CN102373287A

CN102373287A - Method and kit for detecting lung cancer susceptibility gene

Info

Publication number: CN102373287A
Application number: CN201110391964XA
Authority: CN
Inventors: 盛司潼
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-11-30
Filing date: 2011-11-30
Publication date: 2012-03-14
Anticipated expiration: 2031-11-30
Also published as: CN102373287B

Abstract

The invention relates to the field of genetic engineering, and provides a method and a corresponding kit for detecting a lung cancer susceptibility gene. The method for detecting the lung cancer susceptibility gene comprises the following steps of: A, using a specificity primer of the lung cancer susceptibility gene to amplify a plurality of target areas in a sample to be detected, and establishing a sequencing library based on amplified products; B, subjecting the sequencing library to monomolecular amplification to obtain a plurality of monomolecular amplification products corresponding to the plurality of target areas; and C, subjecting the plurality of monomolecular amplification products to high-throughput gene sequencing at the same time so as to obtain sequence information of the plurality of target areas. The method and the corresponding kit are used for sequencing the plurality of areas of the lung cancer susceptibility gene at the same time by using a high-throughput sequencing technology; the sequence information including variation of known mutations and unknown mutations in the areas can be obtained accurately; the kit has high detection sensitivity, and can be used for further detecting mainly samples at the same time.

Description

A kind of method of detection of lung cancer tumor susceptibility gene and test kit

Technical field

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

Background technology

Tumor susceptibility gene is meant with specified disease to have certain related gene or allelotrope.Tumor susceptibility gene to complex disease detects; Obtain its concrete sequence information; Combine the statistical study of follow-up further molecular biology test, clinical trial, clinical observation and integrated data then; Set up certain disease model, can 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, and promptly a plurality of polygenic effects add up each other and then form the obvious phenotypes combined effect.Therefore; A plurality of tumor susceptibility genes and mutational site to disease (particularly are directed against topmost genetic mutation form---SNP (Single Nucleotide Polymorphisms in the genome; SNP), it account for genetic mutation in the whole genome 92%) detect most important.

Lung cancer betides the tunica mucosa bronchiorum epithelium, also claims lung bronchogenic carcinoma.Along with industrial development, topsoil aggravation all increases progressively with 4.5% speed in lung cancer incidence and mortality ratio every year in the world fast.Lung cancer becomes the highest a kind of cancer of mortality ratio, is that medicine and regimen are not still arranged at present especially on the one hand, and the low curative ratio of 10-15% be that lung cancer finds it generally all is late period on the other hand, even transfer has taken place cancer cells.Clinical showing, find that more early the patients with lung cancer survival rate is high more.

The lung cancer tumor susceptibility gene is that preliminary study possibly have certain related gene with lung cancer, and wherein to have height to have low for the relational degree of each tumor susceptibility gene.At present; The method of detection of lung cancer tumor susceptibility gene is modal to be the Sanger PCR sequencing PCR; This method can be carried out the zone to the lung cancer tumor susceptibility gene and detected, but the Sanger PCR sequencing PCR can only check order order-checking cost height to a certain section zone of a sample at every turn; Sensitivity low (20%) can't draw the precise information that morphs in each mutational site.

In addition; Though the achievement in research to detection of lung cancer tumor susceptibility gene and SNP site is a lot; But prior art is applicable to single SNP site and detects and analyze that be inappropriate for joint-detection and analysis are carried out in many SNP site, detected result is all not accurate and comprehensive enough.

Therefore the novel method that needs a kind of detection of lung cancer tumor susceptibility gene; Can detect simultaneously a plurality of zones of lung cancer tumor susceptibility gene; Accurately draw these regional sequence informations; The variation situation that comprises each mutational site of known mutations and unknown mutation, detection sensitivity is high, and can be further simultaneously to a large amount of sample detection.

Summary of the invention

The object of the present invention is to provide a kind of method and reagent corresponding box of detection of lung cancer tumor susceptibility gene; Can detect simultaneously a plurality of target areas of lung cancer tumor susceptibility gene; Draw the accurate sequence information in these zones; The variation situation that comprises each mutational site of known mutations and unknown mutation, detection sensitivity is high, and can further detect a large amount of samples simultaneously.

