CN1264989C - Gene macrofragment, single-copy deficiency detection chip and use thereof - Google Patents
Gene macrofragment, single-copy deficiency detection chip and use thereof Download PDFInfo
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- CN1264989C CN1264989C CN 03131741 CN03131741A CN1264989C CN 1264989 C CN1264989 C CN 1264989C CN 03131741 CN03131741 CN 03131741 CN 03131741 A CN03131741 A CN 03131741A CN 1264989 C CN1264989 C CN 1264989C
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Abstract
The present invention discloses a detection chip and an application thereof for gene Klenow fragments and single-copy deletion, which solves the defects of difficulty in clinic conventional detection on gene Klenow fragment deletion of hereditary diseases due to the limitation of technical methods or sample flows, and low detectable rate of related genes in the prior art, and provides a detection chip and a detection method with high sample energy and high gene detectable rate for gene Klenow fragments and single copy deletion. The present invention is characterized in that the detection method comprises the following steps: selecting a detected gene: using a gene exon as a unit, according to the gene genome DNA structure, selecting a sequence with an approximate Tm value length of 30 to 50 basic groups in the exon and synthesizing a probe; fixing a probe sample application on a slide and making the distribution of the probe meet the statistic requirements; acidizing a DNA sample to be tested and preparing a DNA fragment with a certain size; adding a primer in the DNA sample to be tested, making low amplification and labeling the sample with fluorescence; hybridizing the DNA sample to be tested with the chip and analyzing fluorescence signals of hybridization probe points with special software to determine whether the target gene of the DNA to be tested has Klenow fragment deletion.
Description
Technical field
The present invention relates to a kind of detection chip and application thereof, more specifically to the big fragment of a kind of gene, single copy disappearance detection chip and application thereof.
Background technology
The problem of the work of puzzlement gene diagnosis at present has the recall rate of 1. some heredopathia pathogenesis related genes sudden change not high, 2. for the recessive inheritance disease, account for the detecting of mutator gene carrier of more ratios among the crowd, its reason may mainly contain two aspects: 1. whether the analytical technology of Cai Yonging can be finished 2. the analysis of all kinds transgenation is finished contradiction between the sample flux of full gene screening that diagnosis need accomplish and existing analytical technology.
Complete mutator gene analytical system should comprise the analytical technology that can effectively detect all kinds of sudden changes, current research shows, it is unusual to go back the another kind of gene structure of ubiquity between heredity chromosome abnormalty and gene micromutation, i.e. the large fragment deletion of DNA (Deletion) and duplicate (Duplication).The big fragment structure variation of this class DNA (sudden change) accounts for 1/3 of transgenation, and shows as the unusual (diseased individuals of dominant hereditary disease of single copy in cell more; The mutator gene carrier of recessive hereditary disease).Detect " having " and " nothing " general easy discriminating of sequence in the genetic analysis, what be difficult to differentiate is the variation of the big fragment structure of DNA allelotrope copy number when unusual, and particularly allelic single copy lacks or single copy duplicates.We have developed quantitative multiple PCR technique for this reason, and this technology can effectively detect the big fragment structure variation of genomic dna, but are limited to its sample flux, and the analysis of large sample is difficult to finish in a short time.
In recent years rise be based upon biochip technology on the molecular hybridization principle be a kind of height parallel, on a large scale, bioinformation detection scheme fast, but existing gene chip product is applied to the single of gene more or several bases change detection and expression of gene analysis etc., and the big fragment variation that can't carry out gene detects.Developing and improving the inspiration that obtains in the quantitative multiple PCR technique, in conjunction with chip technology, the notion of introducing amount in design and detection is developed the big fragment structure variation of genomic dna detection chip according to us, and the big fragment that is used for the examination disease related gene makes a variation.
