CN101845502A

CN101845502A - Probe amplification method with multiple connection and extending independency and kit thereof

Info

Publication number: CN101845502A
Application number: CN 201010183920
Authority: CN
Inventors: 陶炯; 孙维; 陈炜; 周华
Original assignee: XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Current assignee: XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Priority date: 2010-05-25
Filing date: 2010-05-25
Publication date: 2010-09-29

Abstract

The invention relates to a probe amplification method with multiple connection and extending independency, comprising the following steps that: (1) a DNA target sequence is denatured and is hybridized with an MLEPA probe; (2) extension and connection reaction is carried out; (3) PCR reaction is carried out; (4) PCR products are separated by capillary electrophoresis; (5) data processing is carried out on electrophoresis results; and the extension and connection reaction uses DNA polymerase and ligase simultaneously. The invention also provides a kit which can amplify and detect a plurality of DNA sites. The invention has the benefit of being capable of carrying out detection on the DNA sites simultaneously in primary reaction. Due to flexible design of the probe, the MLEPA technology can be flexibly and widely applied in detection of various STR sites and conservative DNA sites.

Description

A kind of multiple connection and the probe amplification method and the test kit thereof that extend dependence

[technical field]

The present invention relates to a kind of amplification method, specifically, is about a kind of multiple connection and the probe amplification method and the test kit thereof that extend dependence.

[background technology]

At present, (Multiplex Ligation dependent ProbeAmplification MLPA) is the Protocols in Molecular Biology in a plurality of DNA of a detection site of widely used middle isoflux in scientific research and Clinical Laboratory to the probe amplification technology of multiple join dependency.Generally speaking, MLPA is a kind of technology of multiplex PCR.Can detect the nearly copy numerical abnormality in 50 genomic dna sites simultaneously, minimum even can distinguish the sequence difference of a Nucleotide.MLPA is provided with the probe about 50 pairs in reaction, one section target sequence of the detection of every pair of probe specificity through PCR reaction, detects the dosage of all probes simultaneously, and and then detect different probe the dosage (or copy number) of corresponding target sequence.

Different with conventional multiplex PCR, in the MLPA reaction process, amplification be not target sequence, but hybridization of those and target sequence renaturation and connected probe.With respect to the multiplex PCR of routine, the PCR step of MLPA reaction only needs a pair of universal primer.The product length of MLPA amplification does not wait from 100bp to 500bp, can distinguish by capillary electrophoresis.By in the electrophoresis result, the fragment length of different bands and peak value can be judged the copy number variation of the detection site of correspondent probe correspondence simultaneously.

The MLPA whole test is crossed title can be divided into 5 steps: 1) DNA sex change and hybridize with the MLPA probe; 2) ligation; 3) PCR reaction; 4) the PCR product passes through capillary electrophoresis separation; 5) electrophoresis result is carried out data processing.

MLPA designs a pair of oligonucleotide probe at each target sequence, is called left and right probe.5 ' end of left side probe is for universal primer X, and 3 ' end is the hybridization region of itself and target sequence.5 ' end of right probe is its hybridization region with target sequence, and 3 ' holds and be the complementation district of universal primer Y.The hybridization region of left and right probe closely links to each other on target sequence, and the ligase enzyme that MLPA uses can only connect hybridization in same sequence and two sections closely continuous sequences, therefore, have only when corresponding left and right sides probe all correctly hybridizes to corresponding target sequence district, left and right sides probe just can be connected in the ligation.Can insert padding sequence between universal primer district and hybridization region to regulate the probe sequence length overall.MLPA is different with padding sequence at different its hybridization regions of target sequence designed probe, but all carries identical universal primer district X and Y.By the selection and the adjustment of padding sequence, can make different probes that length overall is had nothing in common with each other, in follow-up capillary electrophoresis, can separate like this according to the length difference.

In MLPA reaction, at first, the template DNA sex change, and mixes to hatch to hybridize with the MLPA probe and spend the night.In this process, corresponding probe and the hybridization of target sequence renaturation.After hybridization is finished, add ligase enzyme, have only the left and right sides probe of correct hybridization just can be connected.In ensuing PCR process, add fluorescently-labeled universal primer X, do not add fluorescently-labeled universal primer Y and DNA polymerase, those probes that do not connect are because only contain a universal primer district, in the PCR process, can not be amplified, can not contain corresponding fluorescent signal yet, and have only the probe of connection to be amplified, so connecting the quantity of product, probe is directly proportional with the quantity of target sequence in the template DNA is corresponding.Pcr amplification product passes through capillary electrophoresis separation.