In order to realize goal of the invention, a kind of method of detection of lung cancer tumor susceptibility gene may further comprise the steps:

A. utilize lung cancer tumor 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, said steps A comprises:

A1. utilize lung cancer tumor 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, 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 the 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 corresponding mark is done 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.

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

Wherein, the complementary fully or part complementation in steps A described lung cancer tumor susceptibility gene Auele Specific Primer and target area.

Further; 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 has second sequence label, is used for the process in the amplification target area, and the target area amplified production of different testing samples is done corresponding mark.Said second sequence label is preferably the nucleic acid molecule that has particular sequence, and its base number is not limit.

Further, the said second sequence label base number is not limit, and is preferably 3～20, and more preferably 4～10.

Wherein, said lung cancer tumor susceptibility gene comprises at least one among APE1, CASP7, CASP8, CASP9, CHEK2, COX-2, CYP1A1, CYP2E1, ERCC1, ERCC2, ERCC6, Exo1, GSTP1, Hmlh1, IL-1 β, MDM2, MPO, MTHFR, NQO1, OGG1, P73, RASSF1, TERT, TGFB1, TP53, TP63, XPC and the XRCC1.

Further, the Auele Specific Primer of said APE1 is SEQ ID NO:1 and SEQ IDNO:2; The Auele Specific Primer of said CASP7 is SEQ ID NO:3 and SEQ ID NO:4; The Auele Specific Primer of said CASP8 is SEQ ID NO:5 and SEQ ID NO:6; The Auele Specific Primer of said CASP9 is SEQ ID NO:7 and SEQ ID NO:8; The Auele Specific Primer of said CHEK2 is SEQ ID NO:9 and SEQ ID NO:10; The Auele Specific Primer of said COX-2 is SEQ ID NO:11 and SEQ ID NO:12; The Auele Specific Primer of said CYP1A1 comprises at least one pair of among SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and the SEQ IDNO:16; The Auele Specific Primer of said CYP2E1 is SEQ ID NO:17 and SEQ IDNO:18; The Auele Specific Primer of said ERCC1 is SEQ ID NO:19 and SEQ ID NO:20; The Auele Specific Primer of said ERCC2 comprises 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 ERCC6 is SEQ ID NO:25 and SEQ ID NO:26; The Auele Specific Primer of said Exo1 is SEQ ID NO:27 and SEQ ID NO:28; The Auele Specific Primer of said GSTP1 is SEQ ID NO:29 and SEQ ID NO:30; The Auele Specific Primer of said Hmlh1 is SEQ ID NO:31 and SEQ ID NO:32; The Auele Specific Primer of said IL-1 β is SEQ IDNO:33 and SEQ ID NO:34; The Auele Specific Primer of said MDM2 is SEQ ID NO:35 and SEQ IDNO:36; The Auele Specific Primer of said MPO is SEQ ID NO:37 and SEQ ID NO:38; The Auele Specific Primer of said MTHFR is SEQ ID NO:39 and SEQ ID NO:40; The Auele Specific Primer of said NQO1 is SEQ ID NO:41 and SEQ ID NO:42; The Auele Specific Primer of said OGG1 is SEQ IDNO:43 and SEQ ID NO:44; The Auele Specific Primer of said P73 comprises at least one pair of among SEQ ID NO:45 and SEQ IDNO:46, SEQ ID NO:47 and the SEQ ID NO:48; The Auele Specific Primer of said RASSF1 is SEQ ID NO:49 and SEQ ID NO:50; The Auele Specific Primer of said TERT is SEQ IDNO:51 and SEQ ID NO:52; The Auele Specific Primer of said TGFB1 is SEQ ID NO:53 and SEQ IDNO:54; The Auele Specific Primer of said TP53 is SEQ ID NO:55 and SEQ ID NO:56; The Auele Specific Primer of said TP63 is SEQ ID NO:57 and SEQ ID NO:58; The Auele Specific Primer of said XPC is SEQ ID NO:59 and SEQ ID NO:60; The Auele Specific Primer of said XRCC1 comprises at least one pair of among SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ IDNO:66, SEQ ID NO:67 and the SEQ ID NO:68.

