CN102363806A - Gene chip for forecasting curative effect of maze surgery on atrial fibrillation - Google Patents
Gene chip for forecasting curative effect of maze surgery on atrial fibrillation Download PDFInfo
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Abstract
The invention discloses a gene chip for forecasting a curative effect of a maze surgery on atrial fibrillation. The gene chip comprises an oligonucleotide probe designed according to an rs2200733 site, an rs10033464 site, an rs13376333 site, an rs6843082 site, an rs17042171 site, an rs2106261 site and an rs7193343 site and a specific primer capable of specifically amplifying DNA segments comprising the single nucleotide polymorphism (SNP) sites, wherein the 5' end of the specific primer is marked by a signal substance in advance. The gene chip can detect a plurality of SNP sites obviously related to atrial fibrillation, can screen the common people quickly and conveniently, is high in accuracy and low in cost, can be applied to batch detection, and can completely meet the large-scale gene screening requirement.
Description
Technical field
The present invention relates to a kind of gene chip, specifically is a kind of gene chip that is used to predict maze operation treatment atrial fibrillation curative effect.
Background technology
Atrial fibrillation (atrial fibrillation) is called for short " atrial fibrillation ", and it is a kind of common irregular pulse, and Chang Bingfa is in rheumatic heart disease, coronary heart disease and congenital heart disease.Atrial fibrillation can inspire heart failure or haemodynamics obstacle; Fatal arrhythmia such as bring out to quiver in chamber speed or chamber; Cause thrombus and embolism incident; Increase basic cardiopathic mortality ratio, make five times of the danger increases of apoplexy generation, four times of danger raisings in heart failure, dead or dull-witted danger raising twice, atrial fibrillation itself can also cause cardiac dilatation.
According to the definition of the World Health Organization, the symptom of atrial fibrillation shows as cardiac muscle and has lost the activity of contracting of normally clocklike relaxing, and replaces quick and inharmonic faint wriggling; Cause the atrium to lose normal effectively contraction; Cause irregular, the rambling electrical activity in atrium, atrial tachycardia, the P ripple disappears on the electrocardiogram(ECG; Replace little f ripple; Frequency about 350-600 time/minute, the f wave that promptly shape, size, direction and time-histories differ on the baseline, Ventricular Rate does not wait fully under not height or dissociation situation.
Divide by the time; Atrial fibrillation is divided into acute AF and chronic atrial fibrillation; Chronic atrial fibrillation is divided into paroxysmal, persistence and permanent atrial fibrillation again; Paroxysmal, the terminated atrial fibrillation is the paroxysmal atrial fibrillation voluntarily, and atrial fibrillation continues can not stop voluntarily after above, the outbreak of three weeks but after treatment, can the terminated atrial fibrillation be the persistence atrial fibrillation, after treating, can not the terminated atrial fibrillation be permanent atrial fibrillation.
Whether by with other cardiovascular disordeies and send division, atrial fibrillation can be divided into isolatism atrial fibrillation and typicalness atrial fibrillation, and the isolatism atrial fibrillation refers to not have the atrial fibrillation on clear and definite heart change basis, promptly without the atrial fibrillation of related cardiovascular disease; The typicalness atrial fibrillation refers to the atrial fibrillation with related cardiovascular disease, and the cardiovascular underlying diseases relevant with atrial fibrillation comprises valvular heart disease (being mitral valve property mostly), coronary heart disease, hypertension, ischemic heart disease and congestive heart failure etc.
Atrial fibrillation has become a kind of new prevailing disease in the worldwide; According to statistics, U.S.'s atrial fibrillation sickness rate is 0.4%-0.9%, with advancing age; Sickness rate sharply increases; The over-65s person then up to 3%-5%, and Europe the atrial fibrillation sickness rate and the U.S. similar, the atrial fibrillation sickness rate in Asia is compared lower slightly with the former two.However, also existing 1,000 ten thousand patients with atrial fibrillation of China, and its sickness rate fast rise, and the trend of oriented youngster transfer.Atrial fibrillation is seriously endangering human beings'health, has also brought suitable economical load to society.
Therefore prevention, diagnosis and treatment atrial fibrillation become needs the urgent important medical problem that solves.The purpose of atrial fibrillation therapy is to recover heart normal rhythm property to beat, or controls rational ventricular rates, prevents the appearance of severe complications such as apoplexy etc.