Like this, in MLPA reaction, can have that nearly 50 target sequences are detected, with related software analytical electrophoresis result, the disappearance of the target sequence that different probe detected as can be known, amplification and copy number are unusual.MLPA is as simple, practical, a stable middle isoflux DNA detection technology, is widely used in clinical and the scientific research.

But, because the principle features of MLPA technology, the MLPA technology only can be used to detect DNA site conservative, that do not have polymorphism, can not be used to detect DNA site with length polymorphism, as STR site (short tandem repeats, STR), this makes the further application of MLPA technology be restricted.Simultaneously, owing to need to insert padding sequence in the probe of MLPA technology, when probe manufacturing, need to use the M13 phage to cultivate, time-consuming, complexity and cost are higher.

STR extensively is present in human and the other biological body genome.Its core sequence is generally 2-6bp, and the difference of core sequence multiplicity has formed abundant genetic polymorphism.Since STR genetic marker One's name is legion, the heterozygosity height, special and stable, make it become one of most important genetic marker of present genetic analysis, be widely used in recombination mapping, in population genetics and the linkage analysis.Although the application in STR site is so extensive, its main detection method still is the PCR detection in single STR site at present, wastes time and energy.Because there is tumor-necrosis factor glycoproteins in STR, amplification is difficulty comparatively, therefore, does not still have a kind of technology to carry out somatotype at a plurality of STR site neatly at present.If can develop a kind of simple, fast, flexibly in the STR somatotype detection technique of isoflux, its application prospect will be boundless.

[summary of the invention]

The objective of the invention is at deficiency of the prior art, a kind of multiple connection is provided and extends the probe amplification method that relies on.

One purpose more of the present invention is to provide a kind of and can increase and detect a plurality of DNA site kits simultaneously.

For achieving the above object, the technical scheme taked of the present invention is:

A kind of multiple connection and the probe amplification method that extends dependence, described method comprises the steps:

(1) DNA target sequence sex change and hybridize with the MLEPA probe;

(2) extension and ligation;

(3) PCR reaction;

(4) the PCR product passes through capillary electrophoresis separation;

(5) electrophoresis result is carried out data processing;

Described extension and ligation are to use the extension of DNA polymerase and ligase enzyme and ligation simultaneously.

MLEPA probe described in the method steps (1) is according to the oligonucleotide left side probe of DNA target sequence design and the right probe of oligonucleotide, 5 ' end of left side probe is universal primer X district, 3 ' end is the hybridization region of itself and target sequence, 5 ' end of right probe is the hybridization region of itself and target sequence, 3 ' end is the complementation district of universal primer Y, the hybridization region of oligonucleotide left side probe and the right probe of oligonucleotide has a spacer segment sequence on the DNA target sequence, DNA site with length polymorphism to be checked places this intervening sequence, by the repetition situation and the length polymorphism in the site to be checked in this intervening sequence of follow-up reaction detection.

Described DNA to be checked site can be to have length polymorphism STR site.

Described DNA to be checked site also can be does not have a conservative DNA sites of length polymorphism.

It is D5S112, D5S435, D5S557, D5S351 and D5S610 that described STR site is selected.

The selection of described conservative DNA sites is AR gene, sry gene and BRCA2 gene.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is: a kind ofly can increase and detect a plurality of DNA site kits simultaneously, comprise oligonucleotide left side probe, the right probe of oligonucleotide, DNA polymerase, ligase enzyme, described test kit also comprises multiple connection and extends the probe groups that relies on probe amplification method, has two pairs in the described probe groups at least according to site to be checked designed probe.

The present invention is in order to provide a kind of stable, technology flexibly that can detect a plurality of STR site simultaneously, at the defective of MLPA technology in the detection of STR site, we improve and invent out a kind of new detection technique---multiple connection with extend the probe amplification technology that relies on (Multiplex Ligation and Extensiondependent Probe Amplification, MLEPA).With respect to the MLPA technology, this MLEPA technology not only can detect conservative unique dna sequence dna, but also can carry out a plurality of detections with length polymorphism site, in the application in STR site and research very big prospect is arranged.

Present technique can not detect the shortcoming in the DNA site with length polymorphism at traditional MLPA technology, and the technical scheme of solution is:

The primitive reaction and the MLPA of MLEPA technology are similar, comprise 5 steps equally: 1) DNA sex change and hybridize with the MLEPA probe; 2) extension and ligation; 3) PCR reaction; 4) the PCR product passes through capillary electrophoresis separation; 5) electrophoresis result is carried out data processing.