Wherein, said method can also comprise step:

D. the amplimer that utilizes GSTM1 and GSTT1 carries out pcr amplification, the deletion condition of detected through gel electrophoresis GSTM1 and GSTT1 gene to the nucleic acid of testing sample; Step D and steps A, B, C do not have precedence relationship.

Further, the amplimer of said GSTM1 comprises at least one pair of among SEQ ID NO:69 and SEQ ID NO:70, SEQ ID NO:71 and the SEQ ID NO:72; The amplimer of said GSTT1 is SEQID NO:73 and SEQ ID NO:74.

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

Lung cancer tumor 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 corresponding mark is done 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.

Wherein, Described tumor susceptibility gene to be measured has a plurality of; In the Auele Specific Primer corresponding, have at least a primer and target area part complementary, and 5 ' end of this primer have second sequence label with each target area; Be used for process, the target area amplified production of different testing samples is done corresponding mark in the amplification target area.

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

Further, said lung cancer tumor susceptibility gene also comprises at least one among GSTM1, the GSTT1.

Further, the Auele Specific Primer of said APE1 is SEQ ID NO:1 and SEQ ID NO:2; The Auele Specific Primer of said CASP7 is SEQ ID NO:3 and SEQ ID NO:4; The Auele Specific Primer of said CASP8 is SEQ ID NO:5 and SEQ ID NO:6; The Auele Specific Primer of said CASP9 is SEQ ID NO:7 and SEQ ID NO:8; The Auele Specific Primer of said CHEK2 is SEQ ID NO:9 and SEQ ID NO:10; The Auele Specific Primer of said COX-2 is SEQ ID NO:11 and SEQ ID NO:12; The Auele Specific Primer of said CYP1A1 comprises at least one pair of among SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and the SEQ IDNO:16; The Auele Specific Primer of said CYP2E1 is SEQ ID NO:17 and SEQ IDNO:18; The Auele Specific Primer of said ERCC1 is SEQ ID NO:19 and SEQ ID NO:20; The Auele Specific Primer of said ERCC2 comprises 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 ERCC6 is SEQ ID NO:25 and SEQ ID NO:26; The Auele Specific Primer of said Exo1 is SEQ ID NO:27 and SEQ ID NO:28; The Auele Specific Primer of said GSTP1 is SEQ ID NO:29 and SEQ ID NO:30; The Auele Specific Primer of said Hmlh1 is SEQ ID NO:31 and SEQ ID NO:32; The Auele Specific Primer of said IL-1 β is SEQ IDNO:33 and SEQ ID NO:34; The Auele Specific Primer of said MDM2 is SEQ ID NO:35 and SEQ IDNO:36; The Auele Specific Primer of said MPO is SEQ ID NO:37 and SEQ ID NO:38; The Auele Specific Primer of said MTHFR is SEQ ID NO:39 and SEQ ID NO:40; The Auele Specific Primer of said NQO 1 is SEQ ID NO:41 and SEQ ID NO:42; The Auele Specific Primer of said OGG1 is SEQ IDNO:43 and SEQ ID NO:44; The Auele Specific Primer of said P73 comprises at least one pair of among SEQ ID NO:45 and SEQ IDNO:46, SEQ ID NO:47 and the SEQ ID NO:48; The Auele Specific Primer of said RASSF1 is SEQ ID NO:49 and SEQ ID NO:50; The Auele Specific Primer of said TERT is SEQ IDNO:51 and SEQ ID NO:52; The Auele Specific Primer of said TGFB1 is SEQ ID NO:53 and SEQ IDNO:54; The Auele Specific Primer of said TP53 is SEQ ID NO:55 and SEQ ID NO:56; The Auele Specific Primer of said TP63 is SEQ ID NO:57 and SEQ ID NO:58; The Auele Specific Primer of said XPC is SEQ ID NO:59 and SEQ ID NO:60; The Auele Specific Primer of said XRCC 1 comprises at least one pair of among SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ IDNO:66, SEQ ID NO:67 and the SEQ ID NO:68; The Auele Specific Primer of said GSTM1 comprises at least one pair of among SEQ ID NO:69 and SEQ ID NO:70, SEQ ID NO:71 and the SEQ ID NO:72; The Auele Specific Primer of said GSTT1 is SEQ ID NO:73 and SEQ ID NO:74.

