CN102605044A - Method for detecting mutation of related genes of genetic long QT syndrome - Google Patents

Abstract

The invention relates to a method for detecting mutation of related genes of an genetic long QT syndrome, in particular relates to a method for detecting mutation of related genes, including KCNJ2 and CAV3, of the genetic long QT syndrome by combining a multi-oligonucleotide link detection probe with a liquid phase chip. The method comprises the following steps of: a) designing and preparing the multi-oligonucleotide link detection probe, wherein a probe sequence for mutation of the gene KCNJ2 is SEQ ID No.1-SEQ ID No.32, and the probe sequence for the mutation of the gene CAV3 is SEQ ID No.33-SEQ ID No.40; b) carrying out DNA sample denaturation and probe hybridization, and carrying out link reaction; c) carrying out PCR (polymerase chain reaction) amplification on the probe sequences by adopting a general primer; and d) detecting amplification products by utilizing the liquid phase chip. The technology can realize single-tube reaction detection on dozens of mutation sites at most, flux is high, operation is easy and cost is low.

Description

A kind of method that detects heredity long QT syndrome associated gene mutation

Technical field

The invention belongs to biology field; Relate to medical diagnosis and biotechnology; Relate to a kind of method that detects heredity long QT syndrome associated gene mutation, concrete is the method that a kind of multiple oligonucleotide joint detection probe that detects heredity long QT syndrome genes involved KCNJ2 and CAV3 sudden change combines liquid-phase chip.

Background technology

On electrocardiogram(ECG, when normal people's heart rate 60～100 times/timesharing, the QT interval, be generally 320～440 milliseconds, when the QT interval surpasses 440 milliseconds, is considered to the QT interval and prolongs (LQT).The QT interval prolongs syndrome, and (long QT syndrome, LQTs), one group of syndrome of refer to have QT interval prolongation on the electrocardiogram(ECG, ventricular arrhythmia, fainting and dying suddenly maybe be with congenital deafness.LQTs can be geneogenous, also can be acquired.We are called heredity LQTs with one type of LQTs with obvious familial aggregation.This type disease overwhelming majority is a monogenic inheritance, and is the most common with autosomal dominant inheritance, mainly show as the QT interval prolong, the T ripple is unusual and torsade de pointes (TdP), can cause fainting, cardiac events such as sudden cardiac arrest even sudden death.

Nineteen fifty-seven Jervell and Lange-Nielsen have reported first kind of clinical form of heredity LQTs, are called Jervell-Lange-Nielsen (JLN) syndrome.They have described a Norway family, have 4 to suffer from congenital deafness among 6 children, and QT prolongs and faints repeatedly, wherein dies suddenly for 3.Father and mother are healthy, do not have and faint or other medical history.1963, Romano etc. and Ward independently reported the case that hearing is normal but have QT to prolong and faint.This more common clinically heredity LQTs form is called as Romano-Ward (RWS) syndrome.The RWS syndrome patient, the QT interval, prolong on the electrocardiogram(ECG, clinical manifestation possibly also comprise faint, sudden death, epilepsy.Non-heart sexual abnormality (be mellitus, asthma or also refer to) also takes place once in a while.RWS syndrome is the most common, and this is that the ill probability theoretical value of offspring is 50% because most RWS syndrome is autosomal dominant inheritance.And the less relatively generation of JLN syndrome is autosomal recessive inheritance.LQTS is not a common clinical disease, but because this disease incidence is unexpected, sudden death rate height, many with teenager's morbidity again, is paid attention to by clinical.The research of delivering in 1985 to 233 LQTs patients shows that in 126 patients without any treatment or non-anti-suprarenal gland treatment, 1 year case fatality rate is that 20%, 3 year case fatality rate is that 26%, 15 year case fatality rate is 53%; (beta-blocker or LCSD patients with surgical, its corresponding case fatality rate is respectively 0.9%, 6% and 9% and through anti-suprarenal gland treatment.The probability that the symptom of falling ill for the first time is sudden cardiac arrest is about 7%～8% [1].