Over nearly 20 years, many scholars attempt curing atrial fibrillation through surgical operation, have obtained remarkable progress, and are wherein, best with the maze operation effect.The american heart doctor invented maze operation in 1987, by well-designed circuit in the right atrium, interatrial septum and left atrium along the linear many places of cutting of annular, unite by a seam respectively again; Form a narrow and crooked atrial tissue passage, a promptly artificial labyrinth, prevention is turned back; The abnormal conduction loop that will cause atrial fibrillation interrupts; Atrial fibrillation can not be taken place, but can pass to atrioventricular node from sinus node by pilot pulse, thereby cure atrial fibrillation.Along with the development of various new technologies is perfect (for example according to the maze operation principle; In the atrium, use RF ablation operative treatment atrial fibrillation), maze operation constantly obtains simplifying and improving, and its complication is few; Curative effect improves, and maze operation treatment atrial fibrillation has become the relatively more definite and reliable a kind of method of curative effect.
However, the research of following up a case by regular visits to shows, 15% the patient's easy relapse of still having an appointment after the maze operation treatment.The postoperative recurrence reason of atrial fibrillation obviously is many-sided, like hypertension, and mellitus, heart failure, heart stalk, obesity and vascular conditions etc.
Present research can't be made nosetiology elaboration clearly to the generator of atrial fibrillation, and it is the synthetic disease of Different types of etiopathogenises, comprises cardiovascular disorder and other risk factors.Acute with some, the temporary reason of atrial fibrillation is relevant, comprise drink, surgical operation, electric shock, myocarditis, pulmonary infarction, other lungs disease and hyperthyroidism, atrial fibrillation also is a more common early complication behind myocardial infarction and the cardiac thoracic surgery.
The isolatism atrial fibrillation does not have obvious structural heart disease; The youngster also can suffer from the isolatism atrial fibrillation; The patient of isolatism atrial fibrillation compares with the aged patient of typicalness atrial fibrillation; More possibly fall ill, it is the prominent feature of isolatism atrial fibrillation that familial form is assembled with early falling ill suddenly, and nearly 30% the isolatism atrial fibrillation origin person of family has at least one slightly to suffer from the relatives of this disease.
That the risk factors of typicalness atrial fibrillation comprise is old, fat, hypertension, heart failure, valvular heart disease, hyperthyroidism and h disease, and the individuality that has relatives to suffer from this disease also has the ill risk of higher atrial fibrillation.
Increasing the weight of of a large amount of evidence prompting atrial fibrillation state of an illness is the structural remodeling of continuous accumulation in the atrium and the result of electric physiology reconstruct, and in a single day atrial fibrillation takes place, and the left atrium is just developed down by histology reconstruct, electric reconstruct rule, even if the cause of disease is removed, atrial fibrillation still can take place.We can say that it is fatefulue, is necessary so carry out the prevention of atrial fibrillation.
Nearest research provides the strong evidence of heredity factor to the atrial fibrillation influence, and it possibly be that the relation of gene maybe be more close with patient's self physique that generation and the treatment that has shown atrial fibrillation lapses to.According to difference between individuals judge maze operation treatment atrial fibrillation curative effect, select suitable operation plan and effectively prevent its recurrence, be major issue to be solved in the atrial fibrillation therapy.
Because the direct cause of disease of atrial fibrillation is not clear yet till now; The immediate cause that causes the atrial fibrillation recurrence after the maze operation is also unclear; Therefore prevent the atrial fibrillation recurrence to have certain difficulty; Be that people can't know that the factor of which aspect of postoperative causes the recurrence of atrial fibrillation, also just can't prevent its generation or block a certain link of its recurrence.However, through various research meanses and clinical trial, people still find some with maze operation after the relevant genetics factor of atrial fibrillation prognosis.
People explored atrial fibrillation Disease-causing gene and with the SNP site of atrial fibrillation significant correlation; But report in the past often concentrates on one or a few gene and SNP site; And the postoperative recurrence of atrial fibrillation is the complex process by multifactor adjusting, and research previously is difficult to reflect the whole essence of complex recurrence process.