By on can know; Lung cancer tumor susceptibility gene detection method provided by the invention and test kit thereof; A plurality of zones of detection of lung cancer tumor susceptibility gene accurately draw these regional sequence informations simultaneously, comprise the variation situation in each mutational site of known mutations and unknown mutation; Detection sensitivity is high, and can be further simultaneously to a large amount of sample detection.

Description of drawings

Fig. 1 is the method flow diagram of detection of lung cancer tumor susceptibility gene in the one embodiment of the invention;

Fig. 2 is the joint synoptic diagram of the single protruding terminus joint in the one embodiment of the invention;

Fig. 3 is the structural representation of the two protruding terminus joints 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 y splice in the one embodiment of the invention;

Fig. 6 is the structural representation of the terminal y splice of T in the one embodiment of the invention;

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

Fig. 8 is the structural representation of the two deoxidation y splices in the one embodiment of the invention;

Fig. 9 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 10 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 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 of detection of lung cancer tumor 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 lung cancer tumor susceptibility gene; Can select as required; Include but not limited to the internal sequence of lung cancer tumor susceptibility gene, the outside regulating and controlling sequence of lung cancer tumor susceptibility gene, the internal sequence of said lung cancer tumor susceptibility gene includes but not limited to the intron zone, exon region of lung cancer tumor susceptibility gene, contains the zone of intron and exon simultaneously.

Fig. 1 shows the method flow of a kind of detection of lung cancer tumor susceptibility gene of the present invention, and this method may further comprise the steps:

S1. utilize lung cancer tumor 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 the zone order-checking is carried out in a plurality of zones of lung cancer tumor susceptibility gene simultaneously; Can accurately learn the sequence information that these are regional; The variation situation that comprises each mutational site of known mutations and unknown mutation; Detection sensitivity is high, can be accurately and obtain the sequence information of lung cancer tumor susceptibility gene comprehensively.

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

Need to prove:

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 lung cancer tumor susceptibility gene, also can derive from different lung cancer tumor 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, in the primer corresponding, have at least a primer and target area part complementary, and 5 ' end of this primer have second sequence label with each target area.This second sequence label is used for the process in the amplification target area, and the target area amplified production of different testing samples is done corresponding 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 affinity tag, includes but not limited to: biotin labeling, poly histidine mark, antigen, antibody, thus make that the purifying of target area amplified production is very convenient.

In addition, the amplification in the different target of same testing sample zone 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 increased respectively in each target area respectively; Can be consistent through the molecule number that the amount of measuring amplified production guarantees to be used among the step S1 target area amplified production in order-checking library, establishing target zone; Can not cause the different target zone copy number of same testing sample different because of amplification step, and then influence follow-up sequencing reaction result.

Certainly, big or small close when each target area is when GC content is also close; Through rational design primer; Utilize multiple PCR technique, step S1 can increase to a plurality of target areas simultaneously, and guarantees that the amplification efficiency between each target area keeps basically identical; So just can effectively improve conventional efficient, reduce the cost of reaction.

When testing sample has when a plurality of, the amplification of the target area of different samples must be carried out respectively.

Wherein, the described lung cancer tumor susceptibility gene of step S1 comprises at least a among APE1, CASP7, CASP8, CASP9, CHEK2, COX-2, CYP1A1, CYP2E1, ERCC1, ERCC2, ERCC6, Exo1, GSTP1, Hmlh1, IL-1 β, MDM2, MPO, MTHFR, NQO1, OGG1, P73, RASSF1, TERT, TGFB1, TP53, TP63, XPC and the XRCC1.