Compared to the principle features of traditional MLPA, the variation of MLEPA maximum was second step: MLPA second is gone on foot the ligation of only using ligase enzyme, be improved to extension and the ligation of using DNA polymerase and ligase enzyme simultaneously.This change makes MLEPA not only have ligation in second step, also has extension simultaneously.This also causes the probe design of MLEPA and MLPA to be not quite similar.At each target sequence, MLEPA designs a pair of oligonucleotide probe equally, is called left and right probe.5 ' end of left side probe is for universal primer X, and 3 ' end is the hybridization region of itself and target sequence.5 ' end of right probe is its hybridization region with target sequence, and 3 ' holds and be the complementation district of universal primer Y.But, be different from the MLPA probe design, owing to second step existed extension, the hybridization region of the left and right probe of MLEPA not to need closely to link to each other, but can there be a spacer segment sequence betwixt on target sequence.Just because of the existence of this spacer segment sequence, MLEPA can have a mind to STR to be checked site is placed this intervening sequence when probe design, and repetition situation and length polymorphism by the STR site in this intervening sequence of follow-up reaction detection.Simultaneously,, can make different MLEPA probes that length overall is had nothing in common with each other, in follow-up capillary electrophoresis, can separate like this according to the length difference by the adjustment of intervening sequence.Therefore, MLEPA need not be provided with padding sequence yet and is used to distinguish final product length in probe.

The overall flow of MLEPA reaction is: in the first step, and the template DNA sex change, and mixes to hatch to hybridize with the MLEPA probe and spend the night.In this process, corresponding probe and the hybridization of target sequence renaturation.After hybridization is finished, add DNA polymerase and dna ligase, its left probe of left and right sides probe of correct hybridization is at first extended to right probe 5 ' by the DNA polymerase and locates, and the left probe and the right probe that will extend by ligase enzyme couples together then.In ensuing PCR process, add fluorescently-labeled universal primer X, do not add fluorescently-labeled universal primer Y and DNA polymerase, only finishing the probe that extends connection can be amplified, those probes that do not connect are because only contain a universal primer district, in the PCR process, can not be amplified, also can not contain corresponding fluorescent signal.The PCR product is by capillary electrophoresis separation, in a MLEPA reaction, is provided with manyly to probe at different STR sites, just can detect a plurality of STR site simultaneously in primary first-order equation.

The invention has the beneficial effects as follows:

(1) can in primary first-order equation, carry out the detection in a plurality of STR site simultaneously.

(2) since its probe design flexibly, the MLEPA technology can be applied in the detection in all types of STR site flexibly widely.

(3) on principle, the MLEPA technology also can be similar to the detection that traditional MLPA technology is applied to a plurality of conservative DNA sites copy numbers.

(4) the MLEPA technology is not owing to need the existence of padding sequence in the probe, therefore just can the satisfy the demands demand of its probe manufacturing of artificial synthetic oligonucleotide strand fast, in probe manufacturing, do not need to use the M13 phage to cultivate, with respect to traditional MLPA technology, fast, simply, cost is also lower relatively.

[description of drawings]

Fig. 1 is the reaction principle synoptic diagram of MLEPA.A represents sex change of MLEPA template and probe hybridization step among Fig. 1; B represents that MLEPA extends and Connection Step; C represents the PCR step of MLEPA; D represents that the PCR product of MLEPA carries out the step of capillary electrophoresis and analysis.Among Fig. 1,1. left probe 5 ' end universal primer X district; 2. left probe 3 ' end target sequence hybridization region; 3. right probe 5 ' end target sequence hybridization region; 4. the complementary district of right probe 3 ' end universal primer Y; 5.DNA target sequence; 6.DNA polymerase; 7. ligase enzyme; 8.PCR universal primer Y.

Fig. 2 is family SMA antenatal diagnosis STR linkage analysis MLEPA result to be checked among the embodiment 1.The left side be this family MLEPA product electrophoresis result figure among Fig. 2, and the right side is to diagnose STR linkage analysis figure term according to this family SMA of MLEPA interpretation of result gained.Left side A represents the paternal MLEPA product of this family electrophoresis result figure among Fig. 2; B represents this family maternal MLEPA product electrophoresis result figure; C represents this family propositus MLEPA product electrophoresis result figure; D represents this family fetus MLEPA to be checked product electrophoresis result figure; E represents to represent in the MLEPA electrophoresis result peak value scope in D5S112 site; F represents to represent in the MLEPA electrophoresis result peak value scope in D5S435 site; G represents to represent in the MLEPA electrophoresis result peak value scope in D5S557 site; H represents to represent in the MLEPA electrophoresis result peak value scope in D5S351 site; I represents to represent in the MLEPA electrophoresis result peak value scope in D5S610 site.What right side family member below showed among Fig. 2 is the str locus type, and STR site from last (kinetochore side) to following (telomere side) is respectively D5S435, D5S557, D5S610, D5S351 and D5S112.The arrow indication is the propositus.Represent with black that with the monoploid that disease is chain normal monoploid is represented with white.