If simultaneously the SNP site of multiple tumor susceptibility gene and a plurality of and atrial fibrillation significant correlation is analyzed, then help to illustrate between the various tumor susceptibility genes interaction mechanism and to the influence of atrial fibrillation postoperative recurrence.Still there are not at present enough special, responsive atrial fibrillation recurrence prediction index and predictive model to asian population, domestic and international also not to the application of polygene and a plurality of related SNP site estimation chips of atrial fibrillation postoperative recurrence.
Summary of the invention
Technical problem to be solved by this invention is the defective that overcomes prior art; A kind of gene chip that is used to predict maze operation treatment atrial fibrillation curative effect is provided, and this gene chip can detect a plurality of and the SNP site atrial fibrillation significant correlation, easily the general population is carried out examination rapidly; Accuracy rate is high; Cost is lower, can be used for batch detection, can fully satisfy the requirement of extensive genescreen.
For solving the problems of the technologies described above; The gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of the present invention; It comprises the oligonucleotide probe that designs to rs2200733 site, rs10033464 site, rs13376333 site, rs6843082 site, rs17042171 site, rs2106261 site and rs7193343 site and can specific amplification goes out to comprise the Auele Specific Primer of the dna fragmentation in above-mentioned SNP site that 5 ' end of said Auele Specific Primer all carries out mark with semiochemicals in advance.
As preferably, said oligonucleotide probe has the nucleotide sequence shown in the SEQ ID NO.1-NO.56.
As preferably, said Auele Specific Primer has the nucleotide sequence shown in the SEQ ID NO.57-NO.70.
As preferably, 5 ' end of said Auele Specific Primer all carries out mark with resorcinolphthalein, isotropic substance, vitamin H or digoxin in advance.
As preferably; Said gene chip also comprises solid phase carrier; Said solid phase carrier is process surface chemistry group modified glass matrix or nylon membrane; The end of said oligonucleotide probe has modifies amino, and the chemical group of said solid phase carrier can combine with the modification amino covalence of oligonucleotide probe.
As improvement, said gene chip also comprises a negative control sample and a positive control.
As improvement, said gene chip also comprises hybridizing box and hybridization solution.
As further improvement, said gene chip also comprises DNA sampling reagent, pcr amplification reagent and PCR product purification reagent.
The gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of the present invention, its preparation method is:
(1) uses oligonucleotide synthesizer to prepare said oligonucleotide probe and Auele Specific Primer, 5 ' of Auele Specific Primer is held all carried out mark in advance with semiochemicals;
(2) use gene chip sample applying appearance built in through on the aldehyde group modified solid phase carrier, makes gene chip with said oligonucleotide probe point after the drying.
The gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of the present invention, its method of use is:
(1) use said DNA sampling reagent to extract patient DNA;
(2) use the amplification of said Auele Specific Primer and said pcr amplification reagent to comprise the dna fragmentation in target SNP site;
(3) each pcr amplification product with same sample mixes, and adds behind the hybridization solution and said chip hybridization;
(4) use the scanner scanning hybridization signal.
The present invention has chosen 7 SNP sites of full genome association study (6WAS) and atrial fibrillation significant correlation.
Karyomit(e) 4q25 locus is a locus that is closely related with atrial fibrillation, and rs2200733 site, rs10033464 site, rs6843082 site and rs17042171 site are all in this locus.
Wherein rs2200733 site, rs10033464 site are positioned at the single linkage disequilibrium zone of karyomit(e) 4q25, young patient with suffer among the patient of isolatism atrial fibrillation, the dependency of above-mentioned two SNP sites and atrial fibrillation is particularly remarkable.There is not known in this linkage disequilibrium zone, but comprises EST (DA725631) and two independent exon ESTs (DB324364 and AF017091) of a montage.Polymerase chain reaction to the complementary DNA library of taking from various tissues carries out the reversed transcriptive enzyme mediation does not detect the expression of these ESTs (EST).The PITX2 gene that is positioned at the upper reaches of adjoining in this linkage disequilibrium zone is and these two nearest genes in risk sudden change SNP site.The PITX2 gene plays an important role in heart development through the generation of mediation heart asymmetric configuration; Its encoded protein is paired type homologous region transcription factor 2 (being paired-liked homeodomain transcription factor 2); Adjustable myocardial cell, cardiac nerve protoblast and pharynx bow mesenchymal cell, its transgenation can cause that the signal conduction of the heart left and right sides changes.
