing depth, lower cost and complete information of functional sites. In addition, the method realizes one-time detection of most mutations of disease-related pathogenic genes, balances cost performance, and provides guarantee for treatment and intervention of diseases. In addition, the invention also provides a kit comprising the gene chip and a method for screening gene mutation of a subject by using the gene chip.

Description

Gene chip, kit comprising gene chip and application of gene chip
Technical Field
The invention relates to the field of genetic disease detection, in particular to a gene chip for detecting hereditary anterior ocular segment diseases and hereditary ocular syndromes, a kit comprising the gene chip and a method for screening genetic mutation of a subject by using the gene chip.
Background
The anterior segment refers to the anterior third of the eyeball, including the cornea, iris, ciliary body, lens and anterior part of vitreous body. The hereditary anterior ocular segment disease is a hereditary disease which involves the anterior ocular segment. Hereditary ocular syndromes are a class of diseases with complex and diverse phenotypes, involvement of structures such as anterior segment of the eye and eye muscle, and resulting dysfunction. The clinical phenotype of the hereditary ocular syndrome is diverse and may involve multiple organs, sites.
Hereditary anterior segment diseases and hereditary ocular syndromes include cataract, aphakia, corneal dystrophy, corneal malformation, Peters' abnormality, anterior segment dysplasia, Waardenburg syndrome, mucopolysaccharidosis type VII, Axenfeld-Rieger syndrome, etc., and the hereditary modes can be divided into autosomal dominant inheritance, autosomal recessive inheritance and sex-linked recessive inheritance. The hereditary anterior segment diseases and the hereditary ocular syndromes have extremely strong clinical and genetic heterogeneity, the relationship between the genotype and the phenotype is very complex, the same pathogenic gene can cause different clinical manifestations, and different pathogenic mutations can cause the same clinical phenotype, thereby bringing great challenges to the screening and diagnosis of the pathogenic genes of the diseases. Therefore, an accurate, efficient and low-cost method for assisting molecular genetics is urgently needed to improve the accuracy and the generalization of the diagnosis and screening of the disease genes.
The traditional genetic analysis methods for hereditary anterior segment diseases and hereditary ocular syndromes include linkage analysis, Sanger sequencing, DNA chip technology and the like. Due to its limitations, efficient and accurate detection with scale cannot be performed. With the rapid development of molecular biology, the next generation sequencing technology (NGS) has become an important tool for screening and detecting pathogenic genes of genetic diseases, and is widely applied to genetic diagnosis and screening of hereditary anterior segment diseases and hereditary ocular syndromes. The second generation sequencing technology is mainly divided into three types: whole Genome Sequencing (WGS), whole genome exon sequencing (WES), and region of interest sequencing. Whole genome sequencing is able to detect gene mutations, CNVs and structural variations throughout the genome, covering almost the entire human genome (98%). However, it has limitations such as high cost, high requirement for DNA quality, relatively low sequencing depth, difficulty in bioinformatic analysis and storage analysis of massive data, and the like. The whole genome sequencing has high sequencing cost and relatively low sequencing depth due to the generation of a large amount of sequencing data, cannot realize full coverage, zero omission and high accuracy of important functional gene screening, and has great limitation in screening and diagnosing pathogenic genes of hereditary anterior segment diseases and related syndromes.
Therefore, the development of a sequencing product with high accuracy and low cost aiming at the known pathogenic genes and mutations of the hereditary anterior ocular segment diseases and the hereditary ocular syndromes is particularly important for the molecular gene diagnosis and screening of the diseases.
Disclosure of Invention
In view of the above, the present invention designs a set of gene probes for known pathogenic genes and mutations of hereditary anterior ocular segment diseases and hereditary ocular syndromes, and thus provides a gene chip comprising the gene probes, a kit comprising the gene chip, and a method for screening genetic mutations of a subject using the gene chip.