Fig. 3 is the single STR PCR result of family SMA antenatal diagnosis STR linkage analysis to be checked among the embodiment 1, and this family SMA according to single STR pcr analysis gained of expression diagnoses STR linkage analysis figure term.What the family member below showed is the str locus type, and STR site from last (kinetochore side) to following (telomere side) is respectively D5S435, D5S557, D5S610, D5S351 and D5S112.The arrow indication is the propositus.Represent with black that with the monoploid that disease is chain normal monoploid is represented with white.

Fig. 4 is that the sex chromosome ploidy of 3 routine samples among the embodiment 2 detects MLEPA electrophoresis result peak figure.A represents the blank sample among Fig. 4, and B represents sample No. 1, and C represents sample No. 2, and D represents sample No. 3.Arrow 1 indication is universal primer PCR dimer peak, and arrow 2 indications are for detecting the MLEPA product peak of AR gene, and arrow 3 indications are for detecting the MLEPA product peak of sry gene, and arrow 4 indications are for detecting the MLEPA product peak of BRCA2 gene.

[embodiment]

Below in conjunction with accompanying drawing the specific embodiment of the present invention is elaborated.

Specific embodiment 1:MLEPA is applied to the detection in STR site---and the MLEPA of spinal muscular atrophy prenatal gene diagnosis STR linkage analysis detects

(spinal muscular atrophy is one of modal children's lethality autosomal recessive hereditary diseases SMA) to spinal muscular atrophy, and crowd's sickness rate is about 1/10,000, and Disease-causing gene crowd carrying rate is about 1/50.SMA is main pathological characters with the anterior horn motor neurons sex change, and clinical manifestation is carrying out property, symmetry myasthenia and the myatrophy due to the lower motor neuron lesion.Respiratory system complication due to respiratory muscle is got involved is to cause most infant main causes of death.(survival motor neuron gene 1 SMN1) is this sick Disease-causing gene to be positioned at the survival motor neuronal gene 1 in No. 5 long-armed 5q13.3 of karyomit(e) zones.Have now and studies show that about 95% SMA patient is by due to the SMN1 homozygous deletion.Because father and mother's majority of SMA infant is the SMA carrier, infant mother undergoes another pregnancy and still has 25% probability fertility SMA infant.Therefore fetus at risk being carried out prenatal gene diagnosis, is the effective measure of prevention SMA.

(PCR-restriction fragment lengthpolymorphism PCR-RFLP) directly detects No. 7 exon homozygous deletions of SMN1 gene and judges whether fetus gets involved can to pass through the PCR-enzyme cutting method in the SMA antenatal diagnosis.Yet, carrying the means of situation as getting rid of source of parents pollution, checking PCR-RFLP result and judgement, linkage analysis still has important effect in the SMA antenatal diagnosis.The STR linkage analysis is a kind of traditional prenatal gene diagnosis method, since nineteen ninety-two, have 10 in the bibliographical information surplus kind of a STR site be applied in the SMA antenatal diagnosis.Domestic existing at present many pieces are adopted the STR linkage analysis to cooperate additive method to carry out the report of SMA prenatal gene diagnosis.

We use the STR site (D5S112, D5S435, D5S557, D5S351 and D5S610) of 5 widespread uses in the SMA antenatal diagnosis, at these site designs MLEPA probe groups, use the MLEPA technology these 5 sites are detected simultaneously.We are applied to this cover probe in the STR linkage analysis of one routine SMA antenatal diagnosis, and use traditional single STR PCR the result of MLEPA is verified.

Carrying out in the family of SMA antenatal diagnosis in this example, have the propositus of a SMA, was SMA in my institute's clinical diagnosis once, cut gene diagnosis through the PCR-enzyme and was diagnosed as No. 7 exon homozygous deletions of SMN1.Mother of propositus undergoes another pregnancy, and fetus is SMA high risk population, requires to carry out the SMA prenatal gene diagnosis.SMA family member (paternal, maternal and propositus) extracts peripheral blood 2ml, EDTA anti-freezing.The fetus sample that is used for antenatal diagnosis is extracted under the B ultrasonic guiding by my obstetrics of institute from high risk gravida pregnant 17～19 all amniotic fluid, through the conventional post analysis of cultivating of centrifugal collection.Peripheral blood and cultivation amniocyte use TOPure ^TMBlood DNA extracts test kit and extracts genomic dna (available from Shanghai Gene science company).The family genomic dna sample that extracts is dissolved among the TE, and it is stand-by to be adjusted to concentration 50～100ng/ul.