At present, the clinical diagnosis of LQTs mainly relies on family history, unknown cause faint with electrocardiogram(ECG on the QT interval prolong.1993.International LQTs cooperative groups has been issued the integration type clinical diagnosis standard of revising.About the LQTs diagnosis, the more important thing is ecg characteristics and family line investigation.(0.44s＜QT interval,＜0.47s), need further take exercises test and dynamic ecg recordings examination are to grasp patient information as much as possible to be in the patient of threshold value for the QT interval.In the sporting trial process, LQTs patient is at motion end and/or recover to have the QT significant prolongation of interval in early days, and normal individual does not have this and changes, can differentiate whereby that the QT interval is in the individuality of threshold value.

But means such as electrocardiogram(ECG are subject to the restriction of time window, are difficult to find potential hidden danger at premorbid not.Since early 1990s, the molecular genetics basis of heredity LQTs has been established in a series of landmark researchs.LQTs is first kind and has confirmed the irregular pulse that causes by the transgenation of coding ionic channel.Congenital LQTs incidence is about 1: 2500, and the transgenation by coding or adjusting heart sodium, potassium and calcium channel causes mostly.Most of known transgenation is positioned at the gene of coding potassium-channel.In multiple diagnostic method and standard, gene are learned inspection has become the important means of making a definite diagnosis heredity LQTs, family screening and generaI investigation.

At present found that the gene relevant with heredity LQTs has 14 hypotypes, wherein 12 cause RWS syndrome, and 2 JLN syndromes that cause that the companion is deaf have more than 1200 sudden change.According to the difference of transgenation situation, heredity LQTs is divided into 10 different hypotype LQT1～LQT10 [2].LQT7 wherein, just Andersen-Tawil syndrome is characteristic with periodic paralysis, ectopic cardiac rhythm fast can occur, but torsades de pointes type chamber speed seldom occurs, the afunction due to its generation suddenlys change with KCNJ2 has substantial connection.KCNJ2 is positioned at long-armed 2 districts of human No. 17 karyomit(e)s 3 bands (17q23), coding inward rectifyimg potassium channel Kir2.1 alpha subunit.Kir2.1 comprises the alpha subunit of four identical KCNJ2 codings.Each alpha subunit comprises two membrane spaning domains (M1 and M2), and M1 is linked to each other by about 30 amino acid whose porose area ring buttons loop with M2, and there is porose area in ring button loop central authorities, and porose area is positioned at the cell outside of film, and the recognition sequence " G-Y-G " of potassium ion is contained in this zone.Determined the specificity ion selectivity [3] of potassium channel just because of the existence of recognition sequence G-Y-G.Therefore KCNJ2 sudden change and heredity LQTs, especially genotype LQT7 is closely related.Matteo in 2006 etc. have reported the closely related gene C AV3 of another genotype LQT9 with heredity LQTs, the CAV3 caveolin-3 (caveolin-3) of encoding.Alveole (caveolae) is a kind of cytoplasmic membrane structure of specialization, mainly is made up of protein and lipid, and wherein caveolin (caveolin) is its main structure and adjusting composition.Caveolae not only participates in striding the membrane substance transhipment, and is the hinge of enrichment of cell signal molecule and signal transduction.Caveolin-3 albumen is caveolin family, strides film twice, on the film surface, forms a hairpin structure, is the primary structure albumen in caveolae membrane structure territory.Matteo in 2006 etc. have confirmed 4 focus sudden changes of this gene, and through its pathogenesis of experiment confirm and influence myocardium sodium channel hNav1.5 relevant [4].According to above-mentioned discovery, selection of the present invention and the syndromic genotype LQT7 of heredity LQTs and the closely-related gene KCNJ2 of LQT9 and CAV3 detect, and are diagnosing hereditary LQTs, differentiate the LQTs genotype foundation is provided.The relevant hot mutant site of the LQTs that the present invention detects is as shown in the table:

Because the gene mutation site that heredity LQTs relates to is many.It is how cost-effective that to carry out gene diagnosis be unusual difficulty.Traditional P CR, multiplex PCR, the order-checking of first-generation automatic DNA sequencer DNA all are feasible to the examination of a spot of site.But when relating to so many mutational site in the face of heredity LQTs, these methods just expose the weakness that the time is long, cost is high, operation easier is big.

At present, carry out the U.S. the most widely in the gene diagnosis of heredity cardiovascular diseases, detection company mainly still adopts direct sequence measurement.Though directly order-checking is the most intuitive and accurate detection method, its sense cycle is long, sends report from receiving that sample to be checked detects to smooth completion, needs successive 15-21 hour.As detecting normal working hours, reality needs to accomplish detection 3 working dayss at least, is difficult to satisfy the ageing requirement of direct clinical medication.Therefore, set up a kind of fast, high-throughput, gene diagnosis technology that specificity is good, highly sensitive, easy and simple to handle be to need the major issue that solves at present.

