CN102586420A

CN102586420A - Method and kit for assaying breast cancer susceptibility genes

Info

Publication number: CN102586420A
Application number: CN2011104451567A
Authority: CN
Inventors: 盛司潼
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-12-27
Filing date: 2011-12-27
Publication date: 2012-07-18
Anticipated expiration: 2031-12-27
Also published as: CN102586420B

Abstract

The invention relates to the field of genetic engineering, and provides a method and a kit for assaying breast cancer susceptibility genes. The method for assaying breast cancer susceptibility genes includes the following steps: (A) breast cancer susceptibility gene specific primers are utilized to amplify a plurality of target regions in a sample to be assayed, and a sequencing library is constructed on the basis of amplification products; (B) the single-molecule amplification of the sequencing library is carried out, so that a plurality of single-molecule amplification products corresponding to the target regions are obtained; (C) the high-throughput gene sequencing of the single-molecule amplification products is carried out simultaneously, so that the sequence information of the target regions is obtained. The method and the kit for assaying breast cancer, which are provided by the invention, can assay a plurality of regions of a breast cancer susceptibility gene at the same time, consequently, the efficiency of assay is increased, the assay cost is reduced, and in addition, the sensitivity of assay and the comprehensiveness and accuracy of the assay result are increased as well.

Description

A kind of method and test kit that detects breast cancer susceptibility gene

Technical field

The present invention relates to the genetically engineered field, more particularly, relate to a kind of method and test kit that detects breast cancer susceptibility gene.

Background technology

Tumor susceptibility gene is meant with specified disease to have certain related gene or allelotrope.Tumor susceptibility gene and mutational site to complex disease are detected, and can obtain its concrete gene order information.On this basis; Can combine the statistical study of follow-up further molecular biology test, clinical trial, clinical observation and integrated data; Set up disease model, thereby realize the susceptibility of early stage assess disease and take corresponding preventive measures to reduce the occurrence probability of disease.Big quantity research confirms that inherited genetic factors plays a significant role in the susceptibility of disease, the own alleged occurrence minor gene of the inherited genetic factors of complex disease dosage effect, i.e. and adding up of minor gene effect just can form the obvious phenotypes combined effect.Therefore; A plurality of tumor susceptibility genes and mutational site to disease are detected most important; Particularly to topmost genetic mutation form---SNP (Single Nucleotide Polymorphisms in the genome; SNP), it accounts for 92% of genetic mutation in the whole genome.

Mammary cancer is one of common cancer that has a strong impact on WomanHealth, and the whole world has 1,200,000 women that mammary cancer takes place every year approximately, has 500,000 women to die from mammary cancer, and China women's breast cancer incidence also rises just year by year.Clinical showing, the curative ratio of breast carcinoma of early stage can reach 80～90%, thus early detection and the prevention important effect is played in the treatment of mammary cancer.

Breast cancer susceptibility gene is the gene with tight association with mammary cancer.At present; The common methods that detects breast cancer susceptibility gene is the Sanger PCR sequencing PCR; This method can be carried out the zone to breast cancer susceptibility gene and detected, but the Sanger PCR sequencing PCR can only check order to a certain section zone of a sample at every turn, so detection efficiency is low, cost is high; And because the restriction of sanger order-checking principle, meeting showed double peaks and even multimodal make this sample accurately to identify when detection has the sample signal of heterozygote, cause detection sensitivity low (20%).

In addition; The another kind of domestic method that is used for the breast cancer susceptibility gene detection is a gene chips; This method can only form detection chip to the site design detection probes of or several known mutations, therefore can't be used to detect the unknown mutation site, and the result of detection also just inadequately comprehensively, accurately.

At present on the market based on the formed detection kit of the detection method of prior art, only detect to single zone or known one or several SNP sites, detected result is inadequately comprehensively accurately.

Therefore need a kind of novel method and novel agent box that detects breast cancer susceptibility gene, can improve detection efficiency, reduce cost, can also improve detection sensitivity and accuracy.

Summary of the invention

The object of the present invention is to provide a kind of method and test kit that detects breast cancer susceptibility gene, can detect simultaneously, improve detection efficiency, reduce cost, can also improve detection sensitivity and accuracy a plurality of zones of breast cancer susceptibility gene.

In order to realize goal of the invention, a kind of method that detects breast cancer susceptibility gene may further comprise the steps:

A. utilize the breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in the testing sample, and make up the order-checking library based on amplified production;

B. the unit molecule amplification is carried out in the order-checking library, obtained a plurality of unit molecule amplified productions corresponding with said a plurality of target areas;

C. simultaneously said a plurality of unit molecule amplified productions are carried out the high-throughput gene sequencing, obtain the sequence information of said a plurality of target areas.

Wherein, steps A comprises:

A1. utilize the breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in the testing sample, obtain and the corresponding amplified production in said a plurality of target areas;

A2. utilize joint component, connect, obtain the library of checking order with the corresponding amplified production in said a plurality of target areas; Said joint component adopts at least a in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure.

Wherein, said steps A 2 may further comprise the steps:

A21. the amplified production corresponding with said a plurality of target areas carried out fragmentation, obtain the fragmentation product;

A22. utilize joint component, be connected, make up the order-checking library with said fragmentation product.

Wherein, said steps A 22 may further comprise the steps:

A221. utilize first joint to be connected, the connection product of winning with the two ends of fragmentation product;

A222. cyclisation first connects product, gets cyclisation product;

A223.II s type digestion with restriction enzyme cyclisation product gets enzyme and cuts product;

A224. cut the product two ends at enzyme and connect second joint and the 3rd joint, the library of must checking order.

Wherein, said steps A 22 may further comprise the steps:

A221 '. utilize the 4th joint to be connected, get second and connect product with the fragmentation product;

A222 ' .II s type digestion with restriction enzyme second connects product, must have the endonuclease bamhi of the 4th joint;

A223 '. the endonuclease bamhi that has the 4th joint is connected with the 5th joint, forms the order-checking library.

Wherein, at least one joint in the steps A 2 said joint components includes first sequence label, is used in the library construction process, and mark is carried out in the order-checking library of different testing samples.Said first sequence label is preferably the nucleic acid molecule that has particular sequence, is used to discern different testing samples.The said first sequence label base number is not limit, and is preferably 3～20, and more preferably 4～10.

Wherein, Breast cancer susceptibility gene described in the steps A has a plurality of; In the Auele Specific Primer of the target area correspondence of each mammary gland tumor susceptibility gene at least one and this target area part complementation; And 5 ' end of this primer has second sequence label, is used in amplification target area process, and the target area amplified production of different testing samples is carried out mark.Said second sequence label is preferably the nucleic acid molecule that has particular sequence, and its base number is not limit, and is preferably 3～20, and more preferably 4～10.

Wherein, said tumor susceptibility gene to be measured comprises at least one among FGFR2, GSTP1, IL-1 β and the MTHFR.

Further, the Auele Specific Primer of said FGFR2 is SEQ ID NO:1 and SEQ ID NO:2; The Auele Specific Primer of said GSTP1 is SEQ ID NO:3 and SEQ ID NO:4; The Auele Specific Primer of said IL-1 β is SEQ ID NO:5 and SEQ ID NO:6; The Auele Specific Primer of said MTHFR is SEQ ID NO:7 and SEQ ID NO:8.

Wherein, said method also comprises step:

D. utilize the specificity amplification primer of GSTM1 testing sample to be carried out pcr amplification, the deletion condition of detected through gel electrophoresis GSTM1 gene.

Wherein, all there is not the sequencing relation between step D and steps A, B or the C.

Further, the amplimer of said GSTM1 comprises at least one pair of among SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and the SEQ ID NO:12.