The MLEPA experiment analytical method:

1. the design and fabrication of probe groups and universal primer: at 5 STR sites design MLEPA probes to be checked, principle of design is (designing probe give birth to the worker by Shanghai synthetic) as previously mentioned, and wherein right probe 5 ' end all carries out phosphorylation.5 pairs of MLEPA probes that design are mixed to final concentration 2～5nM, are dissolved among the TE, making becomes MLEPA probe groups mixed solution.Synthetic universal primer X of design and Y, wherein universal primer X is used for the capillary electrophoresis of later stage PCR product in 5 ' end mark 6-FAM fluorescent mark.(table 1)

Table 1 is applied to the MLEPA probe and the universal primer information of SMA antenatal diagnosis STR linkage analysis

L represents MLEPA left side probe in the table, and R represents the right probe of MLEPA.

2.MLEPA reaction: MLEPA reacts hybridization step, reaction buffer that extension and Connection Step are used and ligase enzyme Ligase-65 are all available from MRC-Holland company (Amsterdam, Holland).

1) hybridization step: 98 ℃ of pre-sex change of 5min of 5ul dna sample, then be cooled to 25 ℃, add the MLPA buffer of 1.5ul and the MLEPA probe groups mixed solution of 1.5ul, hybridized 16 hours in 60 ℃ behind 95 ℃ of 2min.

2) extend and to add 32ul in the sample that spends the night with Connection Step: 8ul hybridization and extend and be connected mixed solution (extension and be connected mixed solution by 3ul Ligase-65Buffer A, 3ul Ligase-65 Buffer B, 1ul 10mM dNTPs, 1ul Ligase-65 ligase enzyme, 0.2ul r-Taq archaeal dna polymerase (the precious biotech firm in Dalian) and 23.8ul ddH ₂O forms), behind 54 ℃ of reaction 20min, 98 ℃ of 5min termination reactions obtain 40ul MLEPA extension and are connected product.

3) PCR reaction: 25ul PCR system comprises 5ul MLEPA extension and is connected product, the Betaine of 4ul 5M (available from U.S. Promega company), 0.5ul universal primer X and the Y of 10uM, the 2 * Premix Taq Hot Start Version of 12.5ul (available from the precious biotech firm in Dalian) and 2.5ulddH ₂O.Placing east to win imperial PCR instrument (east, Beijing KingMax new company) reacts.The PCR response procedures is: 98 ℃ of pre-sex change 3min; 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 20min, 4 ℃ of preservations.

4) after the PCR product uses 3730DNA analyser (American AB I company) to carry out capillary electrophoresis, through GeneMapper software analysis PCR product sheet segment length.The different PCR product length in same STR site can be judged as different allelotypes, carries out STR somatotype and family linkage analysis according to the length of PCR product.

Single STR PCR experiment analytical method:

1.PCR design of primers: design single PCR primer (it is synthetic by the living worker in Shanghai to have designed primer) at 5 STR sites to be checked, the forward primer of every pair of primer is used for the capillary electrophoresis of later stage PCR product to its 5 ' end mark 6-FAM fluorescence.(table 2)

Table 2 is applied to the single STR PCR primer information of SMA antenatal diagnosis STR linkage analysis

F represents used forward primer among the PCR in the table, and R represents used reverse primer among the PCR.

2. single STR PCR reaction: comprise genomic dna 2.5～5ng/ul in the 20ul PCR body, each 0.2uM of forward and reverse primer, 0.25mM dNTPs (available from the precious biotech firm in Dalian), 1.875mM Mg ²⁺(available from the precious biotech firm in Dalian), 1 * Gold Buffer (available from American AB I company), 1M Betaine (available from U.S. Promega company), 0.05U/ul Gold Taq polysaccharase (available from American AB I company) places east to win imperial PCR instrument (east, Beijing KingMax new company) and reacts.The PCR response procedures is: 95 ℃ of pre-sex change 8min; 95 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 60s, 35 circulations; 72 ℃ of 10min, 4 ℃ of preservations.After the PCR product uses 3730DNA analyser (American AB I company) to carry out capillary electrophoresis, through GeneMapper software analysis PCR product sheet segment length.The different PCR product length in same STR site can be judged as different allelotypes, carries out STR somatotype and family linkage analysis according to the length of PCR product.