Multiple oligonucleotide joint detection technology in 2002 by report [5] at first such as Schouten, be that grew up in recent years a kind of carries out qualitative and new technology semi-quantitative analysis to dna sequence dna to be checked.This technique to high-efficiency, special can detect numerous target genes in primary first-order equation, be applied to the research of a plurality of fields, multiple disease at present, like numerical abnormalities of chromosomes, the repetition of heredopathia genetically deficient, gene methylation detection etc.

The maximum characteristics of multiple oligonucleotide joint detection technology are the design of probe; Designed a pair of probe to each testing gene target sequence, this a pair of probe comprises a short probe and the long probe that comes through the phage-derived method preparation of M13 through chemosynthesis.Wherein, short probe is about 50-60bp, comprises one and target sequence complete complementary distinguished sequence A and a common sequences X.Long probe is about 60-450bp, comprise one with target sequence complete complementary distinguished sequence B and a common sequences Y, the M13 phage fragment that a segment length different sizes is arranged between two sequences is as stuffer.Can distinguish different probes because stuffer is different in size.

The step of whole multiple oligonucleotide joint detection reaction comprises hybridization, connection, amplification and electrophoresis detection.When probe design; Long and short probe closely links to each other on target sequence with B with the distinguished sequence A of target complement sequence; Used ligase enzyme can only connect hybridization at same sequence and two sections closely continuous distinguished sequences in the multiple oligonucleotide joint detection technology; Therefore have only the corresponding long and short probe of working as all correctly to hybridize to corresponding target sequence district, long and short probe could be connected in the ligation.After long and short probe and the target sequence hybridization complementation; Under the effect of ligase enzyme; Connect into a complete sequence oligonucleotide probe; If wherein the sequence of a probe and target-gene sequence to be measured are not exclusively complementary, even have only a base not complementary, also can make hybridization not exclusively ligation can't be carried out.The PCR reaction is that template is increased by a pair of universal primer with the probe that connects, and it is complementary with the common sequences X of short probe to have fluorescently-labeled universal primer, and the common sequences Y of unlabelled universal primer and long probe is complementary.Those probes that do not connect are because only contain the complementary district of a universal primer; In the PCR process, can not be increased; Can not contain corresponding fluorescent mark yet, and have only the probe of connection to be increased, so the quantity of amplified production is directly proportional with the quantity of target sequence in the template DNA is corresponding; Cause probe to connect if target sequence is undergone mutation, also just can not get corresponding amplified production.The right amplified production of different probe is because the M13 phage fragment length that contains is different, thereby length is different, available agarose gel electrophoresis or separate through capillary electrophoresis.Electrophoresis result is through computer software analysis, and like Genemapper software, the amplified peak fluorescent signal of probe lacks, increases, reduces representes that all unusual the or target gene of corresponding target gene copy number undergos mutation.But the characteristics of classical multiple oligonucleotide joint detection method and difficult point all are the design of probe.The general phage-derived legal system of M13 that adopts is equipped with long probe, but complicated steps and consuming time.And capillary electrophoresis detects, and deposition condition, pH damping fluid etc. all can influence detected result; Can not realize high-throughput.

The present invention improves the multiple oligonucleotide joint detection of classics technology, replaces M13 phage fragment with the specificity sequence label, prepares probe through chemical synthesis, has simplified the design and the preparation process of probe.And detect pcr amplification product with liquid-phase chip, and having improved detection resolution, single tube reaction reduces pollutes, and has really realized the robotization and the high-throughput that detect, for scientific research and clinical application provide bright prospects.

Reference:

[1]Schwartz，PJ，Locati，E.The?idiopathic?long?QT?syndrome：pathogenetic?mechanisms?and?therapy.Eur?Heart?J?1985；6?Supp1?D：103.

[2]Splawski?I，Shen?J，Timothy?KW，et?al.Spectrum?of?mutations?in?long?QT?Syndrome?genes?KVLQT1，HERG，SCN5A，KCNE1?and?KCNE2.Circulation，2000，102(10)：1178～1185.