A kind of test kit that can be used in any detection breast cancer susceptibility gene method of the present invention comprises:

The breast cancer susceptibility gene Auele Specific Primer is used for being increased in a plurality of target areas of testing sample;

Joint component is used for combining with amplified production making up the order-checking library.

Wherein, said joint component adopts at least a in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure.

Wherein, at least one joint in the said joint component includes first sequence label, is used in the library construction process, and mark is carried out in the order-checking library of different testing samples.

Said first sequence label is preferably the nucleic acid molecule that has particular sequence, and its base number is not limit, and is preferably 3～20, and more preferably 4～10.

Wherein, Said breast cancer susceptibility gene to be measured has a plurality of; Have at least a primer and target area part complementary in the corresponding Auele Specific Primer; And 5 ' end of this primer has second sequence label, and said second sequence label is used for the process in the amplification target area, and the target area amplified production of different testing samples is carried out mark.

Said second sequence label is the nucleic acid molecule that has particular sequence, and the base number is not limit, and is preferably 3～20, more preferably 4～10.

Wherein, said breast cancer susceptibility gene comprises at least one among FGFR2, GSTP1, IL-1 β and MTHFR and the GSTM1.

Further, the Auele Specific Primer of said FGFR2 is SEQ ID NO:1 and SEQ ID NO:2; The Auele Specific Primer of said GSTP1 is SEQ ID NO:3 and SEQ ID NO:4; The Auele Specific Primer of said IL-1 β is SEQ ID NO:5 and SEQ ID NO:6; The Auele Specific Primer of said MTHFR is SEQ ID NO:7 and SEQ ID NO:8; The Auele Specific Primer of said GSTM1 comprises at least one pair of among SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and the SEQ ID NO:12.

By on can know that the method for detection breast cancer susceptibility gene provided by the invention and test kit can detect a plurality of zones of breast cancer susceptibility gene simultaneously, therefore improved detection efficiency, reduced the detection cost; This method also can break through the restriction of traditional detection method in addition, can discern the sample that includes heterozygote, has therefore improved detection sensitivity; Can also detect the variation situation that comprises known mutations and unknown mutation site in addition, therefore improve the comprehensive and accuracy of detected result.

Description of drawings

Fig. 1 is the method flow diagram that detects breast cancer susceptibility gene in the one embodiment of the invention;

Fig. 2 is the method flow diagram that the present invention is used to make up the order-checking library;

Fig. 3 is the structural representation of the protruding terminus joint in the one embodiment of the invention;

Fig. 4 is the structural representation of the joint of the band loop-stem structure in the one embodiment of the invention;

Fig. 5 is the structural representation of the joint of the band loop-stem structure in the another embodiment of the present invention;

Fig. 6 is the structural representation of the used y splice of the present invention;

Fig. 7 is the structural representation of the y splice in the one embodiment of the invention;

Fig. 8 is the structural representation of the y splice in the another embodiment of the present invention;

Fig. 9 is the structural representation of the y splice in the another embodiment of the present invention;

Figure 10 utilizes fragmentation product and joint component to make up the method flow diagram in order-checking library in the one embodiment of the invention;

Figure 11 utilizes fragmentation product and joint component to make up the method flow diagram in order-checking library in the another embodiment of the present invention;

Figure 12 is the method flow diagram that detects breast cancer susceptibility gene in the another embodiment of the present invention.

Embodiment

In order to make the object of the invention, technical scheme and advantage clearer,, the present invention is further elaborated below in conjunction with accompanying drawing and embodiment.

Target area of the present invention; Be the arbitrary sequence on the breast cancer susceptibility gene; Can select as required; Include but not limited to the regulation and control zone, outside of internal sequence, the gene of gene, said gene internal sequence includes but not limited to the intron zone, exon region of gene, contains the zone of intron and exon simultaneously.

Fig. 1 shows a kind of method flow that detects breast cancer susceptibility gene of the present invention, and this method may further comprise the steps:

S1. utilize the breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in the testing sample, and make up the order-checking library based on amplified production;

S2. the unit molecule amplification is carried out in the order-checking library, obtained a plurality of unit molecule amplified productions corresponding with said a plurality of target areas;

S3. simultaneously said a plurality of unit molecule amplified productions are carried out the high-throughput gene sequencing, obtain the sequence information of said a plurality of target areas.

Present method utilizes high throughput sequencing technologies that breast cancer susceptibility gene is carried out the zone order-checking, can detect simultaneously a plurality of zones of breast cancer susceptibility gene, improves detection efficiency, reduces cost; Utilize high-throughput gene sequencing technological breakthrough traditional detection technology limitation simultaneously, improve detection sensitivity; Simultaneously can also detect the variation situation that comprises known mutations and unknown mutation site, obtain comprehensive and accurate detected result.

Detect the breast cancer susceptibility gene sequence information that obtains through present method; Can combine the statistical study of follow-up further molecular biology test, clinical trial, clinical observation and integrated data; Set up certain breast cancer disease model, realize the susceptibility of early stage assessment mammary cancer.

Need to prove:

Wherein, testing sample described in the step S1 is the sample that can extract the arbitrary form of nucleic acid, includes but not limited to: whole blood, serum, blood plasma and tissue sample; Said tissue sample includes but not limited to: paraffin-embedded tissue, flesh tissue and frozen section.

A plurality of target areas described in the step S1, they can derive from same breast cancer susceptibility gene, also can derive from different breast cancer susceptibility genes.

In the step S1 gained order-checking library; There is multiple order-checking library molecule; The unit molecule amplification is carried out in the order-checking library, promptly be meant, with the multiple library molecule in the order-checking library; Form with denier (even unit molecule) is spatially isolated (but these library molecules still belong to same reaction system on the whole), and in space separately, realizes amplification.

In the prior art, the Sanger sequencing technologies can only check order to a certain section zone of a sample at every turn, realize the order-checking to a plurality of target areas, can only be to realize through repeatedly reacting.And in the present invention; After each molecule in the order-checking library increases through unit molecule; Each order-checking library molecule all forms unit molecule copy array, and each unit molecule copy array is in different positions when carrying out the high-throughput gene sequencing, make sequencing primer and unit molecule copy the hybridization between the array; And the extension under the enzyme effect can carry out simultaneously, do not disturb mutually each other.Therefore, can be simultaneously a large amount of (millions of up to ten million, even more) unit molecule copy array be carried out sequencing reaction simultaneously, then through gathering corresponding signal, and then obtain required sequence information, and the sensitivity of order-checking is higher than Sanger.

Wherein, the complementary fully or part complementation in described Auele Specific Primer of step S1 and target area.

Further, every pair to have at least in the Auele Specific Primer of target area one complementary wholly or in part with the target area, and 5 ' end of this primer has second sequence label.This second sequence label is used in amplification target area process, and the target area amplified production of different testing samples is carried out mark.This second sequence label is preferably the nucleic acid molecule that has particular sequence, and its base number is not limit.The base number of this second sequence label is preferably 3～20, and more preferably 4～10.

In addition, said Auele Specific Primer also can have other marks, includes but not limited to: biotin labeling and other resistance markers are used for amplified production is carried out separation and purification.

In addition, when breast cancer susceptibility gene to be measured has when a plurality of, the amplification of the different breast cancer susceptibility genes of same testing sample can be carried out simultaneously or independently carry out respectively or part is carried out simultaneously.In concrete experimentation, can select for use above-mentioned any scheme to carry out as required.If respectively when increasing, the amount that needs to guarantee to be used to make up the amplified production in order-checking library is consistent.When testing sample has when a plurality of, the amplification of the breast cancer susceptibility gene of different samples must be carried out respectively.

Wherein, the described breast cancer susceptibility gene of step S1 comprises at least one among rs10795668, MMP2, SMAD7, ADH2, ALDH2, CYP1A2, MMP-1, MTHFR, TP53, VEGF, COX-2, DNMT3B, hMLH1, LOC727677, MMP9, MTRR and the TGF-β 1.

