CN102586420B - Method and kit for assaying breast cancer susceptibility genes - Google Patents

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 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 refers to specified disease to have certain associated gene or allelotrope.The tumor susceptibility gene of complex disease and mutational site are detected, can obtain its concrete gene order information.On this basis, can be in conjunction with 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.Studies confirm that in a large number inherited genetic factors plays a significant role in the susceptibility of disease, the own alleged occurrence minor gene of the inherited genetic factors dosage effect of complex disease, i.e. adding up of minor gene effect just can form obvious phenotype combined effect.Therefore, to a plurality of tumor susceptibility genes of disease and mutational site, detect most important, particularly for topmost genetic mutation form in genome---single nucleotide polymorphism (Single Nucleotide Polymorphisms, SNP), it accounts for 92% of genetic mutation in whole genome.

Mammary cancer is one of common cancer having a strong impact on WomanHealth, and the whole world approximately has 1,200,000 women that mammary cancer occurs every year, 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 prevention the treatment of mammary cancer is played to important effect.

Breast cancer susceptibility gene is and the tight associated gene of having of mammary cancer.At present, the common methods that detects breast cancer susceptibility gene is Sanger sequencing, the method can be carried out region detection to breast cancer susceptibility gene, but Sanger sequencing can only check order to a certain section of region of a sample at every turn, so detection efficiency is low, cost is high; And due to the restriction of sanger order-checking principle, when the sample signal detecting with heterozygote, there will be bimodal and even multimodal, and this sample cannot accurately be identified, cause detection sensitivity low (20%).

In addition, the another kind of common method detecting for breast cancer susceptibility gene is gene chips, the method can only form detection chip for the site design detection probes of or several known mutations, therefore cannot be for detection of unknown mutation site, and the result of detection is also just not comprehensively, accurately.

The current formed detection kit of the detection method based on prior art on the market, only detects for single region or known one or several SNP site, and detected result not comprehensively accurately.

Therefore need a kind of novel method and novel agent box that detects breast cancer susceptibility gene, can improve detection efficiency, prevent from occurring from the phenomenon connecting at the process center tap that builds sequencing library.

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 a plurality of regions of breast cancer susceptibility gene simultaneously, improve detection efficiency, prevent from occurring from the phenomenon connecting at the process center tap that builds sequencing library.

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

A. utilize breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in testing sample, and build sequencing library based on amplified production;

B. sequencing library is carried out to unit molecule amplification, obtain a plurality of unit molecule amplified productions corresponding with described a plurality of target areas;

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

Wherein, steps A comprises:

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

A2. utilize joint component, the amplified production corresponding with described a plurality of target areas connects, and obtains sequencing library; Described joint component adopts flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

Wherein, described steps A 2 comprises the following steps:

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

A22. utilize joint component, be connected with described fragmentation product, build sequencing library.

Wherein, described steps A 22 comprises the following steps:

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

A222. cyclisation first connects product, obtains cyclisation product;

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

A224. at enzyme, cut product two ends and connect the second joint and the 3rd joint, obtain sequencing library.

Wherein, described steps A 22 comprises the following steps:

A221 '. utilize the 4th joint to be connected with fragmentation product, obtain the second connection product;

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

A223 '. the endonuclease bamhi with the 4th joint is connected with the 5th joint, forms sequencing library.

Wherein, at least one joint described in steps A 2 in joint component includes the first sequence label, in library construction process, the sequencing library of different testing samples is carried out to mark.Described the first sequence label, is preferably the nucleic acid molecule with particular sequence, for identifying different testing samples.Described the 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 steps A has a plurality of, at least one complementary with this target area part in Auele Specific Primer corresponding to the target area of each mammary gland tumor susceptibility gene, and 5 ' end of this primer is with the second sequence label, for in amplification target area process, the target area amplified production of different testing samples is carried out to mark.Described the second sequence label is preferably the nucleic acid molecule with particular sequence, and its base number is not limit, and is preferably 3～20, and more preferably 4～10.

Wherein, described breast cancer susceptibility gene comprises at least one in FGFR2, GSTP1, IL-1 β and MTHFR.

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

Wherein, described method also comprises step:

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

Wherein, between step D and steps A, B or C all without sequencing relation.

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

Can be used in of the present invention any and detect a test kit for breast cancer susceptibility gene method, comprising:

Breast cancer susceptibility gene Auele Specific Primer, increases for a plurality of target areas to testing sample;

Joint component, builds sequencing library for being combined with amplified production.

Wherein, described joint component adopt flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

Wherein, at least one joint in described joint component includes the first sequence label, in library construction process, the sequencing library of different testing samples is carried out to mark.