Rs6843082 site and atrial fibrillation are closely related the most, and the rs17042171 site also has significant correlation property with atrial fibrillation apart from the about 150kb telomere of transcription factor gene PITX2.
The rs13376333 site is positioned on the karyomit(e) 1q21, and the dependency of it and isolatism atrial fibrillation is particularly remarkable, and is also relevant with the typicalness atrial fibrillation, though the dependency undertighten.Rs13376333 on the intron between KCNN3 gene first and second exons, it not with the KCNN3 gene on any known common non-synonym SNP site linkage disequilibrium.The KCNN3 genes encoding participate in the potassium channel protein of atrial repolarization; This albumen belongs to weak conduction calcium activated potassium channel protein family; The maximum characteristics of this albumen are the electric discharge types that influence excitable cell, for example in some neurones, influence the phase type discharge, and influence the successive type discharge at other neurones; Thereby this albumen in many excitable tissures by being expressed, comprise brain and heart.
Rs2106261 site and rs7193343 site all are positioned at ZFHX3 gene on the karyomit(e) 16q22, and (zinc refers to homoeobox gene; Also claim AT mode binding silver; Be ATBF1) in, these two sites are the intron SNP site of ZFHX3 gene, all are closely related with isolatism atrial fibrillation and typicalness atrial fibrillation.The transcription factor of a kind of Atbf1 by name of ZFHX3 genes encoding, this albumen are that maximum known dna is conjugated protein, and be relevant with the adjusting of the growth of some tissues and differentiation, is to regulate muscle-derived to break up with nervosa specifically.ZFHX3 is expressed in many tissues, for example heart, liver, lung, kidney, pituitary body and brain.
Atbf1 is POU class 1 homology frame 1 (POU1F1) gene early transcription activated desirable proteins, and the POU1F1 gene is a member of regulating the POU-homeodomain transcription factor family of mammalian pituitary cytodifferentiation and hormone expression.POU1F1 and PITX2 gene interaction; Promote DNA to combine and transcriptional activity; Interaction between these two genes is significant; Because the sudden change of the atrial fibrillation on fixed before karyomit(e) 4q25 SNP site is positioned at the position of adjoining with PITX2, and PITX2 gene pairs heart development has keying action.
Gene pleiomorphism is meant among the crowd otherness of gene nucleotide series between the Different Individual; SNP (SNP) then is meant in the gene nucleotide series variability of single Nucleotide on certain site; If there are two or more allelotrope in same site in the colony; And minimum gene frequency >=1% promptly thinks to have SNP.SNP extensively exists in human genome, and mean density is about 1/1000bp, and its sum can reach 3,000,000 even more.SNP is modal a kind of in human heritable variation, account for more than 90% of all known polymorphums, so SNP can be used as the genetic marker of the third generation.
The polymorphum that SNP showed only relates to the variation of single base, and this variation mainly causes by the conversion (transition) or the transversion (transversion) of single base, also can be by due to the insertion (insertion) or disappearance (deletion) of base.
In genomic dna, any base all might morph, so SNP both might be in encoding sequence, also might be on non-coding sequence.SNP is the principal mode of genomic dna sequence variation, is the core information of decision human diseases (especially multigenic disease) susceptibility and drug responsiveness difference between individuals.The frequency of quite a few SNP has significant difference in not agnate, crowd.SNP itself only slightly or not changes the function of channel protein, does not directly cause a disease usually, but changes individual susceptibility to disease.
SNP can influence the pathogenic of other mutator genes, same transgenation, and pathogenic possibility is different under different polymorphum backgrounds.The biological action that it should be noted that SNP possibly not come to light, and especially in multigenic disease, other a lot of additional factor actings in conjunction have influenced the phenotype of disease, thereby have hidden the effect of polymorphum.
In addition, the problem of genetic heterogeneity also can not be ignored.Same a kind of polymorphum of same gene is in not agnate or different quantities crowd, and the conclusion that draws maybe be different even opposite fully.
7 selected SNP sites of the present invention are the atrial fibrillation significant correlation SNP site to the Asia ethnic group.
In the technical scheme of the present invention, DNA extraction, pcr amplification, PCR product purification and gene chip etc. are conventional Protocols in Molecular Biology.