Summary of the invention
The invention solves and be subjected to the restriction of technological method or sample flux in the prior art and be difficult to the problem that the routine clinical gene large fragment deletion of carrying out heredopathia detects, a kind of sample energy height, the big fragment of gene that the gene recall rate is high, single copy disappearance detection chip, preparation method and the application in gene diagnosis thereof are provided, and the present invention can improve genes involved and recessive inheritance disease carrier's recall rate greatly.
Technical scheme of the present invention is as follows:
The big fragment of gene of the present invention, single copy disappearance detection chip include slide glass and probe, it is characterized in that:
1) for the heredopathia that single candidate gene is only arranged, described probe comprises the detection gene that is no less than 3, one of them is a Disease-causing gene, all the other are reference gene, and be unit with the gene extron, according to the structural dna sequence of gene, by having close Tm value in the exon, length is the dna sequence dna synthetic of 30-50 base;
Perhaps
2) for having a plurality of candidate gene heredopathias, described probe comprises and is no less than 3 disease related gene, and is unit with the gene extron, according to the structural dna sequence of gene, by having close Tm value in the exon, length is the dna sequence dna synthetic of 30-50 base.
Described reference gene is the conservative dna fragmentation of human genome inner structure sequence; The distribution of described probe on chip is to be arranged in order in proper order with its pairing exon, wherein comprised all exons of Disease-causing gene or disease related gene, and each probe repeats to be no less than 5 times.
The big fragment of gene of the present invention, the application of single copy disappearance detection chip in the gene genetic large fragment deletion detects.
The preparation method of said chip comprises the steps:
(1) modification of slide glass;
(2) probe design, synthetic and modification
(3) probe is fixing
The principal element of the DNA large fragment deletion detection chip performance that influence prepares
1. the processing of chip carrier-slide: can the probe that surface of glass slide chemically modified situation is directly connected to a cloth effectively be incorporated into surface of glass slide by the molecule arm, makes the probe binding capacity of each point stable.
2. probe design: according to each exon sequence of testing goal gene,, adjust probe length, make each probe hybridization condition close, to guarantee the stable of follow-up chip hybridization signal by selecting (A+T)/(G+C) value in the probe.
3. the reference gene probe is selected on the chip: the conserved sequence of the single copy of gene Selection; The probe sequence design makes its hybridization conditions close with the testing goal gene probe.
4. each distribution of probe and multiplicity on the chip: determine the multiplicity of each probe according to the hybrid stability of each probe, make the hybridization signal intensity comprehensive treating process of this probe points after the sample copy number become positive correlation.
Utilizing DNA large fragment deletion detection chip to treat the condition that the sample product analyze can be optimized in the following areas:
1. the adjustment in cracking dna sample pH value to be checked (4.0-4.5) and treatment time thereof, the dna fragmentation size after the assurance cracking is in the 500-1000 base.
2. the minuent of dna sample amplification, by adjusting DNA concentration, Oligonucleolide primers amount and DNA synthetic cycle index, under the prerequisite that the original ratio of each gene fragment copy number is constant in guaranteeing dna sample, the increase of each gene fragment copy number can make the hybridization of chip obtain clear and definite signal.
3. chip hybridization temperature, the ionic strength of post-hybridization washing damping fluid and rinsing time, remove the hybrid context signal as much as possible, guarantee the clear of hybridization signal, each probe hybridization signal synthesis is handled after, its intensity becomes positive correlation with the number of probes that forms hybridization.
The invention has the beneficial effects as follows:
Along with going deep into of functional genome research, more and more Duo disease related gene obtains separating, and it is important that molecular medicine effect clinically more shows.At present, gene diagnosis and genetic counseling is progressively in clinical expansion, the gene diagnosis before heredopathia antenatal, sick, the control of genetic correlation disease, early diagnosis of tumor, work such as the individual examination of mutator gene carrier and high-risk morbidity more obtain paying attention to.In this process, efficiently, the all kinds heredity sudden change that intactly detects pathogenesis related genes is the key of carrying out above-mentioned work, we introduce quantitative analysis in high-throughout chip technology, the design and the arranged distribution of probe on chip of probe parameter have been improved, this detection in Gene Mutation chip at the big fragment structure Exception Type of ubiquitous DNA of development, the perfect analytical system of mutator gene will produce good society and economic benefit.