This example family is through behind STR linkage analysis of MLEPA, and fetus str locus type is identical with propositus in the family, can judge that definite its fetus is the SMA patient (Fig. 2) that gets involved.The interlocking analytical results identical (Fig. 3) of the single STR PCR of this result and routine.STR linkage analysis practice by this example SMA antenatal diagnosis illustrates that MLEPA can finish the STR somatotype fully rapidly and accurately, and is applied in the linkage analysis.

In the STR linkage analysis of SMA antenatal diagnosis, use the MLEPA technology only need to family to be checked carry out 4 PCR reactions both can, and the single STR PCR that produces effect same need carry out 20 PCR reactions.As seen SMA antenatal diagnosis STR linkage analysis by this family is put into practice, and the MLEPA technology is carried out STR analytical results easy, reliable (with widely used single STR PCR result is in full accord at present).By the method for MLEPA, can simplify the experimental procedure of STR linkage analysis, make that the application in STR site and research are quick more, rapid.

Specific embodiment 2:MLEPA is applied to the detection of conservative DNA sites copy number---and the MLEPA of sex chromosome ploidy detects

MLEPA not only can detect the STR site with length polymorphism, simultaneously, similar MLPA, the copy number that also can carry out conservative DNA sites detects.

In order to detect the sex chromosome ploidy situation of sample, at the special conservative AR gene of X chromosome, the special conservative sry gene of Y chromosome and No. 13 special conservative BRCA2 gene design MLEPA probes of karyomit(e), and preparation becomes the MLEPA probe groups that the sex chromosome ploidy detects, set up the MLEPA detection method of sex chromosome ploidy, and be applied to verify its reliability in the different sample of the known sex chromosome ploidy of caryogram.

3 routine experiment samples all show band analysis through Chromosome G, and caryogram is known.Wherein No. 1 sample is normal women, and caryogram is 46, XX; No. 2 samples are normal male, and it is 46 that G shows the band caryogram, XY; No. 3 samples are the unusual male sex of caryogram, and caryogram is a Y chromosome equiarm dicentric chromosome, 46, and X, idic (Y) is (p11.3).The theoretical value of the X of this 3 routine sample, Y chromosome and No. 13 karyomit(e) dose ratio was respectively 2: 0: 2,1: 1: 2 and 1: 2: 2.3 routine sample sources are peripheral blood 2ml, EDTA anti-freezing.Use TOPure ^TMBlood DNA extracts test kit and extracts genomic dna (Shanghai Gene science company).The genomic dna sample that extracts is dissolved among the TE, and it is stand-by to be adjusted to concentration 50～100ng/ul.

The MLEPA experiment analytical method:

The design and fabrication of probe groups and universal primer: at 3 gene locus to be checked (AR genes, sry gene and BRCA2 gene) design MLEPA probe, principle of design is (designing probe is synthetic by the living worker in Shanghai) as previously mentioned, and wherein right probe 5 ' end all carries out phosphorylation.5 pairs of MLEPA probes that design are mixed to final concentration 2～5nM, are dissolved among the TE, making becomes MLEPA probe groups mixed solution.Synthetic universal primer X of design and Y, wherein universal primer X is used for the capillary electrophoresis of later stage PCR product in 5 ' end mark 6-FAM fluorescent mark.(table 3)

Table 3 is applied to MLEPA probe and the universal primer information that the sex chromosome ploidy detects

1.MLEPA reaction: MLEPA reacts hybridization step, reaction buffer that extension and Connection Step are used and ligase enzyme Ligase-65 are all available from MRC-Holland company (Amsterdam, Holland).

2) extend and to add 32ul in the sample that spends the night with Connection Step: 8ul hybridization and extend and be connected mixed solution (extension and be connected mixed solution by 3ul Ligase-65Buffer A, 3ul Ligase-65Buffer B, 1ul 10mM dNTPs, 1ul Ligase-65 ligase enzyme, 0.2ul r-Taq archaeal dna polymerase (the precious biotech firm in Dalian) and 23.8ul ddH ₂O forms), behind 54 ℃ of reaction 20min, 98 ℃ of 5min termination reactions obtain 40ul MLEPA extension and are connected product.

3) PCR reaction: 25ul PCR system comprises 5ul MLEPA extension and is connected product, universal primer X and the Y of 0.5ul 10uM, the 2 * Premix Taq Hot Start Version of 12.5ul (available from the precious biotech firm in Dalian) and 6.5ul ddH ₂O.Placing east to win imperial PCR instrument (east, Beijing KingMax new company) reacts.The PCR response procedures is: 98 ℃ of pre-sex change 3min; 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 20min, 4 ℃ of preservations.