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

Wherein, described breast cancer susceptibility gene to be measured has a plurality of, in corresponding Auele Specific Primer, have at least a primer and target area part complementary, and 5 ' end of this primer is with the second sequence label, described the second sequence label, in amplification target area process, carries out mark to the target area amplified production of different testing samples.

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

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

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

As from the foregoing, the method for detection breast cancer susceptibility gene provided by the invention and test kit can detect a plurality of regions of breast cancer susceptibility gene simultaneously, have therefore improved detection efficiency, have reduced testing cost; The method also can break through the restriction of traditional detection method in addition, can identify including the sample of heterozygote, has therefore improved detection sensitivity; In addition can also detect the variation situation that comprises known mutations and unknown mutation site, therefore improve the comprehensive and accuracy of detected result.

Accompanying drawing explanation

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

Fig. 2 is that the present invention is for building the method flow diagram of sequencing library;

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

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

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

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

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

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

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

Figure 10 utilizes fragmentation product and joint component to build the method flow diagram of sequencing library in one embodiment of the invention;

Figure 11 utilizes fragmentation product and joint component to build the method flow diagram of sequencing library in another embodiment of the present invention;

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

Embodiment

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

Target area of the present invention, for the arbitrary sequence on breast cancer susceptibility gene, can select as required, include but not limited to the internal sequence of gene, the regulation and control region, outside of gene, described gene internal sequence, includes but not limited to the intron region, exon region of gene, the region of simultaneously containing intron and exon.

Fig. 1 shows a kind of method flow that detects breast cancer susceptibility gene of the present invention, and the method comprises the following steps:

S1. utilize breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in testing sample, and build sequencing library based on amplified production;

S2. sequencing library is carried out to unit molecule amplification, obtain a plurality of unit molecule amplified productions corresponding with described a plurality of target areas;

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

Present method utilizes high throughput sequencing technologies to carry out region order-checking to breast cancer susceptibility gene, can detect a plurality of regions of breast cancer susceptibility gene simultaneously, improves detection efficiency, reduces costs; Utilize the restriction of high-throughput gene sequencing technological breakthrough traditional detection technology simultaneously, improve detection sensitivity; Can also detect the variation situation that comprises known mutations and unknown mutation site, obtain comprehensive and accurate detected result simultaneously.

By present method, detect the breast cancer susceptibility gene sequence information obtaining, can be in conjunction with 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.

It should be noted that:

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

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

In step S1 gained sequencing library, there is multiple sequencing library molecule, sequencing library is carried out to unit molecule amplification, refer to, by the multiple library molecule in sequencing library, form with denier (even unit molecule) is spatially isolated (but these library molecules still belong to same reaction system on the whole), and realizes amplification in space separately.

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

Wherein, the Auele Specific Primer described in step S1 and target area complete complementary or part are complementary.

Further, in every pair of Auele Specific Primer for target area, have one and target area complementation wholly or in part at least, and 5 ' end of this primer is with the second sequence label.This second sequence label, in amplification target area process, carries out mark to the target area amplified production of different testing samples.This second sequence label, is preferably the nucleic acid molecule with 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, described Auele Specific Primer also can, with other marks, include but not limited to: biotin labeling and other resistance markers, and for amplified production is carried out to 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 as required above-mentioned any scheme to carry out.If while increasing respectively, need to guarantee that for building the amount of the amplified production of sequencing library be 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 breast cancer susceptibility gene described in step S1 comprises at least one in FGFR2, GSTP1, IL-1 β and MTHFR.

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

In one embodiment, the specific implementation process of step S1 is:

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

S12. utilize joint component, the amplified production corresponding with described a plurality of target areas connects, and obtains sequencing library; Described joint component adopts flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

It should be noted that:

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

In step S12, the mode of connection of described joint component and amplified production, can adopt various ways to realize, and comprises that joint component is directly connected with amplified production, or connects after amplified production is processed again.

Joint component described in step S12, for building sequencing library, can comprise one or more joints.

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

This first sequence label is preferably the nucleic acid molecule with 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; Consider various situations, its base number more preferably 4～10.

By the combination of the second sequence label and the 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 sequencing 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 then successfully connecting with the digestion with restriction enzyme that only cuts methylate DNA, thus guarantee that enzyme cuts the unicity of product.

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

In the first embodiment, joint component adopts flat end fitting, and described flat end fitting is the nucleic acid molecule of double-stranded complete complementary.

In a second embodiment, joint component adopts protruding terminus joint, and described protruding terminus joint is double chain acid molecule, and this double chain acid molecule at least comprises a protruding terminus.The base number of this protruding terminus, without concrete restriction, is preferably 1～10 base.According to the structure of this double chain acid molecule, can be divided into two classes, be respectively single protruding terminus joint, two protruding terminus joint.