Embodiment
Embodiment 1: the preparation of hereditary nonpolyposis large bowel cancer (HNPCC) pathogenesis related genes large fragment deletion detection chip
1) cleaning of slide and surface chemical modification: according to the quantity of institute's point probe, select the slide of suitable dimension, 1: 1 methanolic hydrochloric acid solution cleans, and distilled water is cleaned, and the vitriol oil soaks, and 95% ethanol fully washes, dry air.95% ethanol and silane become silylating reagent by 49: 1 proportional arrangement, the slide immersion that is placed in one, and distilled water is cleaned, and the vertically-arranged slide from up to down dries up, 110 ℃ of bakings.95% ethanol or Virahol soak, and distilled water is cleaned and dried up, immersions that be placed in one of the glutaraldehyde solution of the PBS solution allocation 5% of Ph7.0,0.01M, slide, and the PBS solution of 0.01M cleans, and distilled water is clean, dries up.Scanner scanning selects the more weak slide of fluorescence background as pretreated slide, is used for next step experiment.
2) probe is synthetic: sequence is selected from 45 exons of MLH1, MSH2, MSH6 gene, and probe length 30-50 base has close Tm and plant, and 3 ' end is modified in amination.
3) probe fixing and distributing: carbonate buffer solution dilution probe, under the atomization condition,, equidistantly to put successively on pretreated slide by the order of its pairing exon, the point sample repeat number of each probe is no less than 5 on every chip.Slide places enclosed environment, and room temperature is placed and to be gone in the moist cell 37 ℃ of water-baths two hours after one hour.The 0.1%SDS aqueous solution embathes, and distilled water is cleaned and dried up.Slide places sodium borohydride reduction liquid to soak, and the aldehyde radical on surface is reduced to hydroxyl.
Embodiment 2: the application of the chip among the embodiment 1 in hereditary nonpolyposis large bowel cancer (HNPCC) pathogenesis related genes heredity large fragment deletion is analyzed and detected
1) preparation of nucleic acid samples: extract suspicious patient's peripheric venous blood DNA, acidification, the crack DNA molecule is diluted to 100ng/ul (sample to be checked) to the 500-1000 base.Low 8 the cyclic amplification samples to be checked of random priming participate in fluorescent substance in amplification procedure, finish the mark of sample DNA.
2) hybridization: 95 ℃ of denaturing treatment of fluorescently-labeled sample DNA, under the specified temp, 4 * SSC+1%BSA prehybridization, hybridization.Under the room temperature, 2 * SSC+0.1%SDS, the rinsing of 0.1 * SSC+0.1%SDS lucifuge, distilled water is cleaned and is dried up.
3) hybridization signal detects: hybridization signal is collected in the scanning of fluorescent scanning instrument, finishes the digitizing output of strength of signal simultaneously.
4) analysis of hybridization signal and result judge: at first with the known positive contrast of different positive sample with the big fragment structure variation of gene DNA, with the stable standard that detects this sample list copy variation as system stability, data to output are carried out quantitative analysis, judge whether to exist big pulsating variation of gene and position thereof according to the numerical value and the pairing exon sequence number of quantitative analysis.
Embodiment 3: the preparation of duchenne muscular dystrophy pathogenesis related genes large fragment deletion detection chip
Probe synthetic: sequence is selected from 3 exons of 79 exons, reference gene β-actin gene of DMD Disease-causing gene and 3 exons of reference gene GAPDP gene, reference gene GAPDP brings to the dna sequence dna that the segment structure between 3 bands (8q2.1-8q2.3) is guarded for being positioned at long-armed 2 districts 1 of No. 8 karyomit(e) of human genome, probe length 30-50 base, have close Tm and plant, 3 ' end is modified in amination.