According to a first aspect of the present invention, there is provided a gene chip for detecting hereditary anterior ocular segment disease and hereditary ocular syndrome, the gene chip comprising gene probes specific for the genes shown in Table 1.
According to a second aspect of the present invention, there is provided a kit for detecting hereditary anterior ocular segment disease and hereditary ocular syndrome in a subject, the kit comprising the gene chip provided by the first aspect of the present invention.
According to a third aspect of the present invention, there is provided a method of screening for genetic mutations in a subject, the method comprising:
1) extracting the genomic DNA of the subject, and breaking the genomic DNA into a range of 200-300 bp;
2) preparing a DNA fragment library from the fragmented genomic DNA;
3) hybridizing the DNA fragment library with the gene chip of the first aspect of the invention or the kit of the second aspect of the invention, and capturing genes;
4) amplifying the product captured in the step 3) by adopting PCR (polymerase chain reaction) and taking SEQ ID NO.69 and SEQ ID NO.70 as primers to obtain an amplification product;
5) performing on-machine sequencing on the amplification product obtained in the step 4) to obtain sequencing data of the gene;
6) comparing the sequencing data of step 5) with a human reference genome, thereby obtaining a single nucleotide polymorphism, insertion or deletion different from the reference genome, i.e., the detected gene mutation.
The invention diagnoses and screens the molecular genes of the hereditary anterior ocular segment diseases and the hereditary ocular syndromes by designing the known pathogenic genes and the mutated probes aiming at the hereditary anterior ocular segment diseases and the hereditary ocular syndromes, and detects the known pathogenic genes by the target sequencing of the target region, thereby having the advantages of strong pertinence, high correctness, deep sequencing depth, lower cost and complete information of functional sites. Compared with whole genome sequencing, the method greatly saves the required sequencing data amount. In addition, the method realizes one-time detection of most mutations of disease-related pathogenic genes, balances cost performance, and provides guarantee for treatment and intervention of diseases.
Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. It is to be understood that the described embodiments are merely a subset of the present invention and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments disclosed herein are within the scope of the present invention.
In the present invention, the human reference genome is HG 19.
In the present invention, mutations are expressed by common expression methods in the art. For example, among the mutations (c.1072C > T; p.P358S), "C" represents cDNA, "1072C > T" represents a mutation from nucleotide C to T at position 1072, "P" represents a protein, "P358S" represents a mutation from proline P to serine S at amino acid 358 of the protein sequence, and a mutation at the DNA level (c.1072C > T) corresponds to a mutation at the protein level (p.P358S).
In the present invention, as understood by those skilled in the art, a "gene sequence" or "sequence" actually includes either one, or both, of the complementary double strands. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. For example, reference to the sequence of a gene probe includes the sequence and its complement. For example, reference to SEQ ID NO 1 actually includes the complementary sequence thereof. One skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.
In the present invention, the gene sequence includes a DNA form or an RNA form, and one is disclosed, meaning that the other is also disclosed. For example, reference to the sequence of a gene probe actually includes the corresponding RNA sequence.