Biochip technology is a forward position Protocols in Molecular Biology that in recent years develops rapidly, and its be otherwise known as DNA chip or dna microarray is a kind of of biochip.It through with the combination of computer image analysis, can be in experiment once to sample in the quality or the quantity of target molecule carry out fast, detect accurately and efficiently.Specifically; Gene chip is meant nucleic acid fragment a large amount of synthetic or that use conventional Protocols in Molecular Biology acquisition as probe; Said probe comprises specific nucleotide sequence; Adopt methods such as micro-sampling, said probe is solidified in the surface of upholder (like carriers such as slide, silicon chip, plastic sheet, a polyacrylamide gel piece of writing or nylon membranes) according to specific arrangement mode and specific means, form intensive two-dimentional molecular arrangement.Then with the biological sample to be measured of mark in target molecule hybridize; Through specific scanner such as optical scanner, laser confocal scanning appearance or electric charge coupling photographic camera (CCD) hybridization signal is carried out fast, walks abreast, analyzes efficiently, thus the quality and quantity of target molecule in the judgement sample.
PCR (being the polymerase chain reaction, Polymerase Chain Reaction) is one of the most frequently used Protocols in Molecular Biology, is a kind of method by vitro enzyme catalysis amplifying specific DNA.Typical PCR generally comprises high temperature makes template DNA sex change, primer and template annealing, primer extend the circulation that three-step reaction is formed along template, through above-mentioned repeatedly circulating reaction, makes target DNA be able to a large amount of rapidly amplifications.
Oligonucleotide probe of the present invention is according to each the SNP site and the flanking sequence design of atrial fibrillation postoperative recurrence genes involved, and to each SNP site, positive and negative 2 DNA chains design 8 probes altogether; And then be designed for the upstream and downstream primer of pcr amplification according to the farther flanking sequence at two ends, SNP site; 5 ' end to one of every pair of primer carries out the signal mark in advance, and said mark is preferably fluorescent mark, isotopic labeling, biotin labeling or digoxigenin labeled.
Those skilled in the art can carry out probe through this area ordinary method and synthesize; And carry out amido modified or the signal mark; Extract the DNA of detected sample then; With to the upstream and downstream primer of each SNP to be detected site design to carrying out pcr amplification, make the DNA of micro-detected sample increase in a large number, and in amplification procedure, introduce signal mark such as fluorescent mark.The testing sample nucleic acid target molecule that contains signal tracer after the amplification is through the oligonucleotide probe hybridization on base complementrity pair principle and the chip; After this carry out chip scanning; Obtain the signal in each site; Analyze with having or not hybridization signal is strong and weak, get final product the quantity and the character of target gene in the judgement sample.The technology that the present invention adopts pcr amplification and gene chip to combine, make to detect highly sensitive, flux is big.
Above-mentioned DNA extraction reagent, pcr amplification reagent, PCR product purification reagent, hybridizing reagent all can use all ingredients that in these operating process, needs use well-known to those skilled in the art; These reagent can be included in the gene chip reagent in case of necessity; Also can its prescription be listed in the specification sheets of gene chip reagent, prepare voluntarily by the indication of user in to specifications.
Therefore, the gene chip that is used to predict maze operation treatment atrial fibrillation of the present invention compared with prior art, has the following advantages:
(1) designed to enough special, responsive atrial fibrillation recurrence prediction index and the predictive model of asian population, established the corresponding relation of SNP pattern and atrial fibrillation postoperative recurrence;
(2) different approaches to the atrial fibrillation postoperative recurrence detects a plurality of relevant genes and pleomorphism site thereof; Can more accurately predict the ill risk of the different ill approach of person under inspection all sidedly; Can carry out examination to the general population easily rapidly, according to difference between individuals judge maze operation treatment atrial fibrillation curative effect, select suitable operation plan and effectively prevent its recurrence;
(3) accuracy rate is high, and cost is lower, can be used for batch detection, can fully satisfy the requirement of extensive genescreen;
(4) step of chip manufacturing is succinctly convenient, and detection sensitivity is high, and flux is big.
Embodiment
Below in conjunction with specific embodiment the present invention is done further detailed explanation; But content of the present invention is not limited to following examples; The technician of same area can propose other embodiment in technical scheme framework of the present invention, but these embodiment include in protection scope of the present invention.