Further, the Auele Specific Primer of said rs10795668 is at least one pair of among SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and the SEQ ID NO:4; The Auele Specific Primer of said MMP2 is at least one pair of among SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and the SEQ ID NO:8; The Auele Specific Primer of said SMAD7 is at least one pair of among SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and the SEQ ID NO:12; The Auele Specific Primer of said ADH2 is at least one pair of among SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and the SEQ ID NO:16; The Auele Specific Primer of said ALDH2 is at least one pair of among SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and the SEQ ID NO:20; The Auele Specific Primer of said CYP1A2 is at least one pair of among SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and the SEQ ID NO:24; The Auele Specific Primer of said MMP-1 is at least one pair of among SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and the SEQ ID NO:28; The Auele Specific Primer of said MTHFR is at least one pair of among SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and the SEQ ID NO:32; The Auele Specific Primer of said TP53 is at least one pair of among SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and the SEQ ID NO:36; The Auele Specific Primer of said VEGF is at least one pair of among SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and the SEQ ID NO:40; The Auele Specific Primer of said COX-2 is at least one pair of among SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:43 and the SEQ ID NO:44; The Auele Specific Primer of said DNMT3B is at least one pair of among SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and the SEQ ID NO:48; The Auele Specific Primer of said hMLH1 is at least one pair of among SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51 and the SEQ ID NO:52; The Auele Specific Primer of said LOC727677 is at least one pair of among SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:55 and the SEQ ID NO:56; The Auele Specific Primer of said MMP9 is at least one pair of among SEQ ID NO:57 and SEQ ID NO:58, SEQ ID NO:59 and the SEQ ID NO:60; The Auele Specific Primer of said MTRR is at least one pair of among SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and the SEQ ID NO:64; The Auele Specific Primer of said TGF-β 1 is at least one pair of among SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:67 and the SEQ ID NO:68.

In one embodiment, the concrete implementation procedure of step S1 is:

S11. utilize the breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in the testing sample, obtain and the corresponding amplified production in said a plurality of target areas;

S12. utilize joint component, connect, obtain the library of checking order with the corresponding amplified production in said a plurality of target areas; Said joint component adopts at least a in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure.

Need to prove:

Among the step S11,, can carry out simultaneously or independently carry out respectively or partly carry out simultaneously the amplification of a plurality of target areas in the same testing sample.Can be according to practical situation, as: the annealing temperature of the used breast cancer susceptibility gene Auele Specific Primer that increases, the size of the target area of amplification, GC content, the quantity of the target area of amplification etc. are adjusted accordingly.The amplification of the target area of different testing samples must be carried out respectively.

Among the step S12, the mode of connection of said joint component and amplified production can adopt multiple mode to realize, comprises that joint component directly is connected with amplified production, or amplified production is connected after handling again.

The said joint component of step S12 is used for making up the order-checking library, can comprise one or more joints.

Wherein, at least one joint in the said joint component includes first sequence label, and this first sequence label is used in the library construction process, and mark is carried out in the order-checking library of different testing samples.Like this, after obtaining order-checking library, target area respectively, the order-checking library, target area of different testing samples may be combined in the same reaction system, carries out the unit molecule amplified reaction, and then carries out high-flux sequence simultaneously.Improve the efficient of sequencing reaction, reduced the cost of sample detection.

This first sequence label is preferably the nucleic acid molecule that has particular sequence, and its base number is not limit.The base number of this first sequence label is preferably 3～20, like this, and at every turn at least can be simultaneously to 4 ³Individual sample detects; Take all factors into consideration various situation, its base number more preferably 4～10.

Through the combination of second sequence label and first sequence label, invention of the present invention can detect at least 4 at every turn ³* 4 ³Individual sample, i.e. 4096 samples.

The modification mode of joint has multiple, includes but not limited to: can or methylate by biotinylation, or simultaneously by biotinylation with methylate.In one embodiment, this joint is by biotinylation, and is connected the separation and purification that has the order-checking library that is beneficial to structure of vitamin H with not biotinylated fragmentation product.In another embodiment, this joint is methylated, and is connected with unmethylated fragmentation product, the connection product that successfully connects with the digestion with restriction enzyme that only cuts methylate DNA then, thus guarantee that enzyme cuts the unicity of product.

The structure formation of joint component also has multiple, includes but not limited to: the joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure.Make up and only to use a kind of in the process of order-checking library or use multiple joint simultaneously.Wherein, the joint and the y splice of protruding terminus joint, band loop-stem structure can prevent effectively in connection procedure that all a plurality of joints are from the generation that connects phenomenon.Above-mentioned joint form to joint below will provide a plurality of embodiment.

In first embodiment, joint component adopts flat end fitting, and said flat end fitting is double-stranded complementary nucleic acid molecule fully.

In a second embodiment, joint component adopts the protruding terminus joint, and said protruding terminus joint is a double chain acid molecule, and this double chain acid molecule comprises a protruding terminus at least.The base number of this protruding terminus does not have concrete restriction, is preferably 1～10 base.According to the structure of this double chain acid molecule, can be divided into two types, be respectively single protruding terminus joint, two protruding terminus joint.

Single protruding terminus joint as shown in Figure 2, the one of which end is for flat terminal, and the other end is a protruding terminus.The single protruding terminus joint that wherein has y splice can prevent that joint is from connecting.In order to prevent that the end from connecting for flat terminal single protruding terminus joint certainly, can modify (including but not limited to use the amino-terminated hydroxyl) to 3 ' OH on the flat end, the 5 ' phosphate group that maybe will put down on the end is removed.

Two protruding terminus joints as shown in Figure 3, it contains two protruding terminuses, and these two protruding terminuses can be on a nucleotide chain, and (Fig. 3 is a) or on different nucleotide chains (Fig. 3 b).When these two protruding terminuses were on different nucleic acid chains, they were not complementary each other, in case joint when connecting, occurs from connecting.

In the 3rd embodiment; Joint component adopts the joint of band loop-stem structure, and the joint of said band loop-stem structure (as shown in Figure 4) is a single stranded nucleic acid molecule, and this single stranded nucleic acid molecule comprises that (Fig. 4 a) for the first complementary pairing district 1, stem ring district 2 and the second complementary pairing district 3; The first complementary pairing district 1 can with the second complementary pairing district, 3 complementary pairings; And the complementary pairing district that they form comprises at least one restriction enzyme enzyme recognition site, and cuts recognition site through this enzyme, and specific endonuclease capable cuts stem ring district or excision; Thereby single stranded nucleic acid molecule is become double chain acid molecule, so that follow-up operation.In one embodiment of the invention, shown in Fig. 4 b, the joint of band loop-stem structure also can have protruding terminus 4, and this protruding terminus can be positioned at the 5 ' end or the 3 ' end of single stranded nucleic acid molecule.The existence of protruding terminus 4 can prevent further that joint is from the generation that connects phenomenon.This protruding terminus is preferably T.

In the 4th embodiment, joint component adopts y splice, and said y splice is a double chain acid molecule, and is as shown in Figure 5, comprises complementary district and crotch region, and two strands of said crotch region respectively comprise at least one amplimer binding site.In one embodiment of the invention, the complementation district of said y splice comprises at least one restriction enzyme enzyme recognition site, and this enzyme is cut recognition site can cut the formation end by enzyme in building the storehouse process, so that carry out follow-up operation.

The bifurcation design of this y splice can avoid a plurality of joints from connecting the appearance of phenomenon building the storehouse process; The amplimer binding site that comprises on the said crotch region can directly be used to combine amplimer, carries out amplified reaction.

Wherein, every chain of the crotch region of said y splice contains M Nucleotide; Preferably, 9≤M≤30.Wherein, the Nucleotide logarithm of the collochore complementary pairing of said y splice is not limit; Preferably, the Nucleotide logarithm of complementary pairing is 7～15, and is preferred, and the Nucleotide logarithm of complementary pairing is 9～13.