4) after the PCR product uses 3730DNA analyser (American AB I company) to carry out capillary electrophoresis, through GeneMapper software analysis PCR product sheet segment length and respective segments electrophoresis fluorescence peak value (F).

2. copy number analysis calculation method: in 3 samples, establish wherein that No. 2 samples (normal male) are normal sample for reference R, all the other two samples are sample Tn to be checked, n=1, and 2, wherein No. 1 sample is T ₁, No. 3 samples are T ₂AR, the fluorescent value of SRY and three detection site of BRCA2 is A, S and B.Three detection site fluorescent values of sample for reference R are respectively R _A, R _SAnd R _BThe fluorescent value of three detection site of sample Tn to be checked is Tn _A, Tn _SAnd Tn _B

1) probe normalization method: because sample for reference R is the normal male sample, its X chromosome: Y chromosome: No. 13 chromosomal theoretical dose ratio is 1: 1: 2.Therefore, treat sample detection site fluorescent value originally by following formula normalization method, the value after the normalization method is Tn ' _A, Tn ' _SAnd Tn ' _B:

Tn’ _A＝Tn _A×R _S×R _B

Tn’ _S＝Tn _S×R _A×R _B

Tn’ _B＝2×Tn _B×R _A×R _S

2) sample dyeing body copy number to be checked is analyzed: sample MLEPA to be checked detects the gained X chromosome: Y chromosome: No. 13 chromosomal dosage ratio is calculated as follows:

X chromosome copy number: Y chromosome copy number: No. 13 chromosome copies number=Tn ' _A: Tn ' _S: Tn ' _B

These 3 samples are after MLEPA analyzes, and results peaks figure as shown in Figure 4.Its photoluminescence peak data see Table 4.

Three sample MLEPA of table 4 product capillary electrophoresis result

With No. 2 samples (normal male) is normal sample for reference R, calculates the X chromosome of all the other two samples: Y chromosome: No. 13 chromosomal dosage ratios, and the MLEPA analytical results is:

The X chromosome copy number of No. 1 sample: Y chromosome copy number: No. 13 chromosome copies numbers=1.96: 0: 2

The X chromosome copy number of No. 3 samples: Y chromosome copy number: No. 13 chromosome copies numbers=0.89: 1.82: 2

This result, very approaching with X, Y chromosome and No. 13 theoretical ratios of karyomit(e) dosage of No. 1 sample and No. 3 samples.This explanation, MLEPA also can be applicable to special conservative dna sequence dna and detects, and can finish the copy number check of target sequence, and its result also is reliable.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

SEQUENCE?LISTING

＜110〉Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ.

＜120〉a kind of multiple connection and the probe amplification method and the test kit thereof that extend dependence