Single protruding terminus joint as shown in Figure 2, its one end is flat end, the other end is protruding terminus.Wherein the single protruding terminus joint with y splice can prevent that joint is from connecting.For prevent one end be single protruding terminus joint of flat end from connecting, can 3 ' OH on flat end be modified (including but not limited to amino sealing hydroxyl), maybe 5 ' phosphate group on flat 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 are in different nucleic acid chains, they are not complementary each other, in case occur that when connecting joint is from connecting.

In the 3rd embodiment, joint component adopts the joint with loop-stem structure, the described joint with loop-stem structure (as shown in Figure 4) is single stranded nucleic acid molecule, this single stranded nucleic acid molecule comprises the first complementary pairing district 1, (Fig. 4 a) for Jing Huan 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 cut recognition site by this enzyme, specific endonuclease capable Jiang Jinghuan cuts in district or excision, thereby single stranded nucleic acid molecule is become to double chain acid molecule, so that follow-up operation.In one embodiment of the invention, as shown in Figure 4 b, also can be with protruding terminus 4 with the joint of loop-stem structure, this protruding terminus can be positioned at 5 ' end or the 3 ' end of single stranded nucleic acid molecule.The existence of protruding terminus 4 can further prevent 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 described y splice is double chain acid molecule, as shown in Figure 5, comprises complementary district and crotch region, and two strands of described crotch region respectively comprise at least one amplimer binding site.In one embodiment of the invention, the complementary district of described y splice comprises at least one restriction enzyme enzyme recognition site, and this enzyme is cut recognition site can cut formation end by enzyme in building 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 storehouse process; The amplimer binding site comprising on described crotch region, can be directly used in conjunction with amplimer, carries out amplified reaction.

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

Wherein, the 3 ' end in the complementary district of described y splice is protruding terminus or flat end.Preferably, the 3 ' end in the complementary district of described y splice is protruding terminus, this protruding terminus can with the sticky end complementary pairing of described fragmentation product, improved joint efficiency, be beneficial to build carrying out smoothly of sequencing library reaction.

Preferred, described y splice is T end y splice, and the 3 ' end in the complementary district of this joint is protruding terminus, and last base of protruding terminus is T; Example T end y splice as shown in Figure 6, in figure, N is any in A, T, C, G base.

Preferred, the 3 ' end in the complementary district of described y splice is protruding terminus, and the Nucleotide of protruding terminus comprises universal base; Example y splice as shown in Figure 7, in figure, N is any in A, T, C, G base, X is universal base.

Preferably, described y splice is two deoxidation joints, and the 3 ' end in the complementary district of this joint is flat end, and last Nucleotide of 3 ' end is the Nucleotide with two deoxidation bases; Example two deoxidation y splices as shown in Figure 8, in figure, N is any in A, T, C, G base, dd represents that this last Nucleotide of 3 ' end is for the cytidylic acid(CMP) with two deoxidation bases.

It 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 sequencing library.

In another embodiment, according to the needs of high throughput sequencing technologies, when target area amplified production is larger or the target area sequencing library need building hour, after amplified production being carried out to fragmentation processing, be connected with joint component again, build sequencing library, as shown in Figure 9, step S12 comprises the following steps:

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

S122. utilize joint component, be connected with fragmentation product, build sequencing library.

This step is processed by fragmentation, and different amplified productions is become to the similar fragmentation product of length, can contribute to follow-up unified order-checking.

It should be noted that:

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

Described fragmentation also can comprise the separation and purification of fragmentation product and end modified step after processing.According to the fragment length needs of order-checking, the nucleic acid fragment obtaining for fragmentation, carries out the separation and purification of object nucleic acid fragment, and separation method can adopt common method, as gel electrophoresis, saccharose gradient or cesium chloride gradient sedimentation, column chromatography for separation etc.According to used fragmentation method, further end modified to the object 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 realizing the prerequisite of the order-checking of breast cancer susceptibility gene, along with shortening of the breast cancer susceptibility gene fragment length containing in sequencing library molecule, 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 darker, more to the order-checking number of times of each base position of breast cancer susceptibility gene, sequencing result is more accurate, just sensitiveer to the detection of a small amount of sudden change in sample; So just can effectively prevent because on the low side with the ratio of breast cancer susceptibility gene of sudden change in sample, and cause the absolute value of order-checking signal of this sudden change on the low side, the inaccurate phenomenon of sequencing result occurs.

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

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

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

In another embodiment of the present invention, directly at fragmentation product two ends, connect y splice, form sequencing library.The technical program 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, as shown in figure 10, step S122 specifically can be realized by following steps:

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

S1222. cyclisation first connects product, obtains cyclisation product;

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

S1224. at enzyme, cut product two ends and connect the second joint and the 3rd joint, obtain sequencing library.