Wherein, 3 ' of the collochore of said y splice end is a protruding terminus or flat terminal.Preferably, 3 ' end of the collochore of said y splice is a protruding terminus, this protruding terminus can with the sticky end complementary pairing of said fragmentation product, improved joint efficiency, be beneficial to make up order-checking library successful reaction and carry out.

Preferred, said y splice is the terminal y splice of T, and 3 ' end of the collochore of this joint is a protruding terminus, and last base of protruding terminus is T; The terminal y splice of T for example shown in Figure 6, N is any in A, T, C, the G base among the figure.

Preferred, 3 ' end of the collochore of said y splice is a protruding terminus, and the Nucleotide of protruding terminus comprises universal base; Y splice for example shown in Figure 7, N is any in A, T, C, the G base among the figure, X is a universal base.

Preferably, said y splice is two deoxidation joints, and 3 ' end of the collochore of this joint is for flat terminal, and 3 ' terminal last Nucleotide is the Nucleotide that has pair deoxidation bases; Two deoxidation y splices for example shown in Figure 8, N is any in A, T, C, the G base among the figure, dd representes that this 3 ' terminal last Nucleotide is the cytidylic acid(CMP) that has two deoxidation bases.

Should be noted that above-mentioned joint component is part embodiment, does not do any concrete restriction for protection scope of the present invention.

Implementation about step S12:

In one embodiment, step S12 adopts joint component and the direct-connected mode of amplified production, constructs the order-checking library.

In another embodiment; According to the needs of high throughput sequencing technologies, when the target area amplified production is big or the order-checking library, target area that needs to make up hour, then amplified production is carried out after fragmentation handles; Be connected with joint component again; Make up the order-checking library, as shown in Figure 9, step S12 may further comprise the steps:

S121. the amplified production corresponding with said a plurality of target areas carried out fragmentation, obtain the fragmentation product;

S122. utilize joint component, be connected, make up the order-checking library with the fragmentation product.

This step is handled through fragmentation, and different amplified productions is become the similar fragmentation product of length, can help follow-up unified order-checking.

Need to prove:

Among the step S121, the method for said fragmentation amplified production has multiple, can select to adopt prior art as required, includes but not limited to: atomizing, ultrasonication, the broken fragmentation of mechanical shearing, endonuclease bamhiization, chemistry and thermal induction fragmentation.

After handling, said fragmentation also can comprise the separation and purification of fragmentation product and end modified step.According to the fragment length needs of order-checking, the nucleic acid fragment for fragmentation obtains carries out the separation and purification of purpose nucleic acid fragment, and separation method can adopt domestic method, like gel electrophoresis, saccharose gradient or cesium chloride gradient sedimentation, column chromatography for separation etc.According to employed fragmentation method, further end modified to the purpose nucleic acid fragment of gained, include but not limited to: phosphorylation or dephosphorylation, end-filling and end add A, so that follow-up joint component connects.Above-mentioned purpose nucleic acid fragment length is not limit, and is preferably 25bp～500bp, more preferably 30bp～200bp, more preferably 40～100bp.Under the prerequisite of realization to the order-checking of breast cancer susceptibility gene, along with shortening of the breast cancer susceptibility gene fragment length that contains in the molecule of order-checking library, the order-checking degree of depth to breast cancer susceptibility gene of high throughput sequencing technologies is deepened; And the order-checking degree of depth is dark more, and promptly the order-checking number of times to each base position of breast cancer susceptibility gene is many more, and sequencing result is accurate more, and is just sensitiveer to the detection of a small amount of sudden change in the sample; So just can effectively prevent because have the ratio of breast cancer susceptibility gene of sudden change in the sample on the low sidely, and cause the absolute value of order-checking signal of this sudden change on the low side, the inaccurate phenomenon of sequencing result takes place.

According to above-mentioned joint component, to step S122, the present invention provides different embodiment.

In one embodiment of the invention, directly connect joint and form the order-checking library at the two ends of fragmentation product.

Said joint can adopt at least a in joint and the y splice of above-mentioned flat end fitting, protruding terminus joint, band loop-stem structure.

In another embodiment of the present invention, directly connect y splice, form the order-checking library at fragmentation product two ends.The present technique scheme can be utilized the characteristic of y splice, prevents between a plurality of joints from the generation that connects phenomenon.

In another embodiment of the present invention, shown in figure 10, step S122 specifically can be realized by following steps:

S1221. utilize first joint to be connected, the connection product of winning with the two ends of fragmentation product;

S1222. cyclisation first connects product, gets cyclisation product;

S1223.II s type digestion with restriction enzyme cyclisation product gets enzyme and cuts product;

S1224. cut the product two ends at enzyme and connect second joint and the 3rd joint, the library of must checking order.

Among the step S1221, said first joint can adopt a kind of in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure, and said first joint includes II s type digestion with restriction enzyme recognition site; Described II s type restriction enzyme is the restriction enzyme of cleavage site outside recognition sequence; Include but not limited to: Acu I, Alw I, Bbs I, BbV I, Bcc I, BceA I, BciV I, BfuA I, Bmr I, Bpm I, BpuE I, Bsa I, BseM II, BseR I, Bsg I, BsmA I, BsmB I, BsmF I, BspCN I, BspM I, BspQ I, BtgZ I, Ear I, Eci I, EcoP15 I, Fau I, Fok I, Hga I, Hph I, HpyAV, Mbo II, Mly I, Mme I, Mnl I, NmeAIII, Ple I, Sap I, SfaN I and TspDT I are preferably Acu I, Bsg I, EcoP15 I or Mme I.

If the fragmentation product is repaired through the terminal repair enzyme, and end adds the A reaction, and said first joint is preferably the terminal y splice of band T.If the fragmentation product is just repaired through the terminal repair enzyme, with the end-filling of fragmentation product, the preferred two deoxidation joints of then said first joint.

When said first joint was y splice, this II s type digestion with restriction enzyme recognition site was positioned at complementary district; When said first joint is the joint of band loop-stem structure, the distance between restriction enzyme enzyme recognition site and the loop-stem structure, near than the distance between II s type digestion with restriction enzyme recognition site and the loop-stem structure.

Among the step S1223, the enzyme that utilization can be discerned on first joint is cut recognition site, and cutting cyclisation product (DNA) but the enzyme that do not cut first joint carries out enzyme cuts.Enzyme on said first joint is cut recognition site and included but not limited to: Mme I enzyme cuts that recognition site, Acu I enzyme are cut recognition site, Bsg I enzyme is cut recognition site.

Among the step S1224, said second joint can be a kind of in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure, can have biotin labeling.Said the 3rd joint can be a kind of in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure.Second joint and the 3rd joint can be identical or different.Preferably, said second joint is identical with the 3rd joint, is y splice.Preferred, said y splice is the terminal y splice of T or two deoxidation y splice.

Should be noted that above embodiment is merely several kinds of specific embodiments among the present invention, not in order to restriction protection scope of the present invention.

What need to specify is also can comprise step between step S1222 and the S1223: rolling circle amplification cyclisation product, the cyclisation product after must increasing, this step can guarantee that follow-up enzyme cuts step S1223 enough cyclisation product are arranged.

Wherein, also can comprise step between step S1221 and the S1222: utilize amplimer to connect product and increase, get amplified production to first.Said amplimer is connected the joint sequence complementation at product two ends respectively with first.

Wherein, said first joint is preferably y splice.

Wherein, said complementary district can comprise at least one enzyme and cut recognition site, and two strands of said crotch region respectively comprise at least one amplimer binding site.

Wherein, said amplimer is preferably the biotinylation primer.

Wherein, said amplimer has at least one specific enzymes and cuts recognition site.