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<400>6

caacactctg?tgtttcactt?gttttttcct?tatgtcaggt?cgtagatagc?gtagcccg 58

<210>7

<211>55

<212>DNA

＜213〉artificial sequence

<400>7

gggtagcgtt?gcgaatgatg?tctggagttt?gagaccagtc?taggcaacac?agcga 55

<210>8

<211>57

<212>DNA

＜213〉artificial sequence

<400>8

ctaaagtggc?aatgctcatc?agcattttcg?gtctcaggtc?gtagatagcg?tagcccg 57

<210>9

<211>54

<212>DNA

＜213〉artificial sequence

<400>9

gggtagcgtt?gcgaatgatg?tctgtgcttc?agtttggata?gttaaaattg?tcct 54

<210>10

<211>43

<212>DNA

＜213〉artificial sequence

<400>10

gcactttagg?aggccaaggt?ggggtcgtag?atagcgtagc?ccg 43

<210>11

<211>21

<212>DNA

＜213〉artificial sequence

<400>11

gggtagcgtt?gcgaatgatg?t 21

<210>12

<211>20

<212>DNA

＜213〉artificial sequence

<400>12

cgggctacgc?tatctacgac 20

<210>13

<211>20

<212>DNA

＜213〉artificial sequence

<400>13

tacaaagtcc?tgagccaaat 20

<210>14

<211>21

<212>DNA

＜213〉artificial sequence

<400>14

accagtctat?ggcaacacag?c 21

<210>15

<211>19

<212>DNA

＜213〉artificial sequence

<400>15

acacacatgc?acgctctct 19

<210>16

<211>22

<212>DNA

＜213〉artificial sequence

<400>16

caagagcaca?gtttggagtg?ag 22

<210>17

<211>21

<212>DNA

＜213〉artificial sequence

<400>17

aagtgaaaca?cagaggttga?c 21

<210>18

<211>21

<212>DNA

＜213〉artificial sequence

<400>18

ggtgaatgtt?tgatgaccct?a 21

<210>19

<211>25

<212>DNA

＜213〉artificial sequence

<400>19

ggagtttgag?accagtctag?gcaac 25

<210>20

<211>25

<212>DNA

＜213〉artificial sequence

<400>20

tgagaccgaa?aatgctgatg?agcat 25

<210>21

<211>21

<212>DNA

＜213〉artificial sequence

<400>21

tcctgtcctc?aagtgaccct?c 21

<210>22

<211>27

<212>DNA

＜213〉artificial sequence

<400>22

gtttggatag?ttaaaattgt?cctgttt 27

<210>23

<211>58

<212>DNA

＜213〉artificial sequence

<400>23

gggtagcgtt?gcgaatgatg?ttgggaatac?tcagcagtat?cttcagtgct?cttgcctg 58

<210>24

<211>51

<212>DNA

＜213〉artificial sequence

<400>24

cgctgtcgtc?tagcagagaa?cctttgcatt?cgtcgtagat?agcgtagccc?g 51

<210>25

<211>52

<212>DNA

＜213〉artificial sequence

<400>25

gggtagcgtt?gcgaatgatg?tgactctggc?acctttcaat?tttgtcgcac?tc 52

<210>26

<211>55

<212>DNA

＜213〉artificial sequence

<400>26

tgctatgtta?agcgtattca?acagcgatga?ttacagtcgt?agatagcgta?gcccg 55

<210>27

<211>50

<212>DNA

＜213〉artificial sequence

<400>27

gggtagcgtt?gcgaatgatg?tctctggatt?tagtacagca?agtggaaagc 50

<210>28

<211>51

<212>DNA

＜213〉artificial sequence

<400>28

ctgagcatag?tcttcactat?tcacctacgt?cgtcgtagat?agcgtagccc?g 51

<210>29

<211>21

<212>DNA

＜213〉artificial sequence

<400>29

gggtagcgtt?gcgaatgatg?t 21

<210>30

<211>20

<212>DNA

＜213〉artificial sequence

<400>30

cgggctacgc?tatctacgac 20

Claims

A multiple connection with extend the probe amplification method that relies on, it is characterized in that described method comprises the steps:

(1) DNA target sequence sex change and hybridize with the MLEPA probe;

(2) extension and ligation;

(3) PCR reaction;

(4) the PCR product passes through capillary electrophoresis separation;

(5) electrophoresis result is carried out data processing;

Described extension and ligation are to use the extension of DNA polymerase and ligase enzyme and ligation simultaneously.
2. method according to claim 1, MLEPA probe described in the method steps (1) is according to the oligonucleotide left side probe of DNA target sequence design and the right probe of oligonucleotide, 5 ' end of left side probe is universal primer X district, 3 ' end is the hybridization region of itself and target sequence, 5 ' end of right probe is the hybridization region of itself and target sequence, 3 ' end is the complementation district of universal primer Y, it is characterized in that, the hybridization region of oligonucleotide left side probe and the right probe of oligonucleotide has a spacer segment sequence on the DNA target sequence, if site to be checked is the DNA site with length polymorphism, the dna sequence dna that then will have length polymorphism places this intervening sequence, repetition situation and length polymorphism by the site to be checked in this intervening sequence of follow-up reaction detection, if site to be checked is a conservative DNA sites, then regulates final PCR product length and check corresponding site by follow-up electrophoresis by the length adjustment of intervening sequence.
3. method according to claim 2 is characterized in that, described DNA to be checked site can be the STR site with length polymorphism.
4. method according to claim 2 is characterized in that, described DNA to be checked site also can be does not have a conservative DNA sites of length polymorphism.
5. method according to claim 3 is characterized in that, it is D5S112, D5S435, D5S557, D5S351 and D5S610 that described STR site is selected.
6. method according to claim 3 is characterized in that, it is the AR gene that described conservative DNA sites is selected, sry gene and BRCA2 gene.
7. one kind can be increased simultaneously and detects a plurality of site kits, comprise oligonucleotide left side probe, the right probe of oligonucleotide, DNA polymerase, ligase enzyme, it is characterized in that, described test kit also comprises multiple connection and extends the probe groups that relies on probe amplification method, has two pairs in the described probe groups at least according to site to be checked designed probe.