In step S1221, described the first joint can adopt flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice, and described the 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, NmeA III, Ple I, Sap I, SfaN I and TspDT I, be preferably Acu I, Bsg I, EcoP15 I or Mme I.

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

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

In step S1223, the enzyme that utilization can be identified on the first joint is cut recognition site, and cuts cyclisation product (DNA) but the enzyme that do not cut the first joint carries out enzyme cuts.Enzyme on described the first joint is cut recognition site and is 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.

In step S1224, described the second joint can be flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice, can be with biotin labeling.Described the 3rd joint can be flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice.The second joint and the 3rd joint can be identical or different.Preferably, described the second joint is identical with the 3rd joint, is y splice.Preferred, described y splice is T end y splice or two deoxidation y splice.

It should be noted that above embodiment is only for several specific embodiments in the present invention, not in order to limit the scope of the invention.

It should be noted that, between step S1222 and S1223, also can comprise step: rolling circle amplification cyclisation product, obtain the cyclisation product after amplification, this step can guarantee that follow-up enzyme cuts step S1223 and have enough cyclisation product.

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

Wherein, described the first joint is preferably y splice.

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

Wherein, described amplimer is preferably biotinylation primer.

Wherein, described amplimer is cut recognition site with at least one specific enzymes.

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

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

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

S1221 '. utilize the 4th joint to be connected with fragmentation product, obtain the second connection product;

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

S1223 '. the endonuclease bamhi with the 4th joint is connected with the 5th joint, forms sequencing library.

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

In step S1222 ', the enzyme that utilization can be identified on the 4th joint is cut recognition site, and cuts cyclisation product (DNA) but the enzyme that do not cut the 4th joint carries out enzyme cuts.Enzyme on described the 4th joint is cut recognition site and is 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.

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

In another embodiment of the present invention, step S122 specifically comprises the following steps:

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

S1222 ". utilize restriction enzyme, the fragmentation product Jing Huan district with loop-stem structure is cut or excision, thereby form sequencing library.

The technical program utilization, with the joint of loop-stem structure, has prevented that a plurality of joints are from the generation that connects phenomenon.

Should illustrate, above embodiment is only wherein several specific embodiments of step S122, not in order to limit the scope of the invention.

Wherein, unit molecule amplification described in step S2 refers to each molecule in sequencing 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 described unit molecule amplification includes but not limited to: emulsion-based PCR (Emulsion PCR, EPCR), bridge-type PCR.

Described EPCR is injected into the aqueous solution that comprises all reacted constituents of PCR on the mineral oil surface of high speed rotating, and aqueous solution moment forms the numerous little water droplet being wrapped up by mineral oil.These little water droplets have just formed independently PCR reaction compartment.Under perfect condition, each little water droplet is only containing a DNA profiling (sequencing library molecule) and a magnetic bead, on magnetic bead, contain the primer complementary with the consensus sequence (being introduced by joint component) of sequencing library molecule, after PCR reaction, magnetic bead surfaces is just fixed with the DNA profiling amplified production in the same source of copy huge amount.EPCR concrete steps can 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 described bridge-type PCR is, the primer of bridge-type PCR is fixed on solid phase carrier, in PCR process, pcr amplification product can be fixed on solid phase carrier, and pcr amplification product can with solid phase carrier on primer complementary pairing, Cheng Qiaozhuan, then the primer of complementary pairing be take and increased as template with the amplified production of its Cheng Qiao.The amount adding by controlling original template, after bridge-type PCR react, amplified production form with cluster bunch on solid phase carrier exists, and the amplified production of every cluster be the DNA profiling amplified production of originating together.The principle that it is concrete and embodiment can be with reference to Publication about Document: CN20061009879.X, US6227604.

As previously mentioned, in prior art, Sanger sequencing, due to the technical limitation of self, can only check order to a certain section of region of a sample at every turn, and detection efficiency is low, cost is high.For disposable realization is to detecting when a plurality of region in sample, the present invention takes high-throughput gene order surveying method on sequence measurement.The relative Sanger sequencing of high-throughput gene sequencing detect sequence information more efficient, facilitate sensitive and cost is low, each molecule in sequencing library is after unit molecule amplification, each sequencing library molecule all forms unit molecule copy array, each unit molecule copy array when carrying out high-throughput gene sequencing in different positions, make the hybridization between sequencing primer and unit molecule copy array, and the extension under enzyme effect can carry out simultaneously, do not interfere with each other each other.Therefore, can to a large amount of (millions of up to ten million, even more) unit molecule copy arrays, carry out sequencing reaction, then by gathering corresponding signal simultaneously simultaneously, and then obtain accurately required sequence information, and the sensitivity of order-checking is higher compared with Sanger.Especially the amplified production of a plurality of target areas has been carried out to fragmentation processing, 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, described in step S3, high throughput sequencing technologies includes but not limited to: the synthetic sequencing based on polysaccharase and the connection sequencing based on ligase enzyme.