If on the amplimer with specific enzymes to cut recognition site be the uridylic base, then utilize uridylic specificity excision reagent to carry out enzyme among the step S1222 and cut, and then connect cyclisation.

If it is the restriction enzyme enzyme recognition site that specific enzymes that amplimer is with is cut recognition site, then utilize corresponding restriction enzyme to carry out enzyme among the step S1222 and cut, connecting cyclisation then.

In another embodiment of the present invention, shown in figure 11, step S122 specifically can be realized by following steps:

S1221 '. utilize the 4th joint to be connected, get second and connect product with the fragmentation product;

S1222 ' .II s type digestion with restriction enzyme second connects product, must have the endonuclease bamhi of the 4th joint;

S1223 '. the endonuclease bamhi that has the 4th joint is connected with the 5th joint, forms the order-checking library.

Among the step S1221 ', said the 4th joint can adopt a kind of in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure, and said the 4th joint includes II s type digestion with restriction enzyme recognition site; When said the 4th joint was y splice, this II s type digestion with restriction enzyme recognition site was positioned at complementary district; When said the 4th joint is the joint of band loop-stem structure, the distance between restriction enzyme enzyme recognition site and the loop-stem structure, near than the distance between II s type digestion with restriction enzyme recognition site and the loop-stem structure.If the fragmentation product is repaired through the terminal repair enzyme, and end adds the A reaction, and said the 4th joint is preferably the terminal y splice of band T.If the fragmentation product is just repaired through the terminal repair enzyme, the preferred two deoxidation joints of then said the one or four joint.

Among the step S1222 ', the enzyme that utilization can be discerned on the 4th joint is cut recognition site, and cutting cyclisation product (DNA) but the enzyme that do not cut the 4th joint carries out enzyme cuts.Enzyme on said the 4th joint is cut recognition site and included but not limited to: Mme I enzyme cuts that recognition site, Acu I enzyme are cut recognition site, Bsg I enzyme is cut recognition site.

Among the step S1223 ', said the 5th joint can be a kind of in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure, can have biotin labeling.

In another embodiment of the present invention, step S122 specifically may further comprise the steps:

S1221 ". directly connect the joint of band loop-stem structure, form the fragmentation product of band loop-stem structure at the two ends of fragmentation product;

S1222 ". utilize restriction enzyme, the stem ring district of the fragmentation product of being with loop-stem structure is cut or excision, thereby form the order-checking library.

The joint of present technique scheme utilization band loop-stem structure has prevented that a plurality of joints are from the generation that connects phenomenon.

Should explain that above embodiment is merely wherein several kinds of specific embodiments of step S122, not in order to restriction protection scope of the present invention.

Wherein, The described unit molecule amplification of step S2 is meant each molecule in the order-checking library; Form with denier (even unit molecule) is spatially isolated (but these library molecules still belong to same reaction system on the whole); And in space separately, realize amplification, to promote the signal of various molecules in follow-up sequencing reaction.The method of said unit molecule amplification includes but not limited to: and emulsion PCR (Emulsion PCR, EPCR), bridge-type PCR.

The aqueous solution that said EPCR will comprise all reacted constituents of PCR is injected into the MO surface of high speed rotating, and aqueous solution moment forms numerous by the little water droplet of MO parcel.These little water droplets have just constituted independently PCR reaction compartment.Under the perfect condition; Each little water droplet only contains a dna profiling (order-checking library molecule) and a magnetic bead; Contain consensus sequence (introducing) complementary primer on the magnetic bead by joint component with order-checking library molecule; After the PCR reaction, magnetic bead surfaces just is fixed with the dna profiling amplified production in the same source of copy huge amount.But EPCR concrete steps reference: BEAMing:single-molecule PCR on microparticles in water-in-oil emulsions, Frank Diehl, Meng Li; Yiping He, nature methods, Vol.3; No.7, July 2006.

The ultimate principle of said bridge-type PCR is; The primer of bridge-type PCR is fixed on the solid phase carrier; Pcr amplification product can be fixed on the solid phase carrier in the PCR process; And pcr amplification product can with the primer complementary pairing on the solid phase carrier, Cheng Qiaozhuan, the primer of complementary pairing is that template increases with the amplified production with its Cheng Qiao then.Through the amount that the control original template adds, after bridge-type PCR reaction was accomplished, amplified production form with cluster bunch on solid phase carrier existed, and the amplified production of each bunch be with the dna profiling amplified production of originating.Principle that it is concrete and embodiment can be with reference to following document: CN20061009879.X, US6227604.

As previously mentioned, in the prior art, the Sanger PCR sequencing PCR is because the technical limitation of self can only check order to a certain section zone of a sample at every turn, and detection efficiency is low, cost is high.For disposable realization to detecting a plurality of zone the time in the sample, the present invention takes the high-throughput gene order surveying method on sequence measurement.The relative Sanger PCR sequencing PCR of high-throughput gene sequencing detect sequence information more efficient, make things convenient for sensitivity and cost low; After each molecule in the order-checking library increases through unit molecule; Each order-checking library molecule all forms unit molecule copy array, and each unit molecule copy array is in different positions when carrying out the high-throughput gene sequencing, make sequencing primer and unit molecule copy the hybridization between the array; And the extension under the enzyme effect can carry out simultaneously, do not disturb mutually each other.Therefore, can be simultaneously a large amount of (millions of up to ten million, even more) unit molecule copy array be carried out sequencing reaction simultaneously, then through gathering corresponding signal, and then obtain required sequence information accurately, and the sensitivity of order-checking is higher than Sanger.Especially the amplified production to a plurality of target areas has carried out the fragmentation processing, and being equivalent to has increased the order-checking number of times of each base of the target area molecule of identical sequence, can further improve the sensitivity of order-checking.

Wherein, the said high throughput sequencing technologies of step S3 includes but not limited to: based on the synthetic PCR sequencing PCR of polysaccharase and the PCR sequencing PCR that is connected based on ligase enzyme.

Synthetic PCR sequencing PCR is based on band can remove that the Nucleotide of mark carries out, and in each building-up reactions, each template strand can only extend once at the most.A kind of roughly flow process of synthetic PCR sequencing PCR is following:

A. sequencing primer is combined on the total known array of unit molecule amplified production (this unit molecule amplified production is fixed on primer-solid phase carrier mixture) through complementary pairing; Under the effect of archaeal dna polymerase; The Nucleotide that can remove mark with band carries out the single-basic extension building-up reactions; Collect the marking signal that this time adds Nucleotide, can obtain base sequence information with the next bit of the unit molecule amplified production (being fixed on primer-solid phase carrier mixture) of sequencing primer 3 ' least significant end base complementrity.

B. excision can be removed mark; Then under the effect of archaeal dna polymerase; Can remove the Nucleotide of mark with band and proceed the single-basic extension building-up reactions; Collect the marking signal that adds Nucleotide, can obtain following two base sequence information with sequencing primer 3 ' terminal bases complementary unit molecule amplified production.

Repeat the b step, till can not proceeding building-up reactions, thus the full sequence information of acquisition unit molecule amplified production.

A kind of PCR sequencing PCR that connects is based on and has that fluorescently-labeled oligonucleotide probe carries out; This oligonucleotide probe has n base; (the difference between on the same group is not: the specific position that different fluorescent marks are corresponding is different for the group of h≤n), the Different Alkali basic sequence of the corresponding same specific position of different fluorescent marks of same group of oligonucleotide probe to be divided into h; Because 3 ' end of this oligonucleotide probe has carried out specific modification; Can not directly interconnect between the oligonucleotide probe, each ligation, each unit molecule amplified production can only connect an oligonucleotide probe.This roughly flow process that connects PCR sequencing PCR is following:

A. sequencing primer is combined on the total known array of unit molecule amplified production on (this unit molecule amplified production is fixed on primer-solid phase carrier mixture) through complementary pairing; (the base position that fluorescent mark is corresponding is x to utilize in the above-mentioned oligonucleotide probe one group; X≤h); Under the effect of ligase enzyme, nucleic probe is connected with above-mentioned oligonucleotide chain, gather fluorescent signal then; Can obtain 3 ' terminal back or 5 ' terminal preceding x bit base sequence information with the total known array of single-stranded amplification product, the sex change from the unit molecule amplified production of sequencing primer and the oligonucleotide probe that connected thereof is eluted.