All the other method stepss are with embodiment 1
Embodiment 4: the chip among the embodiment 3 is analyzed and the application that detects at duchenne muscular dystrophy pathogenesis related genes large fragment deletion
1) preparation of nucleic acid samples: extract suspicious disease gene carrier peripheric venous blood DNA, acidification, the crack DNA molecule is diluted to 100ng/ul (sample to be checked) to the 500-1000 base.Low 9 the cyclic amplification samples to be checked of random priming participate in fluorescent substance in amplification procedure, finish the mark of sample DNA.
All the other method stepss are with embodiment 2
Embodiment 5: the preparation of pku genes involved large fragment deletion detection chip
Probe synthetic: sequence is selected from 3 exons of 13 exons, reference gene β-actin gene of PAH gene and 3 exons of reference gene GAPDP gene, reference gene GAPDP brings to the dna sequence dna that the segment structure between 3 bands (8q2.1-8q2.3) is guarded for being positioned at long-armed 2 districts 1 of No. 8 karyomit(e) of human genome, probe length 30-50 base, have close Tm and plant, 3 ' end is modified in amination.
All the other method stepss are with embodiment 1
Embodiment 6: the application of the chip among the embodiment 5 in pku genes involved large fragment deletion is analyzed and detected
1) preparation of nucleic acid samples: extract suspicious patient's peripheric venous blood DNA, acidification, the crack DNA molecule is diluted to 100ng/ul (sample to be checked) to the 500-1000 base.Low 10 the cyclic amplification samples to be checked of random priming participate in fluorescent substance in amplification procedure, finish the mark of sample DNA.
All the other method stepss are with embodiment 2.
Claims (4)
1, the big fragment of a kind of gene, single copy disappearance detection chip include slide glass and probe, it is characterized in that:
1) for the heredopathia that single candidate gene is only arranged, described probe comprises the detection gene that is no less than 3, one of them is a Disease-causing gene, all the other are reference gene, and be unit with the gene extron, according to the structural dna sequence of gene, by having close Tm value in the exon, length is the dna sequence dna synthetic of 30-50 base;
Perhaps
2) for heredopathia with a plurality of candidate genes, described probe comprises and is no less than 3 disease related gene, and is unit with the gene extron, according to the structural dna sequence of gene, by having close Tm value in the exon, length is the dna sequence dna synthetic of 30-50 base.
2, the big fragment of gene according to claim 1, single copy disappearance detection chip is characterized in that described reference gene is the conservative dna fragmentation of human genome inner structure sequence.
3, the big fragment of gene according to claim 1, single copy disappearance detection chip, it is characterized in that the distribution of described probe on chip is to be arranged in order in proper order with its pairing exon, all exons that wherein comprised Disease-causing gene or disease related gene, and each probe repeats to be no less than 5 times.
4, claim 1, the application of 2 or 3 described chips in the gene genetic large fragment deletion detects.
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| CN 03131741 CN1264989C (en) | 2003-07-25 | 2003-07-25 | Gene macrofragment, single-copy deficiency detection chip and use thereof |
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| CN 03131741 CN1264989C (en) | 2003-07-25 | 2003-07-25 | Gene macrofragment, single-copy deficiency detection chip and use thereof |
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| CN1483837A CN1483837A (en) | 2004-03-24 |
| CN1264989C true CN1264989C (en) | 2006-07-19 |
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| CN100393885C (en) * | 2003-11-21 | 2008-06-11 | 中国医学科学院肿瘤医院肿瘤研究所 | Chip for fast detecting full genome range multigene variation |
| CN110129419B (en) * | 2018-12-18 | 2023-03-31 | 华联生物科技股份有限公司 | Method for detecting copy number variation |
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