Synthetic sequencing is that in each building-up reactions, each template strand can only extend once at the most based on being with the Nucleotide that can remove mark to carry out.A kind of roughly flow process of synthetic sequencing is as follows:

A. sequencing primer is combined in (this unit molecule amplified production is fixed on primer-solid phase carrier mixture) on the total known array of unit molecule amplified production by complementary pairing, under the effect of archaeal dna polymerase, the Nucleotide that can remove mark with band carries out single-basic extension building-up reactions, collect the marking signal that this time adds Nucleotide, can obtain the 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, with band, can remove the Nucleotide of mark and proceed single-basic extension building-up reactions, collection adds the marking signal of Nucleotide, can obtain the base sequence information of lower two with the unit molecule amplified production of sequencing primer 3 ' terminal bases complementation.

Repeat b step, until can not proceed building-up reactions, thus obtain the full sequence information of unit molecule amplified production.

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

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) by complementary pairing, (the base position that fluorescent mark is corresponding is x to utilize in above-mentioned oligonucleotide probe one group, x≤h), under the effect of ligase enzyme, nucleic acid probe is connected with above-mentioned oligonucleotide chain, then gather fluorescent signal, after can obtaining 3 ' end of the known array total with single-stranded amplification product or the front x bit base of 5 ' end sequence information, sequencing primer and oligonucleotide probe sex change from unit molecule amplified production of connecting thereof are eluted.

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

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

Use the primer of comparing the many one or more universal base of 3 ' end or 5 ' end with sequencing primer before instead and react by above-mentioned principle, after can extending 3 ' end of the total known array of the unit molecule amplified production of acquisition or the 5 ' 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 sequencing and tetra-sodium sequencing based on polysaccharase have certain similarity, and therefore, in theory, tetra-sodium sequencing can be applicable to detection method of the present invention equally.But that existing tetra-sodium sequencing adopts in order-checking process is natural dNTP, make it in order-checking process, the mensuration for the treatment of continuous single base repetitive sequence that may exist on sequencing library has difficulties; And Nucleotide in synthetic sequencing based on polysaccharase with the mark removed, can guarantee only to extend a base at every turn; 3 ' end with fluorescently-labeled probe or the 5 ' end of connection based on ligase enzyme order-checking in sending out modified, and guarantees only to connect a fluorescent probe in the fragment of each unit molecule amplified production; Therefore the accuracy of synthetic sequencing based on polysaccharase of the present invention and the connection sequencing based on ligase enzyme is high compared with tetra-sodium order-checking.

In addition, aperture on etching optical fiber slide (PTP plate) in existing tetra-sodium order-checking instrument is large (55 μ m * 44 μ m), for holding the amplified production (amplified production of emulsion-based PCR is fixed on the pearl of 10 μ m) of order-checking emulsion-based PCR gained before, the sequencing throughput that this has limited tetra-sodium sequencing greatly, makes the reagent cost of its sequencing reaction higher.In addition, tetra-sodium sequencing also needs to add in the aperture of etching optical fiber slide (PTP plate) mixture that contains multiple protein to guarantee carrying out smoothly of sequencing reaction in order-checking process, and this will improve the reagent cost of sequencing reaction greatly.

And the product that synthetic sequencing based on polysaccharase and the connection sequencing based on ligase enzyme can fix unit molecule amplification by magnetic bead or the slide of 1 μ m, make its flux higher, and except need to be with removing the Nucleotide of mark and the fluorescently-labeled probe that 3 ' end or 5 ' end have carried out modifying, other required reagent, without particular requirement, greatly reduce the reagent cost of sequencing reaction.Obtaining under the prerequisite of same quantity of data, the order-checking cost of the synthetic sequencing based on polysaccharase and the connection sequencing based on ligase enzyme be tetra-sodium sequencing 1/2000th or still less.Therefore what in the present invention program, adopt is that synthetic sequencing based on polysaccharase or the connection sequencing based on ligase enzyme check order to unit molecule amplified production.

In addition, GSTM1 gene belongs to Thiadiazolidine isomerase family, and Main Function is its active group gsh of catalysis and the multiple electrophilic carcinogens coming through a phase metabolic enzyme metabolism activation, promotes it to excrete.The defunctionalization of its isozyme if the individuality of GSTM1 genetically deficient is encoded, lacks the function of detoxification to above-mentioned poisonous substance, thereby increase, appeals to the susceptibility to such carcinogens effect.Therefore, for breast cancer susceptibility detect more accurately with comprehensively, in the present invention, provided the another kind of more accurate and comprehensive method flow that detects breast cancer susceptibility gene, as shown in figure 12, the method has increased the step that detects GSTM1 genetically deficient on the basis of method shown in Fig. 1, specifically comprises following steps:

S1 '. utilize breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in testing sample, and build sequencing library based on amplified production;

S2 '. sequencing library is carried out to unit molecule amplification, obtain a plurality of unit molecule amplified productions corresponding with described a plurality of target areas;

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

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

Step S4 ' and step S3 ', S2 ', S1 ' are without precedence relationship.