Further, the Auele Specific Primer of said APE1 is SEQ ID NO:1 and SEQ ID NO:2; The Auele Specific Primer of said CASP7 is SEQ ID NO:3 and SEQ ID NO:4; The Auele Specific Primer of said CASP8 is SEQ ID NO:5 and SEQ ID NO:6; The Auele Specific Primer of said CASP9 is SEQ ID NO:7 and SEQ ID NO:8; The Auele Specific Primer of said CHEK2 is SEQ ID NO:9 and SEQ ID NO:10; The Auele Specific Primer of said COX-2 is SEQ ID NO:11 and SEQ ID NO:12; The Auele Specific Primer of said CYP1A1 comprises at least one pair of among SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and the SEQ IDNO:16; The Auele Specific Primer of said CYP2E1 is SEQ ID NO:17 and SEQ IDNO:18; The Auele Specific Primer of said ERCC1 is SEQ ID NO:19 and SEQ ID NO:20; The Auele Specific Primer of said ERCC2 comprises 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 ERCC6 is SEQ ID NO:25 and SEQ ID NO:26; The Auele Specific Primer of said Exo1 is SEQ ID NO:27 and SEQ ID NO:28; The Auele Specific Primer of said GSTP1 is SEQ ID NO:29 and SEQ ID NO:30; The Auele Specific Primer of said Hmlh1 is SEQ ID NO:31 and SEQ ID NO:32; The Auele Specific Primer of said IL-1 β is SEQ IDNO:33 and SEQ ID NO:34; The Auele Specific Primer of said MDM2 is SEQ ID NO:35 and SEQ IDNO:36; The Auele Specific Primer of said MPO is SEQ ID NO:37 and SEQ ID NO:38; The Auele Specific Primer of said MTHFR is SEQ ID NO:39 and SEQ ID NO:40; The Auele Specific Primer of said NQO1 is SEQ ID NO:41 and SEQ ID NO:42; The Auele Specific Primer of said OGG1 is SEQ IDNO:43 and SEQ ID NO:44; The Auele Specific Primer of said P73 comprises at least one pair of among SEQ ID NO:45 and SEQ IDNO:46, SEQ ID NO:47 and the SEQ ID NO:48; The Auele Specific Primer of said RASSF1 is SEQ ID NO:49 and SEQ ID NO:50; The Auele Specific Primer of said TERT is SEQ IDNO:51 and SEQ ID NO:52; The Auele Specific Primer of said TGFB 1 is SEQ ID NO:53 and SEQ IDNO:54; The Auele Specific Primer of said TP53 is SEQ ID NO:55 and SEQ ID NO:56; The Auele Specific Primer of said TP63 is SEQ ID NO:57 and SEQ ID NO:58; The Auele Specific Primer of said XPC is SEQ ID NO:59 and SEQ ID NO:60; The Auele Specific Primer of said XRCC 1 comprises at least one pair of among SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ IDNO:66, SEQ ID NO:67 and the SEQ ID NO:68.

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

S11. utilize lung cancer tumor 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 lung cancer tumor 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. 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 of step S12 includes first sequence label, and this first sequence label is used in the library construction process, and corresponding mark is done 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.Further, the base number of said first sequence label is preferably 3～20, like this, and at every turn at least can be to 4 ³Individual sample detects simultaneously.After taking all factors into consideration various situation, as: the specificity of label, the cost of joint, length of said joint etc., the base number of first sequence label is preferably 4～10.

Through the combination of second label and first 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: by biotinylation or methylate, 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 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 in the process of order-checking library and can use one or more joints.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.Said structure form to joint below will provide a plurality of embodiment.In first embodiment, joint component adopts flat end fitting, and this joint is double-stranded complementary nucleic acid molecule fully.