B. again sequencing primer is combined on the unit molecule amplified production then; (the base position that fluorescent mark is corresponding is y to use the oligonucleotide probe group different with a step instead; Y≤h); Under the effect of ligase enzyme, nucleic probe is connected with above-mentioned oligonucleotide chain, gather fluorescent signal then; Can obtain 3 ' terminal back or 5 ' terminal preceding y bit base sequence information with the total known array of single-stranded amplification product, the sex change from the unit molecule amplified production of sequencing primer and the oligonucleotide probe that connected thereof is eluted.

C. repeating step b carried out a ligation respectively until h group oligonucleotide probe, thereby obtained 3 ' terminal back or 5 ' terminal preceding the 1st, the 2...... of the total known array of unit molecule amplified production, the base sequence information of h position.

Use instead and compare 3 ' primers terminal or 5 ' terminal many one or more universal base with sequencing primer before and react by above-mentioned principle, can prolong the total known array of the unit molecule amplified production of acquisition 3 ' terminal back or 5 ' the proterminal base sequence read long.

This connection order-checking ratio juris and specific embodiments based on ligase enzyme can be with reference to CN200710170507.1.

Synthetic PCR sequencing PCR and tetra-sodium PCR sequencing PCR based on polysaccharase have certain similarity, and therefore, in theory, the tetra-sodium PCR sequencing PCR can be applicable to detection method of the present invention equally.That but existing tetra-sodium PCR sequencing PCR adopts in the order-checking process is natural dNTP, makes it in the order-checking process, and the mensuration of treating continuous single base repetitive sequence that possibly exist on the order-checking library has difficulties; And, can guarantee only to extend a base based on the mark removed that the Nucleotide in the synthetic PCR sequencing PCR of polysaccharase has at every turn; 3 ' end or 5 ' end with fluorescently-labeled probe in sending out based on the connection of ligase enzyme order-checking are modified, and guarantee only to connect a fluorescent probe on the fragment of each unit molecule amplified production; Therefore of the present invention high with the accuracy that is connected PCR sequencing PCR than the tetra-sodium order-checking based on ligase enzyme based on the synthetic PCR sequencing PCR of polysaccharase.

In addition; Aperture on the etching optical fiber slide (PTP plate) in the existing tetra-sodium order-checking instrument is big (55 μ m * 44 μ m); Be used to hold the amplified production (amplified production of emulsion PCR is fixed on the pearl of 10 μ m) of order-checking emulsion PCR gained before; This big limitations the sequencing throughput of tetra-sodium PCR sequencing PCR, make that the reagent cost of its sequencing reaction is higher.In addition, the tetra-sodium PCR sequencing PCR also needs in the aperture of etching optical fiber slide (PTP plate), to add the mixture that contains multiple protein with the carrying out smoothly of assurance sequencing reaction in the order-checking process, and this will improve the reagent cost of sequencing reaction greatly.

And based on the synthetic PCR sequencing PCR of polysaccharase and the product that PCR sequencing PCR can fix the unit molecule amplification through magnetic bead or the slide of 1 μ m that is connected based on ligase enzyme; Make its flux higher; And except needs have the Nucleotide that can remove mark and the fluorescently-labeled probe that 3 ' end or 5 ' end have carried out modifying; Other required reagent do not have particular requirement, greatly reduce the reagent cost of sequencing reaction.Obtaining under the prerequisite of same quantity of data, based on the synthetic PCR sequencing PCR of polysaccharase and the order-checking cost that is connected PCR sequencing PCR based on ligase enzyme be the tetra-sodium PCR sequencing PCR 1/2000th or still less.Therefore the synthetic PCR sequencing PCR that is based on polysaccharase that adopts among the present invention program or the unit molecule amplified production is checked order based on the connection PCR sequencing PCR of ligase enzyme.

In addition, the GSTM1 gene belongs to Thiadiazolidine isomerase family, mainly act as its reactive group gsh of catalysis with through a metabolic enzyme metabolism activation and the multiple electrophilic carcinogens that comes promotes it to excrete mutually.If the defunctionalization of its isozyme of the individuality of GSTM1 genetically deficient coding lacks the function of detoxification to above-mentioned poisonous substance, appeal to susceptibility to such carcinogens effect thereby increase.Therefore; For the detection of breast cancer susceptibility property more accurately with comprehensively; Provided the method flow of another kind of more accurate and comprehensive detection breast cancer susceptibility gene in the present invention; Shown in figure 12, this method has increased the step that detects GSTM1 genetically deficient on the basis of method shown in Figure 1, specifically comprise following steps:

S1 '. utilize the breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in the testing sample, and make up the order-checking library based on amplified production;

S2 '. the unit molecule amplification is carried out in the order-checking library, obtained a plurality of unit molecule amplified productions corresponding with said a plurality of target areas;

S3 '. simultaneously said a plurality of unit molecule amplified productions are carried out the high-throughput gene sequencing, obtain the sequence information of said a plurality of target areas,

S4 '. utilize the amplimer of GSTM1 that testing sample is carried out pcr amplification, the deletion condition of detected through gel electrophoresis GSTM1 and GSTT1 gene.

Step S4 ' and step S3 ', S2 ', the no precedence relationship of S1 '.

The present technique scheme is through high-throughput gene sequencing technology for detection breast cancer susceptibility gene; Confirm known and the unknown mutation site; Carry out gene type, the deletion condition that has also carried out the GSTM1 gene simultaneously detects, thereby more accurately, comprehensively detects breast cancer susceptibility property.

Need to prove, in the present technique scheme, step S1 ' to S3 ' can referring to before step S1 among the embodiment to S3, do not give unnecessary details at this.

The Auele Specific Primer of GSTM1 described in the step S4 ' comprises at least one pair of among SEQ ID NO:9 and SEQ ID NO10, SEQ ID NO:11 and the SEQ ID NO:12.

Except that the target area of amplification GSTM1 gene, also comprise the amplification to the internal control gene target area among the step S4 ', the Auele Specific Primer of said internal control gene is SEQ ID NO:13 and SEQ ID NO:14.

Among the embodiment that the present invention proposes, detect FGFR2, GSTP1, MTHFR and GSTM1 gene in the breast cancer susceptibility gene simultaneously.

Focus sudden change zone design corresponding special primers to FGFR2, GSTP1, MTHFR: F1F (SEQ ID NO:1) and F1R (SEQ ID NO:2), G1F (SEQ ID NO:3) and G1R (SEQ ID NO:4), M1F (SEQ ID NO:7) and M1R (SEQ ID NO:8); Design simultaneously detects the certain Auele Specific Primer of GSTM1 gene: G2F (SEQ ID NO:9) and G2R (SEQ ID NO:10), and Auele Specific Primer M2F of internal control gene (SEQ ID NO:13) and M2R (SEQ ID NO:14).

One, the extraction of DNA in the testing sample

Utilize nucleic acid extraction kit common on the market to extract the DNA of whole blood sample (1 to 10), serum sample (11 to 20), paraffin organization sample (21 to 30) respectively, and carry out mark respectively.