The technical program is by high-throughput gene sequencing technology for detection breast cancer susceptibility gene, determine known and unknown mutation site, carry out gene type, the deletion condition that has simultaneously also carried out GSTM1 gene detects, thereby more accurately, comprehensively detects breast cancer susceptibility.

It should be noted that, in the technical program, step S1 ' can be referring to the step S1 in embodiment before to S3 to S3 ', and therefore not to repeat here.

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

In step S4 ', except the target area of amplification GSTM1 gene, also comprise the amplification to reference gene target area, the Auele Specific Primer of described reference gene is SEQ ID NO:13 and SEQ ID NO:14.

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

For the hot spot mutation region of FGFR2, GSTP1, MTHFR, design corresponding Auele Specific Primer: 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 the Auele Specific Primer M2F of reference gene (SEQ ID NO:13) and M2R (SEQ ID NO:14).

One, the extraction of DNA in testing sample

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

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 as follows:

PCR reaction conditions is as follows:

95℃3min；

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

72℃7min。

Utilize PCR to reclaim test kit, respectively the amplified production of each sample is carried out to separation, remove primer and the dNTP of not amplification, reclaim amplified production.

Three, utilize amplified production to build sequencing library

Described in before, this step can be realized by various ways.In one embodiment of the invention, adopt the method that directly connects joint component at fragmentation product two ends to build sequencing library, specifically comprise:

1. the fragmentation of amplified production

In the present embodiment, adopt sonioation method to carry out fragmentation processing, concrete operations are:

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

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

2. utilize fragmentation product and joint component to build sequencing library

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

1) phosphorylation and end-filling

Reaction system is:

Hatch 20min for 20 ℃, reaction finishes rear utilization recovery test kit and carries out purifying recovery.

2) end adds A tail

Reaction system is:

Hatch 30min for 37 ℃, after reaction finishes, utilize purification kit purifying to reclaim.

3) jointing 1

In the present embodiment, adopt as shown in Figure 6 T end y splice as joint 1, same sample is used identical T end y splice, the corresponding different T end y splices of different samples, according to being with the first sequence label to distinguish on it, the sequence label that different samples are corresponding is as shown in table 1 below.

Table 1. the first sequence label data sheet

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

16 ℃ hatch 4h more than, reaction utilizes after finishing purification kit purifying to reclaim.

4) fragment of pcr amplification belt lacing 1

Amplification system is:

PCR reaction conditions is as follows:

95℃3min；

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

72℃7min。

Utilize PCR cleaning agents box, respectively the amplified production of each sample is cleaned, remove primer and the dNTP of not amplification, reclaim the amplified production of the fragment of belt lacing 1.

5) II s type digestion with restriction enzyme

The amplified production that utilizes the fragment of the belt lacing 1 after Acu I enzyme cuts back to close, reaction system is as follows:

Reaction conditions: hatch 1h for 37 ℃.After reaction finishes, utilize purification kit purifying to reclaim enzyme and cut product.

6) jointing 2

Utilization protruding terminus y splice as shown in Figure 7, as joint 2, is cut product with the enzyme reclaiming and is connected, and obtains sequencing library, and linked system is as follows:

Condition of contact, hatches 2h for 14 ℃.After reaction finishes, utilize purification kit purifying to reclaim, then sex change forms strand, obtains sequencing library.

Four, sequencing library is carried out to unit molecule amplification

Determination step three 30 sequencing libraries concentration separately that obtains, unit molecule amplification is carried out in isoconcentration mixing, obtains unit molecule amplified production, and the method for described unit molecule amplification can adopt EPCR or bridge-type PCR.