In a second embodiment, joint component adopts the protruding terminus joint, and this 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, the protruding terminus joint can be divided into two types, is 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 is as shown in Figure 4.This joint is a single stranded nucleic acid molecule; This single stranded nucleic acid molecule comprise the first complementary pairing district 1, stem ring district 2 and the second complementary pairing district 3 (Fig. 4 a), the first complementary pairing district 1 can with the second complementary pairing district, 3 complementary pairings, and the complementary pairing district that their form comprises at least one restriction enzyme enzyme recognition site; And cut recognition site through this enzyme; 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 is as shown in Figure 5, is double chain acid molecule, 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 N Nucleotide; Preferably, 9≤N≤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.Wherein, the base number of protruding terminus does not have particular restriction, preferably between 1 to 4.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, not in order to restriction 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.

The length of the said fragmentation product of step S121 is not limit, preferably between 25bp to 500bp, more preferably between 30bp to 200bp, more preferably between 40bp to 100bp.Under the prerequisite of realization to the order-checking of target area, along with shortening of the target area fragment length that contains in the molecule of order-checking library, target area, the order-checking degree of depth to the target area 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 target area 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 target area 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, will further explain this step through a plurality of embodiment and accompanying drawing below.

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, EcoP15I, 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.

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.

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.

In step S1222, cyclisation first connects product has multiple implementation.

In one embodiment of this invention, step S1222 may further comprise the steps:

S12221. utilize enzyme to cut primer the first connection product is increased, get amplified production;

S12222. amplified production is carried out enzyme and cut, make amplified production form sticky end, and self loop changes into cyclisation product.

Said enzyme is cut 3 ' of primer and is held two terminal portions complementations that are connected product respectively with first, and 5 ' end all contains the restriction enzyme enzyme recognition site.The amplified production that the amplification of process step S12221 forms; Its two ends all contain the restriction enzyme enzyme recognition site, under the effect of corresponding enzyme, make the two ends of amplified production form sticky end then; And these two sticky end complementations can be carried out recirculation.

In another embodiment of the present invention; Said first joint includes 2 enzymes and cuts recognition site; One of them is the restriction enzyme enzyme recognition site, and the two ends that are used to make first of step S1222 formation connect product form sticky end under the effect of corresponding enzyme; And complementary between them, can carry out recirculation.Another is an II s type digestion with restriction enzyme recognition site, is used at step S1223, utilizes the enzyme identification cyclisation product of this restriction enzyme site of identification, carries out enzyme and cuts, and then obtain enzyme and cut product.

Should explain, more than two kinds of being merely among the present invention of two embodiment realize that cyclisation first connect the embodiments of products, do not do any concrete restriction for protection scope of the present invention.

In 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.

In 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.

What need special instruction is also can comprise step S1222A between step S1222 and the S1223: the rolling circle amplification cyclisation product gets the rolling circle amplification product.Through step S1222A, can guarantee that follow-up enzyme cuts step S1223 enough starting material are arranged.

Or, between step S1221 and S1222, also can comprise step S1221A: utilize amplimer to be connected product and increase first, amplified production.Said amplimer is connected the joint sequence complementation at product two ends respectively with first.Through step S1221A, can guarantee that follow-up cyclisation step has enough starting material.

This programme can avoid after step S1222, carrying out rolling circle amplification, replaces and carry out the common PCR amplification with the step S1221A before the step S1222, can effectively reduce follow-up enzyme to cut in the step consumption of II s type restriction enzyme.

Wherein, said first joint is preferably y splice.

Wherein, the complementation district of said y splice can comprise at least one enzyme and cuts recognition site.Said enzyme is cut recognition site and can be common restriction enzyme enzyme recognition site, also can be II s type digestion with restriction enzyme recognition site.

Wherein, the said amplimer of step S1221A is preferably the biotinylation primer, helps the recovery purifying of amplified production.

Wherein, the said amplimer of step S1221A 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 utilizes corresponding restriction enzyme to carry out enzyme among the step S1222 and cut, and then connect cyclisation.

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.

In 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, with the end-filling of fragment product, the preferred two deoxidation joints of then said the 4th joint.

In 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.

In 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 increases separately to each molecule in the order-checking library, 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 sequencing technologies can only check order to a certain section zone of a sample owing to the technical limitation of self at every turn.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 detects the more convenient sensitivity of sequence information; 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.