Two, the amplification of a plurality of target areas of breast cancer susceptibility gene to be measured

Utilize the specificity amplification primer of above-mentioned tumor susceptibility gene to be measured, increased in the target area of breast cancer susceptibility gene, obtain amplified production.The target area amplification of above-mentioned tumor susceptibility gene to be measured is carried out respectively, and reaction system is following:

Upstream primer (10 μ M) 2 μ L;

Downstream primer (10 μ M) 2 μ L;

DNTP (each 2.5mM) 4 μ L;

Testing sample DNA 20ng;

Ex?Taq(5U/μL) 0.25μL；

10×Ex?Taq?Buffer 5μL；

DdH ₂O adds to 50 μ L.

The PCR reaction conditions is following:

95℃?3min；

94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Repeat 25 circulations;

72℃?7min。

Utilize PCR to reclaim test kit, the amplified production to each sample separates respectively, removes the primer and the dNTP of not amplification, reclaims amplified production.

Three, utilize amplified production to make up the order-checking library

According to before said, this step can be realized by multiple mode.In one embodiment of the invention, be employed in the method structure order-checking library that fragmentation product two ends directly connect joint component, specifically comprise:

1. the fragmentation of amplified production

Adopt sonioation method to carry out fragmentation in the present embodiment and handle, concrete operations are:

Measure the amplified production concentration after reclaiming, mix, get mixed solution, and mark is distinguished by the different breast cancer susceptibility genes target area amplified production that waits mole number with same sample.

The mixed solution 50 μ L of each sample are added among the TE buffer of 400 μ L, ultrasonic 4s under the 430W power condition, 10s 5 times repeatedly, obtains the fragment mixture at interval.Utilize 1% sepharose to carry out separation and purification, select the fragment of 40-100bp size to cut the glue recovery, obtain the fragmentation product.

2. utilize fragmentation product and joint component to make up the order-checking library

For ease of carrying out the connection of joint component, carry out end modified to the fragmentation product.In the present embodiment, carry out phosphorylation, end-filling respectively and add the A end reaction, concrete operations are following:

1) phosphorylation and end-filling

Reaction system is:

The about 2000ng of fragmentation product;

10mM?dNTP 1.5μL；

T4DNA polysaccharase (5U/ μ L) 1 μ L;

Klenow archaeal dna polymerase 0.1 μ L;

T4 polynucleotide kinase (10U/ μ L) 0.5 μ L;

10m?MATP 1.5μL；

T4 DNA connects damping fluid 10 μ L;

Add ddH ₂O to 100 μ L.

Hatch 20min for 20 ℃, reaction finishes the back and utilizes the recovery test kit to carry out purifying and recovering.

2) end adds the A tail

Reaction system is:

The about 1000ng of recovery product behind phosphorylation and the end-filling;

Klenow damping fluid (NEB Buffer2) 10 μ L;

10mM?dATP 2μL；

Klenow enzyme (3 ' to, 5 ' exo minus, 10U/ μ L) 1 μ L;

Add ddH ₂O to 100 μ L.

Hatch 30min for 37 ℃, reaction utilizes the purification kit purifying and recovering after finishing.

3) jointing 1

Adopt the terminal y splice of T as shown in Figure 6 as joint 1 in the present embodiment; Same sample uses the terminal y splice of identical T; The terminal y splice of the corresponding different T of different samples, according to being with first sequence label to distinguish on it, the sequence label that different samples are corresponding is as shown in table 1 below.

The table 1. first sequence label data sheet

Under the effect of T4 ligase enzyme, add A tail recovery product afterwards and be connected with the terminal y splice of T, form the fragment of belt lacing 1, linked system is:

The product 50 μ L (about 500ng) that add purifying and recovering behind the A tail;

Joint 12 μ L (about 3000ng);

10mM?ATP 5μL；

T4DNA ligase enzyme (30U/ μ L) 1 μ L;

10 * T4 ligase enzyme damping fluid, 10 μ L;

Add ddH ₂O to 100 μ L.

Hatch more than the 4h for 16 ℃, reaction utilizes the purification kit purifying and recovering after finishing.

4) fragment of pcr amplification belt lacing 1

Amplification system is:

The about 300ng of the fragment of belt lacing 1;

10×Ex?Taq?Buffer 100μL；

Primer?F(100μM，SEQ?ID?NO：23) 10μL；

Primer?R(100μM，SEQ?ID?NO：24) 10μL；

Ex?Taq(5U/μL) 7.5μL；

DNTP (each 2.5mM) 80 μ L;

ddH ₂O?up?to?1000μL。

The PCR reaction conditions is following:

95℃?3min；

94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Repeat 25 circulations;

72℃?7min。

Utilize PCR cleaning agents box, the amplified production to each sample cleans respectively, removes the primer and the dNTP of not amplification, reclaims the segmental amplified production of belt lacing 1.

5) II s type digestion with restriction enzyme

Utilize the segmental amplified production of the belt lacing 1 after the switchback of Acu I enzyme is received, reaction system is following:

The segmental amplified production 60 μ L (3-5 μ g) of the belt lacing 1 after the recovery;

10×NEB?Buffer?4 8μL；

Acu?I(NEB) 2μL(10U)；

SAM (3.2mM) 1 μ L (final concentration 50 μ M);

ddH ₂O?up?to?80μL。

Reaction conditions: hatch 1h for 37 ℃.Reaction utilizes purification kit purifying and recovering enzyme to cut product after finishing.

6) jointing 2

Utilize protruding terminus y splice as shown in Figure 7 as joint 2, cut product with the enzyme that reclaims and be connected, the library of must checking order, linked system is following:

The enzyme that reclaims is cut product 2 μ g;

Joint 21 μ L (10pM);

10 T4DNA ligase enzymes (3U/ μ L), 1 μ L;

10 * T4 ligase enzyme damping fluid, 2 μ L;

Add ddH ₂O to 100 μ L.

Condition of contact is hatched 2h for 14 ℃.Reaction utilizes the purification kit purifying and recovering after finishing, and sex change forms strand, the library of must checking order then.

Four, the unit molecule amplification is carried out in the order-checking library

Determination step three obtains 30 order-checking library concentration separately, and the amplification of isoconcentration mixing carrying out unit molecule gets the unit molecule amplified production, and the method for said unit molecule amplification can adopt EPCR or bridge-type PCR.

Be preferably the EPCR amplification, the unit molecule amplimer that is fixed on the magnetic bead is preferably: SEQ ID NO:15 and SEQ ID NO:16, idiographic flow is operated by classical EPCR.

Five, the unit molecule amplified production is carried out high-flux sequence

Said sequence measurement can adopt the synthetic PCR sequencing PCR based on synthetic enzyme, also can adopt the connection PCR sequencing PCR based on ligase enzyme.Adopt aforesaid connection PCR sequencing PCR in the present embodiment, obtain the sequencing result of each unit molecule amplified production, then it is carried out bioinformatic analysis, can obtain the sequence information in each mammary cancer tumor susceptibility gene respective objects to be measured zone in the testing sample.

Through the sequencing result analysis, detected result is in the present embodiment: FGFR2 gene occurrence positions chr10:123346190 is transformed into the sudden change of G by base A in the sample 3, and GSTP1 gene occurrence positions chr11:67352689 is by the sudden change of bases G to A; The sudden change of base C to T takes place in the chr1:11856378 position of mthfr gene in the sample 5; GSTP1 occurrence positions chr11:67352689 is by the sudden change of bases G to A in the sample 8; GSTP1 gene occurrence positions chr11:67352689 is by the sudden change of bases G to A in the sample 12, and the sudden change of base C to T takes place in the chr1:11856378 position of mthfr gene; The sudden change of base C to T takes place in the chr1:11856378 position of mthfr gene in the sample 15; The sudden change of base C to T takes place in the chr1:11856378 position of mthfr gene in the sample 24, and GSTP1 gene occurrence positions chr11:67352689 is by the sudden change of bases G to A; All the other are all the wild type gene sequence.