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

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

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

Through sequencing result analysis, in the present embodiment, detected result is: in sample 3, FGFR2 gene occurrence positions chr10:123346190 is transformed into the sudden change of G by base A, and GSTP1 gene occurrence positions chr11:67352689 sudden change to A by bases G; In sample 5 there is the sudden change of base C to T in the chr1:11856378 position of mthfr gene; GSTP1 occurrence positions chr11:67352689 sudden change to A by bases G in sample 8; GSTP1 gene occurrence positions chr11:67352689 sudden change to A by bases G in sample 12, and there is the sudden change of base C to T in the chr1:11856378 position of mthfr gene; In sample 15 there is the sudden change of base C to T in the chr1:11856378 position of mthfr gene; In sample 24 there is the sudden change of base C to T in the chr1:11856378 position of mthfr gene, and GSTP1 gene occurrence positions chr11:67352689 sudden change to A by bases G; All the other are all 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 respectively the amplification of GSTM1 and reference gene, obtain amplified production.PCR reaction system is:

PCR reaction conditions is as follows:

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 180V condition, 0.5h electrophoresis, then with the SYBR dyeing observation that develops the color.Product judging criterion: if having 270bp band in running gel, contain GSTM1 gene, otherwise lack.

Detected result shows: GSTM1 genetically deficient in sample 3, in sample 8, in sample 12, in sample 15, in sample 24; All the other have no GSTM1 genetically deficient.

It should be noted that the present embodiment is a specific embodiment of the present invention, the present invention, without any restriction effect, is applied corresponding Auele Specific Primer and substituted, can be applied to detection method of the present invention equally.

For detection sensitivity of the present invention, the invention provides an embodiment and verified.

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

Prepare respectively saltant type ratio and be 20%, 10%, 5%, 3%, 1%, 0% plasmid mixed solution.The above-mentioned plasmid mixed solution that contains 1000 copy plasmids of take is template, then by the method with reference to above-described embodiment, detects.Detected result is as shown in table 2 below:

Table 2. detection sensitivity verification msg

Result shows, the lowest detection of the method for detection breast cancer susceptibility gene of the present invention is limited to 3% left and right, 5% and above detected result and practical situation basically identical.This shows, detection method of the present invention can break through the restriction of traditional method, can identify including the sample of heterozygote, has improved detection sensitivity.

A detection kit that can be used for above-mentioned any breast cancer susceptibility gene detection method, comprising:

Breast cancer susceptibility gene Auele Specific Primer, increases for a plurality of target areas to testing sample;

Joint component, builds sequencing library for being combined with amplified production.

Wherein, described joint component adopt flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

The test kit of described detection breast cancer susceptibility gene can detect a plurality of regions of breast cancer susceptibility gene simultaneously, improves detection efficiency, reduces costs; 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 described joint component includes the first sequence label, in library construction process, the sequencing library of different testing samples is carried out to mark.Described the first sequence label, is preferably the nucleic acid molecule with specific base sequence, and its base number is not limit.

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

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

The test kit of described detection breast cancer susceptibility gene for detection of breast cancer susceptibility gene comprise at least one in FGFR2, GSTP1, IL-1 β and MTHFR and GSTM1.

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

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

Wherein, described test kit can also comprise reference gene amplimer, and described amplimer is SEQ ID NO:13 and SEQ ID NO:14.

Wherein, described test kit also can comprise the enzyme of cutting recognition site for identifying enzyme on joint component.

It should be noted that the 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 apply method set forth in the present invention.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, in conjunction with follow-up further molecular biology test, clinical trial, clinical observation and other integrated data, carry out statistical study, set up certain breast cancer disease model, can realize the susceptibility of early stage assessment mammary cancer.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.

Claims (24)

1. for a method for the detection breast cancer susceptibility gene of non-medical diagnosis on disease object, it is characterized in that, said method comprising the steps of:

A. utilize breast cancer susceptibility gene Auele Specific Primer, increased in a plurality of target areas in testing sample, and build sequencing library based on amplified production;

B. sequencing library is carried out to unit molecule amplification, obtain a plurality of unit molecule amplified productions corresponding with described a plurality of target areas;

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

Described steps A comprises:

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

A2. utilize joint component, the amplified production corresponding with described a plurality of target areas connects, and obtains sequencing library; Described joint component adopts flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice, and at least adopt this kind of joint of y splice, described y splice is double chain acid molecule, comprise complementary district and crotch region, two strands of described crotch region respectively comprise at least one amplimer binding site, and described complementary district comprises at least one digestion with restriction enzyme recognition site;

Described testing sample is whole blood, serum, blood plasma or tissue sample.

2. the method for detection breast cancer susceptibility gene according to claim 1, is characterized in that, described restriction enzyme enzyme recognition site is II s type digestion with restriction enzyme recognition site.

3. the method for detection breast cancer susceptibility gene according to claim 1, is characterized in that, the 3 ' end in the complementary district of described y splice is protruding terminus or flat end.

4. the method for detection breast cancer susceptibility gene according to claim 3, is characterized in that, the 3 ' end in the complementary district of described y splice is protruding terminus, and this protruding terminus is T.

5. the method for detection breast cancer susceptibility gene according to claim 3, is characterized in that, the 3 ' end in the complementary district of described y splice is flat end, and this last Nucleotide of flat end is the Nucleotide with two deoxidation bases.