The gene chip is one of the great technological advances in high technology field in recent years, and the gene chip described in this patent refers to a liquid phase probe chip, and its main principle is that probes capable of hybridizing with a large number of gene regions in a sample are placed in a test tube in the form of solution, and perform hybridization reaction with an actual sample, and the hybridized product is subjected to high-throughput sequencing analysis to obtain the information of all genes to be detected. The technology has the advantages of high throughput, rapidness, accuracy and the like, provides a new solution for genetic diagnosis of hereditary anterior segment diseases and hereditary eye syndromes, and has important effects on clinical treatment and prognosis evaluation of children patients. Therefore, to diagnose and screen hereditary anterior segment diseases and hereditary eye syndromes by using the gene chip, the gene probe plays a crucial role, and the key to design the gene probe is to accurately master the pathogenic gene causing the diseases.
The inventors have accumulated sequencing data for exome sequences of nearly 1000 cases of hereditary anterior ocular disease and hereditary ocular syndrome in recent years, and the original sequenced fragments (reads) were processed by Illumina base harvesting Software 1.7, decontaminated by filtration, aligned to the reference genome using SOAPaligner 2.20(Li R, Li Y, Kristiansen K, et al, SOAP: short oligonucleotide alignment program. bioinformatics 2008,24(5): 713-714; Li R, Yu C, Li Y, et al, 2: an improved oligonucleotide for short reading alignment. bioinformatics 2009,25(15):1966-1967) to obtain uniquely matched sequenced fragments aligned to the genome. The genotype of the target region was determined by SOAPsnp (Li R, Li Y, Fang X, Yang H, et al, SNP detection for mapping parallel gene re-sequencing. genome Res 2009,19(6): 1124-1132). The results were then filtered through four public databases (dbSNP (v131): http:// hgdownload. cse. ucsc. edu/goldenPath/hg19/database/snp131.txt. gz.; 1000 people: ftp:// ftp. 1000genes. ebi. ac. uk/vol1/ftp or ftp:// ftp-trace. ncbi. niv/1000 genes/ftp; hapmap: ftp. ncbi. nlm. gov/hapmap; and YH database: http:// YH. genomics. org. cn), all nodes known to be unrelated to hereditary pre-ocular diseases and hereditary ocular syndromes and with allelic gene variations in the database above 0.005.
As a result, it was found that the hereditary anterior ocular segment disease and the hereditary ocular syndrome are associated with the genes as listed in the following table 1:
TABLE 1 genes responsible for inherited anterior segment diseases and inherited ocular syndromes
ABHD12 ADAM9 ADAMTS10 ADAMTS18 ADAMTSL4 AGBL1 AGK ALDH1A3
ALMS1 ASB10 ATOH7 B3GALNT2 B3GLCT BBS1 BFSP1 BMP4
CHMP4B ChrX CNGB3 COL11A1 COL18A1 COL2A1 COL4A1 COL8A2
COL9A1 CRB1 CRX CRYAA CRYAB CRYBA1 CRYBA4 CRYBB1
CRYBB2 CRYBB3 CRYGA CRYGB CRYGC CRYGD CRYGS CTDP1
CYP1B1 CYP27A1 CYP4V2 DAG1 DCN DDX58 DHCR7 ELP4
EPG5 EPHA2 ERCC2 ERCC5 ERCC8 FAM126A FBN1 FBN2
FKRP FKTN FLNB FOXC1 FOXE3 FTL FYCO1 GALK1
GCNT2 GDF6 GFER GJA1 GJA3 GJA8 GMPPB GSN
GTF2H5 GUSB HCCS HMX1 HSF4 IARS2 IDUA INPP5K
ITM2B JAG1 JAM3 KERA KRT12 KRT3 LCAT LIM2
LMX1B LRP2 LRP5 LTBP2 MAF MAN2B1 MED25 MFRP
MIP MIPEP MIR204 MITF MPLKIP MSMO1 MT-TS2 MVK
MYH9 MYOC NDP NECTIN1 NF1 NF2 NHS NOD2
NR2E3 NTF4 OAT OPA3 OTX2 P3H2 PAX2 PAX3
PAX6 PEX1 PEX11B PEX16 PEX2 PEX5 PEX7 PIK3R1
PIKFYVE PITX2 PITX3 PLG PLK4 POLG POMGNT1 POMK
POMT1 POMT2 PORCN PRDM5 PRSS56 RAX RBP3 RP2
RP9 RPE65 RPGRIP1 RS1 S1L1 SIX6 SOX10 STRA6
TBK1 TDRD7 TUBGCP6 TYR USH1C USH1G USH2A VCAN
VHL VSX2 WDR36 WRN ZEB1 ZNF469
As will be appreciated by those skilled in the art, the key to accurately detecting inherited anterior segment diseases and inherited ocular syndromes is also the accurate understanding of the mutation sites that lead to these causative genes. The mutation sites of the genes in table 1 are shown in table 2.
TABLE 2 mutation sites of the genes shown in TABLE 1
Figure BDA0002839148120000051
Figure BDA0002839148120000061
Figure BDA0002839148120000071
Figure BDA0002839148120000081
Figure BDA0002839148120000091
Figure BDA0002839148120000101
Figure BDA0002839148120000111
Figure BDA0002839148120000121
Table 2 shows the mutation sites of the genes in table 1 and their positions on the chromosome, for example for the gene mutant ABHD12, "Chr 20" indicates the chromosome, "25282958" indicates the position on the chromosome, c.1054c > T indicates that the 1054 th amino acid was mutated from nucleotide C to T, p.r352 indicates that the 352 nd amino acid of the protein sequence was mutated from arginine R to a stop codon.
A gene probe, i.e., a nucleic acid probe, is a nucleic acid sequence (DNA or RNA) complementary to a gene of interest, with a detectable label and a known sequence. The gene probe is combined with target gene by means of molecular hybridization to produce hybridization signal, so that the target gene can be displayed from vast genome. Therefore, it is necessary to design a sequence of a gene probe capable of specifically binding to a target gene.