[3]Tamargo?J，Caballero?R，Gomez?R，et?al.Pharmacology?of?cardiac?potassium?channels.Cardiovasc?Res，2004，62(1)：9～33.

[4]Matteo?Vatta，PhD；Michael?J.Ackerman，et?al.Mutant?Caveolin-3?Induces?Persistent?Late?Sodium?Current?and?Is?Associated?With?Long-QT?Syndrome.Circulation.2006?Nov?14；114(20)：2104-12.Epub?2006?Oct?23.

[5]Schouten?JP，McElgunn?CJ，Waaijer?R，et?al.Relative?quantification?of?40?nucleic?acid?sequences?by?multiplex?ligation-dependent?probe?amplification[J].Nucleic?Acids?Res，2002，30(12)：e57.

Summary of the invention

The objective of the invention is, provide a kind of multiple oligonucleotide joint detection probe that detects heredity long QT syndrome genes involved KCNJ2 and CAV3 sudden change to combine the method for liquid-phase chip.This method flux is high, can realize nearly tens mutational sites of single tube reaction detection, and is easy and simple to handle, cost is low.

For realizing above-mentioned purpose, the technical scheme that the present invention adopts is: the multiple oligonucleotide joint detection probe of a kind of KCNJ2 of detection and CAV3 transgenation combines the method for liquid-phase chip, may further comprise the steps:

A. design and prepare the multiple oligonucleotide joint detection of the sudden change probe that is used for KCNJ2 and CAV3 gene test;

B. DNA sample to be measured and step a gained probe are added in the reaction tubes, add ligation and the required reagent of pcr amplification reaction, dna sample sex change and probe are hybridized, and activate ligase enzyme through the conditioned reaction temperature, and probe is carried out ligation;

C. the conditioned reaction temperature activates archaeal dna polymerase, uses a pair of universal primer, step b gained is accomplished the probe sequence that connects carry out pcr amplification reaction;

D. the xTAG microballoon in step c gained pcr amplification reaction product and the liquid-phase chip is hybridized, add Streptomycin sulphate avidin-phycoerythrin behind the hybridization and react, then through Lu Mingkesi liquid-phase chip Equipment Inspection fluorescent signal.

For realizing technique scheme; The described multiple oligonucleotide joint detection probe of step a; When design and preparation, comprise a long probe and a short probe; Wherein long probe comprises 5 ' end and target gene specific sequence complementary hybridization sequences, 3 ' end common sequences and the specificity sequence label between hybridization sequences and common sequences, and short sequence comprises 3 ' end and target gene specific sequence complementary hybridization sequences and 5 ' end common sequences;

Wherein, the described multiple oligonucleotide joint detection probe that is used for the KCNJ2 transgenation of step a has comprised the wild-type sequence in 16 mutational sites of No. 2 exon of KCNJ2 gene with target gene specific sequence complementary hybridization sequences.Wherein, 16 mutational sites of No. 2 exon of KCNJ2 gene are 200G＞A, 223A＞G, 224C＞T, 283_294del, 407C＞T, 430G＞A 488_493del, 557C＞T, 646A＞C, 652C＞T, 679G＞T, 899G＞T, 904G＞A 907G＞A, 913A＞G and 940_945del.

Further, the sequence of the multiple oligonucleotide joint detection of the described KCNJ2 of being used for transgenation probe is SEQ ID No.1-SEQ ID No.32 (table 1).

Wherein, the described multiple oligonucleotide joint detection probe that is used for the CAV3 transgenation of step a has comprised the wild-type sequence of 4 nucleotide mutant sites of No. 2 exon of CAV3 gene with target gene specific sequence complementary hybridization sequences.Wherein, 4 mutational sites of No. 2 exon of CAV3 gene are 233C＞T, 253G＞A, 290T＞G, 423C＞G.

Further, the sequence of the multiple oligonucleotide joint detection of the described CAV3 of being used for transgenation probe is SEQ ID No.33-SEQ ID No.40 (table 1)

The described multiple oligonucleotide joint detection probe of step a, the hybridization sequences of long probe 5 ' end closely links to each other on target-gene sequence with the hybridization sequences of short probe 3 ' end;

The all ingredients that adds ligation and pcr amplification reaction among the step b simultaneously, wherein the optimum temperuture of dna ligase is 55 ℃, the optimum temperuture of archaeal dna polymerase is 72 ℃.Therefore can pass through the conditioned reaction temperature; Activate earlier dna ligase and carry out the probe ligation, activate archaeal dna polymerase again and carry out pcr amplification, can guarantee to carry out earlier to carry out after the ligation purpose of amplified reaction; Also accomplished the omnidistance stopped pipe operation of reaction, effectively avoided polluting.