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

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

A22. utilize joint component, be connected with described fragmentation product, build sequencing library.

7. the method for detection breast cancer susceptibility gene according to claim 6, is characterized in that, described steps A 22 comprises the following steps:

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

A222. cyclisation first connects product, obtains cyclisation product;

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

A224. at enzyme, cut product two ends and connect the second joint and the 3rd joint, obtain sequencing library.

8. the method for detection breast cancer susceptibility gene according to claim 6, is characterized in that, described steps A 22 comprises the following steps:

A221 '. utilize the 4th joint to be connected with fragmentation product, obtain the second connection product;

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

A223 '. the endonuclease bamhi with the 4th joint is connected with the 5th joint, obtains sequencing library.

9. according to the method for the detection breast cancer susceptibility gene described in any one in claim 1 to 8, it is characterized in that, at least one joint described in steps A 2 in joint component includes the first sequence label, in library construction process, the sequencing library of different testing samples is carried out to mark.

10. according to the method for the detection breast cancer susceptibility gene described in any one in claim 1 to 8, it is characterized in that, breast cancer susceptibility gene described in steps A has a plurality of, at least one complementary with this target area part in Auele Specific Primer corresponding to the target area of each mammary gland tumor susceptibility gene, and 5 ' end of this primer is with the second sequence label, for in library construction process, the sequencing library of different testing samples is carried out to mark.

11. according to the method for the detection breast cancer susceptibility gene described in any one in claim 1 to 8, it is characterized in that, described breast cancer susceptibility gene comprises at least one in FGFR2, GSTP1, IL-1 β and MTHFR.

The method of 12. detection breast cancer susceptibility genes according to claim 11, it is characterized in that, described breast cancer susceptibility gene Auele Specific Primer comprises: at least one pair of in SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

The method of 13. detection breast cancer susceptibility genes according to claim 1, is characterized in that, described method also comprises step:

D. utilize the specificity amplification primer of GSTM1 testing sample to be carried out to pcr amplification, the deletion condition of detected through gel electrophoresis GSTM1 gene; Between step D and steps A, B or C all without sequencing relation.

The method of 14. detection breast cancer susceptibility genes according to claim 13, is characterized in that, the specificity amplification primer of described GSTM1 is to comprising: at least one pair of in SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

15. 1 kinds of breast cancer susceptibility gene detection kit, is characterized in that, described test kit comprises:

Breast cancer susceptibility gene Auele Specific Primer, increases for a plurality of target areas to testing sample;

Joint component, builds sequencing library for being combined with amplified production;

Described joint component adopts flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice, and at least adopt this kind of joint of y splice, described y splice is double chain acid molecule, comprise complementary district and crotch region, two strands of described crotch region respectively comprise at least one amplimer binding site, and described complementary district comprises at least one digestion with restriction enzyme recognition site.

16. breast cancer susceptibility gene detection kit according to claim 15, is characterized in that, described restriction enzyme enzyme recognition site is II s type digestion with restriction enzyme recognition site.

17. breast cancer susceptibility gene detection kit according to claim 15, is characterized in that, the 3 ' end in the complementary district of described y splice is protruding terminus or flat end.

18. breast cancer susceptibility gene detection kit according to claim 17, is characterized in that, the 3 ' end in the complementary district of described y splice is protruding terminus, and this protruding terminus is T.

19. breast cancer susceptibility gene detection kit according to claim 17, is characterized in that, the 3 ' end in the complementary district of described y splice is flat end, and this last Nucleotide of flat end is the Nucleotide with two deoxidation bases.

20. breast cancer susceptibility gene detection kit according to claim 15, it is characterized in that, at least one joint in described joint component includes the first sequence label, in library construction process, the sequencing library of different testing samples is carried out to mark.

21. breast cancer susceptibility gene detection kit according to claim 15, it is characterized in that, described breast cancer susceptibility gene has a plurality of, in the Auele Specific Primer corresponding with each target area, have at least a primer and target area part complementary, and 5 ' end of this primer is with the second sequence label, for in amplification target area process, the target area amplified production of different testing samples is carried out to mark.

22. test kits according to claim 15 to the detection breast cancer susceptibility gene described in any one in 21, is characterized in that, described breast cancer susceptibility gene comprises at least one in FGFR2, GSTP1, IL-1 β, MTHFR and GSTM1.

23. test kits according to claim 15 to the detection breast cancer susceptibility gene described in any one in 21, it is characterized in that, described breast cancer susceptibility gene Auele Specific Primer comprises: at least one pair of in SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

The test kit of 24. detection breast cancer susceptibility genes according to claim 23, it is characterized in that, described breast cancer susceptibility gene Auele Specific Primer also comprises: at least one pair of in SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.