Therefore, a set of gene probes for detecting hereditary anterior ocular segment disease and hereditary ocular syndrome was designed based on the genes causing hereditary anterior ocular segment disease and hereditary ocular syndrome in table 1 and the mutation sites in table 2, wherein the gene probes are specifically directed to the genes shown in table 1. The sequences of these gene probes are capable of specifically binding to genes responsible for inherited anterior segment diseases and inherited ocular syndromes, thereby detecting these causative genes from the human genome.
In some embodiments, the sequence of the gene probe is 90bp to 120bp in length. In a preferred embodiment, the sequence of the gene probe is 100bp in length.
Specifically, a probe sequence with the length of 90bp-120bp is designed from the first base in the direction from 5 'to 3' of the coding sequence of the gene according to the principle of reverse sequence complementation, for the length of the sequence of the gene probe, greater than 120bp brings difficulties in synthesis, less than 90bp reduces the capture capacity, on the other hand, the number of probes is increased, so that the cost is increased, the balance between the two is very important, and the inventor finds that the sequence length of 100bp is a better value through continuous tests.
In the present invention, the gene probe exerts its detecting action in the form of a gene chip.
Therefore, according to a first aspect of the present invention, there is provided a gene chip for detecting hereditary anterior ocular segment disease and hereditary ocular syndrome, the gene chip comprising gene probes specific for the genes shown in table 1.
In some embodiments, the gene chip further comprises a sequence obtained by replacing the site of the gene probe with a mutant base.
In some embodiments, the gene chip comprises gene probes specific for the genes shown in table 1. The gene chip can detect 97% of hereditary anterior segment diseases and hereditary eye syndrome cases.
The invention adopts liquid phase gene chip technology to carry out relevant detection. As is understood by those skilled in the art, the principle of the liquid phase gene chip technology is that probes capable of hybridizing with a large number of gene regions in a sample are placed in a test tube in the form of solution, hybridization reaction is performed with the actual sample, and the hybridized products are analyzed by high-throughput sequencing to obtain information of all genes to be detected. The invention adopts
Figure BDA0002839148120000131
The liquid phase probe hybridization capture technology consists of
Figure BDA0002839148120000141
Independently research and development, the technology mainly carries out probe design on a target region genome based on a thermodynamic stability algorithm, then synthesizes an effective specific probe, further carries out liquid phase hybridization with genome DNA, captures and enriches a target region sequence, and then carries out high-throughput sequencing by using a mainstream sequencing platform.
In order to simplify the gene chip of the invention and comprehensively consider the cost performance of the probe and the detection rate, the gene chip is optimized. The gene chip comprises probes aiming at the following genes, and can detect 83% of hereditary anterior segment diseases and hereditary ocular syndrome cases: ADAMTS18, ALDH1A3, ALMS1, B3GALNT2, B3GLCT, BFSP1, BMP4, COL2A1, COL4A1, CRX, CRYAA, CRYBB3, DHCR7, ELP4, EPG5, ERCC2, ERCC8, FBN2, FKRP, FK, FLNB, GCNT2, GDF 2, GFER, GJA 2, GMPPB, HCCS, INPP 52, JAG 2, KRT 2, LRP2, MAF, WRRP, MIR204, MPLKIP, MSMO 2, MT-TS2, MVK, MYH 2, MYOC, NF 2, NHS, NOD2, NTNR 2E 2, NTF 2, OAT, OPA 2, OTX2, OTP 2, TYPH 2, TBPIR 2, TFPI 2, TBPIR 2, TFN 2, TFS 2, TFN 2.
The mutation sites of the genes and their locations on the chromosome, as well as the locations on the chromosome corresponding to the probe arrays, are shown in table 3 below.
TABLE 3
Figure BDA0002839148120000142
Figure BDA0002839148120000151
Figure BDA0002839148120000161
In order to further simplify the gene chip of the invention, the cost performance of the probe cost and the detection rate are further considered, and the gene chip is optimized. In some embodiments, the gene chip includes probes for the following genes, which can detect 69% of cases of inherited anterior segment disease and inherited ocular syndrome: AGBL1, B3GLCT, CRYGD, FKRP, GSN, JAM3, MAN2B1, SIX6, VSX2, ZEB 1.