Forward primer 5 ' the end of the said universal primer of step c has biotin labeling, and reverse primer is unmarked, and forward primer is complementary with short probe 5 ' end common sequences, and reverse primer and long probe 3 ' end common sequences are complementary.The forward primer sequence is SEQ ID NO.41, and the reverse primer sequence is SEQ ID NO.42.

The described xTAG microballoon of steps d; Be commercialization reagent; Be coated with the amido modified probe of base sequence from SEQ IDNO.43-SEQ ID NO.62, SEQ ID NO.43-SEQ ID NO.62 sequence has comprised the anti-Tag sequence with SEQ ID NO.1, SEQ ID NO.3, the contained Tag sequence-specific of SEQ ID NO.5...SEQ ID NO.39 probe complementary pairing.Be connected with spacerarm between above-mentioned SEQ ID NO.43-SEQ ID NO.62 base sequence and the amino; Spacerarm can reduce sterically hindered; Improve hybridization efficiency and hybridization specificity, spacerarm sequence commonly used comprises poly dT (poly dT), (CH2) 12, CH2) 6, CH2) 18 etc.The present invention adopts 8 dT of poly as spacerarm.Above-mentioned every kind of microballoon has the different colours coding.The amido modified probe sequence that universal primer and xTAG microballoon encapsulate is as shown in table 2.

The present invention utilizes the sudden change situation of multiple oligonucleotide joint detection technology for detection long QT syndrome genes involved KCNJ2 and CAV3; And the multiple oligonucleotide joint detection of classics method improved; Should technology combine with liquid-phase chip technology; Its characteristics are, the contained M13 phage of the long probe of multiple oligonucleotide joint detection fragment is replaced the same length of each specificity sequence label with one section specificity sequence label; All about 20-30 base; This specific specificity sequence label can with entrained hybridization probe complementary pairing on the xTAG microballoon of liquid-phase chip, we are called Tag with the specificity label on the probe, and the complementary hybridization probe that carries on the microballoon is called Anti-Tag.At first long and short probe and target sequence hybridization are complementary, under the effect of ligase enzyme, connect into a complete sequence oligonucleotide probe; Being that template is another with the probe that connects then carries out the PCR reaction to universal primer; Obtain hybridizing with the microballoon of liquid-phase chip behind the pcr amplification product, the Tag that the amplified production of each probe carries is unique, and the xTAG microballoon of complementary hybridization also is unique with it; Detect through the liquid-phase chip platform subsequently, reach a conclusion.If the target sequence origination point that detects sudden change; So long and short probe just can't connect; Can not carry out pcr amplification subsequently through universal primer; Also just can not detect corresponding fluorescent signal, therefore just can judge according to the change of fluorescent signal whether target sequence has sudden change to exist through the liquid-phase chip platform.

Major advantage of the present invention is:

1) the present invention can carry out the detection in 20 mutational sites of heredity LQTs genes involved KCNJ2 and CAV3 simultaneously in primary first-order equation;

2) detection method of the present invention being connected before the hybridization of amplified production and microballoon, amplified reaction can be realized the stopped pipe operation, can effectively avoid laboratory pollution;

3) detection method step of the present invention is simple, thereby has avoided the many uncertain primer that exists in the complex operations process, has improved detection accuracy rate and stability.

4) detection method required time provided by the present invention is less than sequencing technologies greatly, more meets the clinical detection requirement.

Embodiment

With embodiment the present invention is further described below:

Embodiment one, extract the DNA of sample to be tested by ordinary method

Get sample to be tested (comprising whole blood, blood plasma, serum), with reference to health be century the poba gene group extract test kit explanation in a small amount and extract the DNA in the sample, detailed step is following:

1) adds 20 μ l Protease in 1.5ml centrifuge tube bottom.

2) add 200 μ l samples

3) in sample, add 200 μ l Buffer GL, vortex concussion 15 seconds.Adding 20ul does not have the RnaseA of DNA enzyme.