The enforcement of unreceipted actual conditions is amplified in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of the present invention; It comprises the oligonucleotide probe that designs to rs2200733 site, rs10033464 site, rs13376333 site, rs6843082 site, rs17042171 site, rs2106261 site and rs7193343 site and can specific amplification goes out to comprise the Auele Specific Primer of the dna fragmentation in above-mentioned SNP site that 5 ' end of said Auele Specific Primer all carries out mark with semiochemicals in advance.
As preferably, said oligonucleotide probe has the nucleotide sequence shown in the SEQ ID NO.1-NO.56.
As preferably, said Auele Specific Primer has the nucleotide sequence shown in the SEQ ID NO.57-NO.70.
As preferably, 5 ' end of said Auele Specific Primer all carries out mark with resorcinolphthalein, isotropic substance, vitamin H or digoxin in advance.
As preferably; Said gene chip also comprises solid phase carrier; Said solid phase carrier is process surface chemistry group modified glass matrix or nylon membrane; The end of said oligonucleotide probe has modifies amino, and the chemical group of said solid phase carrier can combine with the modification amino covalence of oligonucleotide probe.
As improvement, said gene chip also comprises a negative control sample and a positive control.
As improvement, said gene chip also comprises hybridizing box and hybridization solution.
As further improvement, said gene chip also comprises DNA sampling reagent, pcr amplification reagent and PCR product purification reagent.
The gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of the present invention, its preparation method is:
(1) uses oligonucleotide synthesizer to prepare said oligonucleotide probe and Auele Specific Primer, 5 ' of Auele Specific Primer is held all carried out mark in advance with semiochemicals;
(2) use gene chip sample applying appearance built in through on the aldehyde group modified solid phase carrier, makes gene chip with said oligonucleotide probe point after the drying.
The gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of the present invention, its method of use is:
(1) use said DNA sampling reagent to extract patient DNA;
(2) use the amplification of said Auele Specific Primer and said pcr amplification reagent to comprise the dna fragmentation in target SNP site;
(3) each pcr amplification product with same sample mixes, and adds behind the hybridization solution and said chip hybridization;
(4) use the scanner scanning hybridization signal.
Present embodiment has been chosen 7 SNP sites of full genome association study (GWAS) and atrial fibrillation significant correlation: rs2200733 site, rs10033464 site, rs13376333 site, rs6843082 site, rs17042171 site, rs2106261 site and rs7193343 site; According to these 7 SNP sites and flanking sequence design oligonucleotides probe and corresponding Auele Specific Primer; To 4 pairs of oligonucleotide probes of each SNP site design; Be that positive and negative two DNA chains design 8 oligonucleotide probes altogether, described 7 pairs of Auele Specific Primers can amplify the dna fragmentation that comprises above-mentioned 7 SNP sites respectively.
Specifically, gene chip preparation method can be carried out according to following steps:
Raw material is prepared---
Aldehyde radical chip (specification is 75.5*25.2*1.0 for polymer three-dimensional substrate D, the Capitalbio of manufacturer)
Gene chip sampling liquid (Capitalbio, 5ml) or 2*Spoting Solution etc.
1, the sequence oligonucleotide probe design is of sequence table, and according to synthetic 5 ' the end amino labeled probe of method well known to those skilled in the art, the corresponding relation in said oligonucleotide probe and each SNP site is as follows:
2, the synthetic probe is dissolved in the gene chip sampling liquid (or mix with 2*Spoting Solution equal-volume probe, making final concentration is 40pmols/ μ l), with point sample instrument (like SmartArray
TM, CapitalBioCorp. or Cartesian Tech., PROSYS 5510A chip manufacturing system) and point is on the aldehyde group modified chip substrate of process, and preserve dry back, and probe puts in order as follows on its chips:
| rs2200733?SNP1 | rs2200733?SNP2 | rs2200733?SNP3 | rs2200733?SNP4 |
| rs10033464?SNP1 | rs10033464?SNP2 | rs10033464?SNP3 | rs10033464?SNP4 |
| rs13376333?SNP1 | rs13376333?SNP2 | rs13376333?SNP3 | rs13376333?SNP4 |
| rs6843082?SNP1 | rs6843082?SNP2 | rs6843082?SNP3 | rs6843082?SNP4 |
| rs17042171?SNP1 | rs17042171?SNP2 | rs17042171?SNP3 | rs17042171?SNP4 |
| rs2106261?SNP1 | rs2106261?SNP2 | rs2106261?SNP3 | rs2106261?SNP4 |
| rs7193343?SNP1 | rs7193343?SNP2 | rs7193343?SNP3 | rs7193343?SNP4 |
| Positive control PC |
3, chip is carried out pre-treatment: chip is placed under 75% relative humidity, the 30 ℃ of conditions fixed in 48-72 hour, with chip boiling water bath 30 seconds, take out the room temperature thorough drying then.