The mutation sites 1 to 24 of the above genes and their positions on the chromosome are shown in Table 4 below.
TABLE 4
Figure BDA0002839148120000171
The position on the chromosome corresponding to the probe columns corresponding to mutations 1-24 is shown in Table 5 below:
TABLE 5
Figure BDA0002839148120000172
Figure BDA0002839148120000181
Figure BDA0002839148120000191
Figure BDA0002839148120000201
Figure BDA0002839148120000211
According to a second aspect of the present invention, there is provided a kit for detecting hereditary anterior ocular segment disease and hereditary ocular syndrome, the kit comprising the gene chip of the first aspect of the present invention.
According to a third aspect of the present invention, there is provided a method of screening a subject for a genetic mutation using a gene chip, the method comprising:
1) extracting the genomic DNA of the subject, and breaking the genomic DNA into a range of 200-300 bp;
2) preparing a DNA fragment library from the fragmented genomic DNA;
3) hybridizing the DNA fragment library with the gene chip of the first aspect of the invention or the kit of the second aspect of the invention, and capturing genes;
4) amplifying the product captured in the step 3) by adopting PCR (polymerase chain reaction) and taking SEQ ID NO.69 and SEQ ID NO.70 as primers to obtain an amplification product;
5) performing on-machine sequencing on the amplification product obtained in the step 4) to obtain sequencing data of the gene;
6) comparing the sequencing data of step 5) with a human reference genome, thereby obtaining a single nucleotide polymorphism, insertion or deletion different from the reference genome, i.e., the detected gene mutation.
Specifically, preparation of a DNA fragment library was performed in step 3) using the Illumina TruSeq DNA library preparation kit.
The invention diagnoses and screens the molecular genes of the hereditary anterior ocular segment diseases and the hereditary ocular syndromes by designing the known pathogenic genes and the mutated probes aiming at the hereditary anterior ocular segment diseases and the hereditary ocular syndromes, and detects the known pathogenic genes by the target sequencing of the target region, thereby having the advantages of strong pertinence, high correctness, deep sequencing depth, lower cost and complete information of functional sites. Compared with whole genome sequencing, the method greatly saves the required sequencing data amount. In addition, most of mutations of disease-related pathogenic genes are detected at one time, so that the cost performance is balanced, and the guarantee is provided for treatment and intervention of diseases.
The present invention will be described in more detail with reference to the following examples, which are merely preferred embodiments of the present invention and are not intended to limit the present invention. All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Examples
Gene probes for the genes were prepared by commercial Gene chip Co.Ltd as gene chips, and the sequence of each gene probe was repeated three times. The design and testing of gene chips comprising all gene probes for these genes designed according to the genes listed in table 1 was performed by agutaikon (beijing) biotechnology limited.
1. Preparation of kit for detecting hereditary anterior segment disease and hereditary eye syndrome of subject
The kit comprises a DNA probe library for the hereditary anterior segment diseases and the hereditary ocular syndrome, which is prepared by the following method:
1) obtaining all coding sequences of the hereditary anterior segment diseases and the hereditary ocular syndromes in the table 1 by combining an Ensembl, CCDS, Gencode, VEGA, SNP and a CytoBand database according to a human reference genome HG 19;
2) aiming at each coding sequence, designing a probe sequence with the length of 100bp from the first base in the direction from 5 'to 3' according to the principle of reverse sequence complementation;
3) adding TAGGTGTGTAGGCGC (SEQ ID NO.65) and GTCAGCTAGTACGCA (SEQ ID NO.66) sequences to the 5 'end and the 3' end of each probe sequence respectively to form a probe sequence list with the same sequences at both ends;
4) adopting oligonucleotide in-situ synthesis technology to synthesize the sequences in the probe sequence list on a chip in a large scale;
5) washing the oligonucleotides on the chip with ammonia, dissolving in 100. mu.l of ultrapure water to form an oligonucleotide mixture;