4) hatched 10 minutes for 56 ℃.

5) add 200 μ l absolute ethyl alcohols, vortex concussion 15 seconds, mixing finishes of short duration centrifugal, makes the liquid on tube wall and the lid focus on the pipe end.

6) Spin Column DM for use is put into Collection Tube; The solution that obtains in the step 5) is carefully changed among the Spin Column DM, and avoid joining Spin Column DM edge, build the pipe lid; 8; Centrifugal 1 minute of 000rpm (6000xg) puts into a new CollectionTube with Spin Column DM, discards the Collection Tube that waste liquid is housed.

7) carefully open the pipe lid; In Spin Column DM, add 500 μ l Buffer GW1, avoid joining Spin Column DM edge, build the pipe lid; 8; Centrifugal 1 minute of 000rpm (6000xg) puts into a new Collection Tube with Spin Column DM, discards the Collection Tube that waste liquid is housed.

8) carefully open the pipe lid, in Spin Column DM, add 500 μ l Buffer GW2, avoid joining Spin Column DM edge, build the pipe lid, centrifugal 3 minutes of maximum speed of revolution is abandoned waste liquid.

9) Spin Column DM is put into a new centrifuge tube, centrifugal 1 minute of maximum speed of revolution.

10) Spin Column DM is changed in the new centrifuge tube, add 100 μ l Buffer GE, room temperature was placed 1 minute, and 8, centrifugal 1 minute of 000rpm (6000xg) obtains dna solution.

11) solution in the centrifuge tube is inhaled back Spin Column DM, room temperature was placed 1 minute, and 8, centrifugal 1 minute of 000rpm (6000xg) obtains dna solution.The DNA product places-20 ℃ of preservations.

12) DNA quality inspection, marked places-80 ℃ of preservations.

Embodiment two, dna sample sex change and probe hybridization, linking probe, and the probe sequence that connects carried out pcr amplification

1) configuration reaction system owing to multiple connection, amplified reaction carry out in single tube, adds DNA sample to be measured and probe in the reaction tubes, in pipe, adds the required reagent of ligation and pcr amplification reaction then.Comprising of per 10 μ l reaction systems: 0.8 μ l dna ligase (5U/ μ l); 1 μ l long probe mixing solutions (50nM), the short probe mixing solutions (50nM) of 1 μ l, 1 μ l

Buffer II (10 *); 0.8 4 kinds of dNTPs mixing solutionss (2.5mM) of μ l; 1.6 μ l magnesium chloride (25mM), 0.1 μ l archaeal dna polymerase, 0.1 μ l Reduced nicotinamide-adenine dinucleotide (50mM); 0.2 the biotin labeled forward universal primer of μ l (10mM); 0.2 the reverse universal primer of μ l (10mM), the dna profiling that 1 μ l extracts according to embodiment one, adding aseptic double-distilled water to whole reaction system at last is 10 μ l.

2) through the conditioned reaction temperature, sample DNA sex change and probe are hybridized, activate dna ligase then, the probe of hybridizing is connected; Activate archaeal dna polymerase at last, the probe sequence that connects is carried out pcr amplification with a pair of universal primer.It is 94 ℃ of preparatory sex change 1.5 minutes that reaction begins, and the condition of carrying out ligation then is 94 ℃ of sex change 25 seconds, and 50 ℃ connect 30 seconds; Totally 30 circulations; The condition of then carrying out pcr amplification is 94 ℃ of preparatory sex change 20 seconds, anneals 20 seconds for 58 ℃, and 72 ℃ were extended 20 seconds; Totally 35 circulations, last 72 ℃ of final extensions 7 minutes.

Embodiment three, amplified production and liquid-phase chip hybridization are also gone up machine testing

The xTAG microspheres solution that 1) will have a corresponding anti-Tag of various Tag sequences mixing 30 seconds on vibrator, supersound process 30 seconds;

2) with 1.5 * Tm hybridization solution preparation xTAG mixing microballoon working fluid, make that every kind of microballoon concentration is 1000/μ l in the solution, mixing is 30 seconds on the vibrator, supersound process 30 seconds;

3) in the end reaction system of per 10 μ l embodiment two, add the above-mentioned mixing microballoon of 5 μ l working fluid, make and finally contain each 5000 of every kind of microballoons in the reaction system;

4) the background contrast is set, adds 15 μ l TE damping fluids (pH 8.0);

5) the hybridization condition is set: 94 ℃ 1 minute, slowly be cooled to 25 ℃ subsequently, rate of temperature fall is 5 ℃/minute.