Or follow these steps to chip is carried out pre-treatment:
(1) chip is placed wet box airtight, 100% humidity, 37 ℃ of overnight treatment;
(2) chip is put in the 0.25%NaBH4 solution, horizontal shaking table (80rpm) is gone up reduction and is handled 5min;
(3) in zero(ppm) water, horizontal shaking table (80rpm) is gone up and is cleaned 5min;
(4) repeating step (3) once;
(5) be positioned over chip in the special centrifuge dripping pipe of 50ml the centrifugal 1min of 2000rpm.
Promptly can be used for hybridization through the chip of as above handling.
In the present embodiment; Select the carrier of glass matrix for use as chip; The substrate of said glass matrix has the activation aldehyde groups of handling through chemically modified, and oligonucleotide probe is according to the oligonucleotide fragment of sequence table design, and its end has amino labeled; Therefore incidental the modifications amino of dna probe can with the activation aldehyde groups covalent attachment of glass matrix, utilize this method dna probe just can be fixed on substrate surface by certain arrangement regulation.Therefore can make required chip according to user's requirement easily, the step of chip manufacturing is succinctly convenient, cost is lower.Also can select the solid phase carrier of activated nylon membrane for use as chip.
The concrete preparation of pcr chip can be carried out according to following steps:
1, the sequences Design of Auele Specific Primer is shown in sequence table; According to the synthetic upstream and downstream primer that has 5 ' end signal mark of method well known to those skilled in the art; Said signal beacon is designated as fluorescent mark or digoxigenin labeled, and the corresponding relation in said Auele Specific Primer and each SNP site is as follows:
2, adopt the form of prefabricated pcr chip, with pcr amplification reagent and each Auele Specific Primer to quantitatively in advance o'clock in 96 hole real-time quantitative PCR plates (like ABI company), in-20 ℃ of preservations.96 hole real-time quantitative PCR plates, every row 12 holes, each gene locus is established multiple hole, and whenever list and index and survey 1 patient in totally 12 holes, can detect 8 patients' same SNP site simultaneously.
The concrete method of use of gene chip can be carried out according to following steps:
1, sample DNA extracts: use DNA extraction reagent to extract sample DNA, as scrape the oral mucosa epithelial cell of getting the person under inspection, add the cell pyrolysis liquid of equal volume; White corpuscle is fully dissolved in cracking twice, abandons supernatant after centrifugal; Add Proteinase K damping fluid and Proteinase K (50ug/ml); Hatched 10 minutes for 65 ℃, isopropanol precipitating, 75% washing with alcohol twice are dissolved in after drying in an amount of TB solution.
2, pcr amplification: in pcr chip, add corresponding person under inspection's DNA extraction liquid during detection, carry out the multiplex PCR amplification, reaction conditions is 95 ℃ of 3min of preparatory sex change, 94 ℃ of 30s, 58 ℃ of 30s, 72 ° of 1min, 40 circulations, 72 ℃ of 5min, 4 ℃ of Hold.
3, PCR product purification: use PCR product purification reagent purified pcr product; As in the PCR product, adding 0.2 μ l Exo I, 1 μ l SAP, 3 μ l 10 * SAP reaction buffers and 1.5 μ l deionized waters; Pcr chip is put into the PCR appearance carry out purifying; Purifying procedure: 37 ℃ of 30min, 96 ℃ of 10min, Hold4 ℃.
4, hybridization: each the 2 μ l of each pcr amplification product that get same sample to be checked mix, and add hybridization solution 15 μ l, and 95 ℃ of denaturing treatment 3min place 2min on ice and are splined on chip after above, and covered places 40 ℃ of wet boxes to hybridize 30min.
5, chip cleans: chip is moved to preheating is housed the chip scavenging solution is arranged (like 0.5*SSC, 0.1%SDS is in 50ml pipe PH7.4), in 42 ℃ of jogs washing 5min.