6) the oligonucleotide mixture is amplified by a PCR method by adopting a forward primer (SEQ ID NO.67: TTAGATAGGTGTGTAGGCGC) with a biotin label at the 5 'end and a reverse primer (SEQ ID NO.68: TAAGGTGCGTACTAGCTGAC) with the same label at the 5' end to form a DNA probe library with the biotin label for the hereditary anterior segment disease and the hereditary ocular syndrome.
The reaction system is as follows:
name of reagent Volume of
KAPA 2G Buffer B 5× 10μl
dNTP(10mM each) 1μl
Forward primer (25. mu.M) 0.5μl
Reverse primer (25. mu.M) 0.5μl
Oligonucleotide mixture 5μl
KAPA 2G robust DNA Taq 0.8μl
H2O 32.2μl
The reaction conditions were as follows:
Figure BDA0002839148120000231
2. kit for screening mutation of hereditary anterior ocular segment disease and hereditary ocular syndrome
1) Taking 1 mu g of genome DNA of a human subject, and breaking the range to 200-300bp by adopting an ultrasonic disruptor;
2) preparing a DNA small fragment library by adopting an Illumina TruSeq DNA library preparation kit;
3) carrying out liquid phase hybridization on the DNA small fragment library and the prepared DNA probe library for the hereditary anterior ocular segment disease and the hereditary ocular syndrome to capture the hereditary anterior ocular segment disease and the hereditary ocular syndrome;
4) amplifying the captured product by adopting PCR (polymerase chain reaction) by taking Illumina PE PCR primer 1.0(SEQ ID NO.69: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and Illumina PE PCR primer 2.0(SEQ ID NO.70: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT) as primers to obtain a sequencing library;
the reaction system is as follows:
Figure BDA0002839148120000241
the reaction conditions were as follows:
Figure BDA0002839148120000242
5) performing on-machine sequencing on the sequencing library by adopting an Illumina high-throughput sequencer Hiseq 4000 to obtain sequencing data of the hereditary anterior ocular segment disease and the hereditary ocular syndrome;
6) sequencing data were aligned to the human reference genome HG19 using BWA MEM software using the parameters: the genome sequence of the genome sequence is bwa mem-M-k 40-t 8-R "@ RG \ tID: Hiseq \ tPL: Illumina \ tSM: sample", so as to obtain single nucleotide polymorphism, insertion or deletion different from that of a reference genome, namely, detected gene mutation.
The results showed that gene probes for the genes listed in table 1 and corresponding mutations in table 2 were designed, and 97% of cases of hereditary anterior ocular segment disease and hereditary ocular syndrome could be detected by a gene chip including the gene probes.
In order to simplify the gene chip, the cost performance of the probe cost and the detection rate are comprehensively considered, and the gene chip is optimized. The gene chip comprises gene probes aiming at the following genes: ADAMTS18, ALDH1A3, ALMS1, B3GALNT2, B3GLCT, BFSP1, BMP4, COL2A1, COL4A1, CRX, CRYAA, CRYBB3, DHCR7, ELP4, EPG5, ERCC2, ERCC8, FBN2, FKRP, FK, FLNB, GCNT2, GDF 2, GFER, GJA 2, GMPPB, HCCS, INPP 52, JAG 2, KRT 2, LRP2, MAF, WRRP, MIR204, MPLKIP, MSMO 2, MT-TS2, MVK, MYH 2, MYOC, NF 2, NHS, NOD2, NTNR 2E 2, NTF 2, OAT, OPA 2, OTX2, OTP 2, TYPH 2, TBPIR 2, TFPI 2, TBPIR 2, TFN 2, TFS 2, TFN 2. The result shows that the gene chip can detect 83% of hereditary anterior segment diseases and hereditary ocular syndrome cases.
In order to simplify the gene chip of the invention, the cost performance of the probe cost and the detection rate are further considered, and the gene chip is optimized. The gene chip comprises gene probes aiming at the following genes: AGBL1, B3GLCT, CRYGD, FKRP, GSN, JAM3, MAN2B1, SIX6, VSX2, ZEB 1. The result shows that the gene chip can detect 69% of cases of hereditary anterior segment diseases and hereditary ocular syndrome.
For the 24 mutations listed in Table 4, in order to achieve the goal that a minimum of 24 gene probes can effectively detect the mutations, the inventors tested the probes SEQ ID NO.1-64 that can be designed for these mutation sites and found that SEQ ID NO.1, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.20, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.34, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.43, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO.54, SEQ ID NO.57, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO.63 can maximally achieve a 64% differentiation between patients with inherited anterior ocular disease and inherited ocular syndrome and normal controls. All probes SEQ ID NO.1-64 can detect 69% of cases of inherited anterior segment diseases and inherited ocular syndromes.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent replacements, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A gene chip for detecting hereditary anterior ocular segment diseases and hereditary ocular syndromes, wherein the gene chip comprises gene probes specifically directed to genes shown in Table 1.