6) with 1 * Tm hybridization solution preparation SA-PE to 2 μ g/ml;

7) with reaction tubes 12, centrifugal 5 minutes of 000g, careful abandoning supernatant.

8) each reaction tubes adds 75 μ l SA-PE working fluid, mixings gently;

9) hybridized 5 minutes for 60 ℃;

10) Lu Mingkesi liquid-phase chip appearance is set to hybridization temperature, detects reading.If the target sequence origination point that detects sudden change; So long and short probe just can't connect; Can not carry out pcr amplification subsequently through universal primer; Also just can not detect corresponding fluorescent signal, therefore just can judge according to the change of fluorescent signal whether target sequence has sudden change to exist through the liquid-phase chip platform.

Claims (6)

1. a method that detects the multiple oligonucleotide joint detection probe combination liquid-phase chip of KCNJ2 and CAV3 transgenation is characterized in that, said method comprising the steps of:

A. design and prepare the multiple oligonucleotide joint detection probe that is used for KCNJ2 and CAV3 detection in Gene Mutation;

B. DNA sample to be measured and step a gained probe are added in the reaction tubes, add ligation and the required reagent of pcr amplification reaction, dna sample sex change and probe hybridization activate ligase enzyme through the conditioned reaction temperature, and probe is carried out ligation;

C. the conditioned reaction temperature activates archaeal dna polymerase, with a pair of universal primer step b gained is accomplished the probe sequence that connects and carries out pcr amplification reaction;

D. the xTAG microballoon in step c gained pcr amplification reaction product and the liquid-phase chip is hybridized, add Streptomycin sulphate avidin-phycoerythrin behind the hybridization and react, then through Lu Mingkesi liquid-phase chip Equipment Inspection fluorescent signal.

2. said according to claim 1; A kind of multiple oligonucleotide joint detection probe that detects KCNJ2 and CAV3 transgenation combines the method for liquid-phase chip; It is characterized in that; The multiple oligonucleotide joint detection probe that is used for the KCNJ2 detection in Gene Mutation has comprised the wild-type sequence in 16 mutational sites of No. 2 exon of KCNJ2 gene, and said 16 mutational sites are 200G＞A, 223A＞G, 224C＞T, 283_294del, 407C＞T, 430G＞A, 488_493del, 557C＞T, 646A＞C, 652C＞T, 679G＞T, 899G＞T, 904G＞A, 907G＞A, 913A＞G and 940_945del.

3. said according to claim 1; A kind of multiple oligonucleotide joint detection probe that detects KCNJ2 and CAV3 transgenation combines the method for liquid-phase chip; It is characterized in that the multiple oligonucleotide joint detection probe sequence that is used for the KCNJ2 detection in Gene Mutation is SEQ ID No.1-SEQ ID No.32.

4. said according to claim 1; A kind of multiple oligonucleotide joint detection probe that detects KCNJ2 and CAV3 transgenation combines the method for liquid-phase chip; It is characterized in that; The multiple oligonucleotide joint detection probe that is used for the CAV3 detection in Gene Mutation has comprised the wild-type sequence of 4 nucleotide mutant sites of No. 2 exon of CAV3 gene, and described 4 mutational sites are 233C＞T, 253G＞A, 290T＞G, 423C＞G.

5. said according to claim 1; A kind of multiple oligonucleotide joint detection probe that detects KCNJ2 and CAV3 transgenation combines the method for liquid-phase chip; It is characterized in that the multiple oligonucleotide joint detection probe sequence that is used for the CAV3 detection in Gene Mutation is SEQ ID No.33-SEQ ID No.40.

6. said according to claim 1; A kind of multiple oligonucleotide joint detection probe that detects KCNJ2 and CAV3 transgenation combines the method for liquid-phase chip; It is characterized in that; Multiple oligonucleotide joint detection probe comprises a long probe and a short probe; Wherein long probe comprises 5 ' end and target gene specific sequence complementary hybridization sequences, 3 ' end common sequences and the specificity sequence label between hybridization sequences and common sequences, and short sequence comprises 3 ' end and target gene specific sequence complementary hybridization sequences and 5 ' end common sequences.