6, be example with the fluoroscopic examination detection---
Scan: chip is positioned over presses blas scanning in the scanner (LuxScan 10K or General Scanning chip signal analytical system Scannarray 3000).
Detecting with the digoxin development process is example---
After the digoxin colouring reagents process chip,, the result is analyzed with corresponding software with optics scanner scanning hybridization signal.
7, the result judges: the hybridization point as contrast can (be a positive control at PC; Positive Control) green fluorescence signal or color signal appear in the position; Point out this to hybridize successfully, the site of negative control fluorescence or color signal should not occur simultaneously, and this moment, result's interpretation was regarded as effectively.
Position by signaling point manifests can conclude whether this patient carries the SNP site relevant with the atrial fibrillation postoperative recurrence.
Claims (10)
1. gene chip that is used to predict maze operation treatment atrial fibrillation curative effect; It is characterized in that: it comprises the oligonucleotide probe that designs to rs2200733 site, rs10033464 site, rs13376333 site, rs6843082 site, rs17042171 site, rs2106261 site and rs7193343 site and can specific amplification goes out to comprise the Auele Specific Primer of the dna fragmentation in above-mentioned SNP site that 5 ' end of said Auele Specific Primer all carries out mark with semiochemicals in advance.
2. the gene chip that is used to predict maze operation treatment atrial fibrillation curative effect according to claim 1, it is characterized in that: said oligonucleotide probe has the nucleotide sequence shown in the SEQ ID NO.1-NO.56.
3. the gene chip that is used to predict maze operation treatment atrial fibrillation curative effect according to claim 1, peculiar levying is: said Auele Specific Primer has the nucleotide sequence shown in the SEQ ID NO.57-NO.70.
4. according to each described gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of claim 1-3, it is characterized in that: 5 ' end of said Auele Specific Primer all carries out mark with resorcinolphthalein, isotropic substance, vitamin H or digoxin in advance.
5. according to each described gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of 1-3; It is characterized in that: said gene chip also comprises solid phase carrier; Said solid phase carrier is process surface chemistry group modified glass matrix or nylon membrane; The end of said oligonucleotide probe has modifies amino, and the chemical group on the said solid phase carrier can combine with the modification amino covalence of oligonucleotide probe.
6. according to each described gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of claim 1-3, it is characterized in that: said gene chip also comprises a negative control sample and a positive control.
7. according to each described gene chip that is used to predict maze operation treatment atrial fibrillation curative effect of claim 1-3, it is characterized in that: said gene chip also comprises hybridizing box and hybridization solution.
8. the gene chip that is used to predict maze operation treatment atrial fibrillation curative effect according to claim 7 is characterized in that: said gene chip also comprises DNA sampling reagent, pcr amplification reagent and PCR product purification reagent.
9. described gene chip preparation method of claim 1, it is characterized in that: it adopts following steps---
(1) uses oligonucleotide synthesizer to prepare said oligonucleotide probe and Auele Specific Primer, 5 ' of Auele Specific Primer is held all carried out mark in advance with semiochemicals;
(2) use gene chip sample applying appearance built in through on the aldehyde group modified solid phase carrier, makes gene chip with said oligonucleotide probe point after the drying.
10. the method for use of the described gene chip of claim 8 is characterized in that: said method is used said DNA sampling reagent for (1) and is extracted patient DNA; (2) use the amplification of said Auele Specific Primer and said pcr amplification reagent to comprise the dna fragmentation in target SNP site; (3) each pcr amplification product with same sample mixes, and adds behind the hybridization solution and said chip hybridization; (4) use the scanner scanning hybridization signal.
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| CN2011103382625A CN102363806A (en) | 2011-10-31 | 2011-10-31 | Gene chip for forecasting curative effect of maze surgery on atrial fibrillation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107083429A (en) * | 2017-04-17 | 2017-08-22 | 江苏苏博生物医学科技南京有限公司 | A kind of SNP parting kits detected for atrial fibrillation tumor susceptibility gene and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107083429A (en) * | 2017-04-17 | 2017-08-22 | 江苏苏博生物医学科技南京有限公司 | A kind of SNP parting kits detected for atrial fibrillation tumor susceptibility gene and its application |
| CN107083429B (en) * | 2017-04-17 | 2019-06-14 | 江苏苏博生物医学科技南京有限公司 | A kind of SNP parting kit and its application for the detection of atrial fibrillation tumor susceptibility gene |
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