Six, pcr amplification and detected through gel electrophoresis GSTM1 gene

1. utilize the Auele Specific Primer of design to carry out the amplification of GSTM1 and internal control gene respectively, obtain amplified production.The PCR reaction system is:

Upstream primer (10 μ M) 2 μ L;

Downstream primer (10 μ M) 2 μ L;

DNTP (each 2.5mM) 4 μ L;

Testing sample DNA 20ng;

Ex?Taq(5U/μL) 0.25μL；

10×Ex?Taq?Buffer 5μL；

DdH ₂O adds to 50 μ L.

The PCR reaction conditions is following:

95℃?3min；

94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 30s; Repeat 30 circulations;

72℃?10min。

2. detected through gel electrophoresis amplified production

Amplified production is with 12% denaturing polyacrylamide gel, and under the 180V condition, the 0.5h electrophoresis is then with the SYBR dyeing observation that develops the color.Product judgement criteria: if having the 270bp band in the running gel, then contain the GSTM1 gene, otherwise then lack.

Detected result shows: GSTM1 genetically deficient in the sample 3, in the sample 8, in the sample 12, in the sample 15, in the sample 24; All the other do not see GSTM1 genetically deficient.

Should be noted that present embodiment is a specific embodiment of the present invention, the present invention is not had any qualification effect, use corresponding special primers and substitute, can be applied to detection method of the present invention equally.

To detection sensitivity of the present invention, the invention provides an embodiment and verify.

Pass through conventional design; Make up the wild-type sequence (SEQ ID NO:17) and the plasmid of mutant nucleotide sequence (SEQ ID NO:18), the wild-type sequence (SEQ ID NO:19) in GSTP1 gene amplification zone and the plasmid of mutant sequence (SEQ ID NO:20) that contain FGFR2 gene amplification zone respectively, and the plasmid of wild-type sequence of mthfr gene amplification region (SEQ ID NO:21) and mutant sequence (SEQ ID NO:22).

Prepare the mutant ratio respectively and be 20%, 10%, 5%, 3%, 1%, 0% plasmid mixed solution.Above-mentioned plasmid mixed solution to contain 1000 copy plasmids is a template, detects by the method with reference to the foregoing description then.Detected result is as shown in table 2 below:

Table 2. detection sensitivity verification msg

The result shows that the lowest detection of the method for detection breast cancer susceptibility gene of the present invention is limited to about 3%, 5% and more than detected result and practical situation basically identical.This shows that detection method of the present invention can break through the restriction of traditional method, can discern, improved detection sensitivity the sample that includes heterozygote.

A kind of detection kit that can be used for above-mentioned any breast cancer susceptibility gene detection method comprises:

The test kit of said detection breast cancer susceptibility gene can detect a plurality of zones of breast cancer susceptibility gene simultaneously, improves detection efficiency, reduces cost; Utilize the restriction of high-throughput gene sequencing technological breakthrough conventional art simultaneously, improve the accuracy and the sensitivity that detect.

Wherein, at least one joint in the said joint component includes first sequence label, is used in the library construction process, and mark is carried out in the order-checking library of different testing samples.Said first sequence label is preferably the nucleic acid molecule that has specific base sequence, and its base number is not limit.

Further, the said first sequence label base number is 3～20, more preferably 4～10.

Wherein, said breast cancer susceptibility gene has a plurality of, have at least in the Auele Specific Primer one fully complementary or part is complementary with breast cancer susceptibility gene to be measured, and 5 ' end of this complementary primer includes second sequence label, is used to discern different testing samples.Said second sequence label is preferably the nucleic acid molecule that has specific base sequence, and its base number is not limit, and is preferably 3～20, and more preferably 4～10.

The breast cancer susceptibility gene that the test kit of said detection breast cancer susceptibility gene is used for detecting comprises at least one of FGFR2, GSTP1, IL-1 β and MTHFR and GSTM1.

Wherein, said test kit can also comprise pcr amplification reagent, and said pcr amplification reagent comprises PCR enzyme, dNTP, PCR damping fluid, Mg ²⁺Solution.

Wherein, said test kit can also comprise the internal control gene amplimer, and said amplimer is SEQ ID NO:13 and SEQ ID NO:14.

Wherein, said test kit can comprise that also the enzyme that is used to discern on the joint component cuts the enzyme of recognition site.

Should be noted that typical application of the present invention but be not limited to the detection of breast cancer susceptibility gene, in other similar gene tests, also can use the method that the present invention sets forth.The method of detection breast cancer susceptibility gene provided by the present invention and test kit; The tumor susceptibility gene that can be applied to extensive sample detects and screening; Obtain its concrete gene order information; Carry out statistical study in conjunction with follow-up further molecular biology test, clinical trial, clinical observation and other integrated data, set up certain breast cancer disease model, can realize assessing in early days the susceptibility of mammary cancer.

The above is merely preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of within spirit of the present invention and principle, being done, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.

Claims

1. a method that detects breast cancer susceptibility gene is characterized in that, said method comprising the steps of:

2. the method for detection breast cancer susceptibility gene according to claim 1 is characterized in that, said steps A comprises:

3. the method for detection breast cancer susceptibility gene according to claim 2 is characterized in that, said steps A 2 may further comprise the steps:

4. the method for detection breast cancer susceptibility gene according to claim 3 is characterized in that, said steps A 22 may further comprise the steps:

A222. cyclisation first connects product, gets cyclisation product;

5. the method for detection breast cancer susceptibility gene according to claim 3 is characterized in that, said steps A 22 may further comprise the steps:

A223 '. the endonuclease bamhi that has the 4th joint is connected with the 5th joint, the library of must checking order.

6. according to the method for each described detection breast cancer susceptibility gene of claim 2 to 5; It is characterized in that; At least one joint in the steps A 2 said joint components includes first sequence label, is used in the library construction process, and mark is carried out in the order-checking library of different testing samples.

7. according to the method for each described detection breast cancer susceptibility gene of claim 1 to 5; It is characterized in that; Breast cancer susceptibility gene described in the steps A has a plurality of, at least one in the Auele Specific Primer of the target area correspondence of each mammary gland tumor susceptibility gene and this target area part complementation, and 5 ' end of this primer has second sequence label; Be used in the library construction process, mark is carried out in the order-checking library of different testing samples.

8. the method for detection breast cancer susceptibility gene according to claim 5 is characterized in that, said tumor susceptibility gene to be measured comprises at least one among FGFR2, GSTP1, IL-1 β and the MTHFR.

9. the method for detection breast cancer susceptibility gene according to claim 1 is characterized in that, said method also comprises step:

D. utilize the specificity amplification primer of GSTM1 testing sample to be carried out pcr amplification, the deletion condition of detected through gel electrophoresis GSTM1 gene; All there is not the sequencing relation between step D and steps A, B or the C.

10. a breast cancer susceptibility gene detection kit is characterized in that, said test kit comprises:

11. breast cancer susceptibility gene detection kit according to claim 10 is characterized in that, said joint component adopts at least a in joint and the y splice of flat end fitting, protruding terminus joint, band loop-stem structure.

12. breast cancer susceptibility gene detection kit according to claim 10; It is characterized in that; At least one joint in the said joint component includes first sequence label, is used in the library construction process, and mark is carried out in the order-checking library of different testing samples.

13. breast cancer susceptibility gene detection kit according to claim 10; It is characterized in that; Said breast cancer susceptibility gene has a plurality of, has at least a primer and target area part complementary in the Auele Specific Primer corresponding with each target area, and 5 ' end of this primer has second sequence label; Be used in amplification target area process, the target area amplified production of different testing samples is carried out mark.

14. the test kit according to each described detection breast cancer susceptibility gene in the claim 10 to 13 is characterized in that, said breast cancer susceptibility gene comprises at least one among FGFR2, GSTP1, IL-1 β, MTHFR and the GSTM1.