2. The gene chip of claim 1, wherein the gene chip comprises gene probes for: ADAMTS18, ALDH1A3, ALMS1, B3GALNT2, B3GLCT, BFSP1, BMP4, COL2A1, COL4A1, CRX, CRYAA, CRYBB3, DHCR7, ELP4, EPG5, ERCC2, ERCC8, FBN2, FKRP, FK, FLNB, GCNT2, GDF 2, GFER, GJA 2, GMPPB, HCCS, INPP 52, JAG 2, KRT 2, LRP2, MAF, WRRP, MIR204, MPLKIP, MSMO 2, MT-TS2, MVK, MYH 2, MYOC, NF 2, NHS, NOD2, NTNR 2E 2, NTF 2, OAT, OPA 2, OTX2, OTP 2, TYPH 2, TBPIR 2, TFPI 2, TBPIR 2, TFN 2, TFS 2, TFN 2.
3. The gene chip of claim 1, wherein the gene chip comprises gene probes for: AGBL1, B3GLCT, CRYGD, FKRP, GSN, JAM3, MAN2B1, SIX6, VSX2, ZEB 1.
4. The gene chip of claim 3, wherein the AGBL1 mutation sites are: c.3220C > T, c.3107G > C; the mutation sites of B3GLCT are: c.347+5G > A, c.459+1G > A, c.660+1G > A, c.1178G > A; the mutation sites of CRYGD are: c.497C > T, c.470G > A; the mutation sites of FKRP are: c.1A > G, c.826C > A; the mutation sites of GSN are: c.520G > A; mutation sites of JAM3 are: c.2T > G, c.346G > A, c.612+1G > T, c.656G > A; the mutation sites of MAN2B1 were: c.2248C > T, c.1830+1G > C, c.1383C > A; the mutation sites of SIX6 are: c.385G > A, c.421C > A; the mutation sites of VSX2 are: c.244G > A, c.299C > A and c.871G > A; the mutation sites of ZEB1 are as follows: c.973C > T.
5. The gene chip of claim 3, wherein the gene chip comprises 1-4 probes selected from the following groups: SEQ ID NO.1-SEQ ID NO.3, SEQ ID NO.4-SEQ ID NO.7, SEQ ID NO.8-SEQ ID NO.9, SEQ ID NO.10-SEQ ID NO.12, SEQ ID NO.13-SEQ ID NO.15, SEQ ID NO.16-SEQ ID NO.17, SEQ ID NO.18-SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22-SEQ ID NO.23, SEQ ID NO.24-SEQ ID NO.26, SEQ ID NO.27-SEQ ID NO.29, SEQ ID NO.30-SEQ ID NO.32, SEQ ID NO.33-SEQ ID NO.35, SEQ ID NO.36-SEQ ID NO.38, SEQ ID NO.39-SEQ ID NO.41, SEQ ID NO.42-SEQ ID NO.44, SEQ ID NO.45-SEQ ID NO.46, SEQ ID NO.47-SEQ ID NO.49, SEQ ID NO.50-SEQ ID NO.52, SEQ ID NO.53, SEQ ID NO.54 to SEQ ID NO.58, SEQ ID NO.59 to SEQ ID NO.61, SEQ ID NO.62 to SEQ ID NO. 64.
6. The gene chip of claim 5, wherein the gene chip comprises the following probes: SEQ ID NO.1, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.20, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.34, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.43, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO.54, SEQ ID NO.57, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO. 63.
7. The gene chip according to any one of claims 1 to 6, wherein the gene chip further comprises a sequence obtained by replacing the site of the gene probe with a mutant base.
8. A kit for detecting hereditary anterior ocular segment disease and hereditary ocular syndrome in a subject, wherein the kit comprises the gene chip of any one of claims 1 to 7.
9. A method for screening a subject for a gene mutation using a gene chip, wherein the method comprises:
1) extracting the genomic DNA of the subject, and breaking the genomic DNA into a range of 200-300 bp;
2) preparing a DNA fragment library from the fragmented genomic DNA;
3) hybridizing the DNA fragment library with the gene chip of any one of claims 1 to 7 or the kit of claim 8, and capturing the gene;
4) amplifying the product captured in the step 3) by adopting PCR (polymerase chain reaction) and taking SEQ ID NO.69 and SEQ ID NO.70 as primers to obtain an amplification product;
5) performing on-machine sequencing on the amplification product obtained in the step 4) to obtain sequencing data of the gene; and
6) comparing the sequencing data of step 5) with a human reference genome, thereby obtaining a single nucleotide polymorphism, insertion or deletion different from the reference genome, i.e., the detected gene mutation.
10. The method according to claim 9, wherein the preparation of the DNA small fragment library is performed in step 2) using the illumina TruSeq DNA library preparation kit.
CN202011485199.3A 2020-12-16 2020-12-16 Gene chip, kit comprising gene chip and application of gene chip Pending CN112626193A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011485199.3A CN112626193A (en) 2020-12-16 2020-12-16 Gene chip, kit comprising gene chip and application of gene chip

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011485199.3A CN112626193A (en) 2020-12-16 2020-12-16 Gene chip, kit comprising gene chip and application of gene chip

Publications (1)

Publication Number Publication Date
CN112626193A true CN112626193A (en) 2021-04-09

Family

ID=75313658

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011485199.3A Pending CN112626193A (en) 2020-12-16 2020-12-16 Gene chip, kit comprising gene chip and application of gene chip

Country Status (1)

Country Link
CN (1) CN112626193A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116732026A (en) * 2023-03-29 2023-09-12 内蒙古农业大学 Development method and application of oat SNP breeding chip

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018204764A1 (en) * 2017-05-05 2018-11-08 Camp4 Therapeutics Corporation Identification and targeted modulation of gene signaling networks
US20190192691A1 (en) * 2016-04-11 2019-06-27 Obsidian Therapeutics, Inc. Regulated biocircuit systems

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190192691A1 (en) * 2016-04-11 2019-06-27 Obsidian Therapeutics, Inc. Regulated biocircuit systems
WO2018204764A1 (en) * 2017-05-05 2018-11-08 Camp4 Therapeutics Corporation Identification and targeted modulation of gene signaling networks

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116732026A (en) * 2023-03-29 2023-09-12 内蒙古农业大学 Development method and application of oat SNP breeding chip
CN116732026B (en) * 2023-03-29 2024-05-28 内蒙古农业大学 Development method and application of oat SNP breeding chip

Similar Documents

Publication Publication Date Title
US20230203573A1 (en) Methods for detection of donor-derived cell-free dna
CN108642160B (en) Method and kit for detecting fetal thalassemia pathogenic gene
CN104561016B (en) New mutation of congenital cataract PITX3 gene
CN110904128A (en) Nucleic acid for encoding TEK gene mutant and application thereof
WO2012125848A2 (en) A method for comprehensive sequence analysis using deep sequencing technology
CN102206701A (en) Identification method for genetic disease-related gene
CN109402132B (en) Nucleic acid for coding SCN1A gene mutant and application thereof
CN104212806A (en) New mutant disease-causing gene of Alport syndrome, encoded protein and application thereof
CN106701925B (en) Cataract pathogenic gene detection kit and detection method thereof
CN103571847A (en) FOXC1 gene mutant and its application
AU2016351311B2 (en) SCAP gene mutant and the application thereof
CN106282369A (en) A kind of probe groups for detecting congenital cataract related gene and test kit
CN112626193A (en) Gene chip, kit comprising gene chip and application of gene chip
CN110551728A (en) FOXC1 gene mutant and application thereof
CN110734974B (en) SNP locus combination and detection primer for cancer chemotherapy drugs
CN111304226A (en) Nucleic acid for coding CYP1B1 gene mutant and application thereof
CN105838720B (en) PTPRQ gene mutation body and its application
CN112442528B (en) LOXHD1 gene mutant and application thereof
CN108504660A (en) ADCY10 gene mutation bodies and its application
CN108531580A (en) C5orf42 gene mutation bodies and its application
CN112626192A (en) Gene chip, kit comprising gene chip and application of gene chip
CN114480411A (en) COL4A5 pathogenic mutant gene of Alport syndrome patient and detection reagent thereof
CN110993024B (en) Method and device for establishing fetal concentration correction model and method and device for quantifying fetal concentration
CN104789572B (en) GPRASP2 mutated genes, its authentication method and detection kit
CN112442503A (en) KCNQ1 gene mutant and application thereof

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