Connecting PCR sequencing PCR 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 connect PCR sequencing PCR principle and specific embodiments can be with reference to CN200710170507.1.

In addition, GSTM1 and GSTT1 gene all belong to Thiadiazolidine isomerase family, play the katabolism hazardous and noxious substances in vivo.The individuality of GSTM1 or GSTT1 genetically deficient is subject to the influence of smoking, and risk of lung cancer takes place to be increased.Therefore; For lung cancer tumor susceptibility gene sequence information further accurately with comprehensively; The present invention provides the method flow of another kind of more accurate and comprehensive detection of lung cancer tumor susceptibility gene; Shown in figure 12, this method has increased the step that detects GSTM1 or GSTT1 genetically deficient on the basis of method shown in Figure 1, specifically comprise following steps:

S1 '. utilize lung cancer tumor 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 '. the amplimer that utilizes GSTM1 and GSTT1 carries out pcr amplification, the deletion condition of detected through gel electrophoresis GSTM1 and GSTT1 gene to the nucleic acid of testing sample.

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

The present technique scheme is confirmed known and the unknown mutation site through detecting lung cancer tumor susceptibility gene to be measured, carries out gene type, and the deletion condition that also carries out GSTM1 and GSTT1 gene simultaneously detects, and can access more accurately and comprehensively tumor susceptibility gene sequence information.

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.

Preferably, the amplimer of GSTM1 described in the step S4 comprises at least one pair of among SEQ ID NO:69 and SEQ IDNO:70, SEQ ID NO:71 and the SEQ ID NO:72; The amplimer of said GSTT1 is SEQ ID NO:73 and SEQ ID NO:74.

Further, step S4 also comprises the amplification to the internal control gene target area, the Auele Specific Primer of said internal control gene is SEQ ID NO:75 and SEQ ID NO:76.

Among the embodiment that the present invention proposes, lung cancer tumor susceptibility genes to be measured such as COX-2, CYP1A1, ERCC2, GSTP1, TP53, XRCC1 and the GSTM1 in the while detection of lung cancer tumor susceptibility gene, GSTT1.

To COX-2; CYP1A1; ERCC2; GSTP1; TP53; The focus sudden change zone design corresponding special primers of XRCC1: C1F (SEQ ID NO:11) and C1R (SEQ ID NO:12); C2F (SEQ ID NO:13) and C2R (SEQ ID NO:14); E1F (SEQ ID NO:21) and E1R (SEQ ID NO:22); G1F (SEQ ID NO:29) and G1R (SEQ ID NO:30); T1F (SEQ ID NO:55) and T1R (SEQ ID NO:56); X1F (SEQ ID NO:63) and X1R (SEQ ID NO:64); Design simultaneously detects the Auele Specific Primer of GSTM1, GSTT1 genetically deficient: G2F (SEQ ID NO:69) and G2R (SEQ ID NO:70), G3F (SEQ ID NO:73) and G3R (SEQID NO:74), and Auele Specific Primer M1F of internal control gene (SEQ ID NO:75) and M1R (SEQID NO:76).

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 do corresponding mark respectively.

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

Utilize the Auele Specific Primer of above-mentioned tumor susceptibility gene to be measured, increased in the target area of lung cancer tumor 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 lung cancer tumor 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；

T4DNA 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：85) 10μL；

Primer?R(100μM，SEQ?ID?NO：86) 10μL；

Ex?Taq(5U/μL) 7.5μL；

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);

10T4DNA ligase enzyme (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 IDNO:77 and SEQ ID NO:78, 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 lung 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: the CYP1A1 gene takes place to be transformed into the I 462 V sudden change that G causes by base A in the sample 2, and the GSTP1 gene takes place to change the I 105 V missense mutation that cause to A by bases G; The base A to C of ERCC2 gene changes and causes K 751 Q missense mutation in the sample 6; The base C to T in COX2 gene 3 ' regulation and control zone suddenlys change in the sample 8; The base C to G of TP53 gene changes and causes P 72 R missense mutation in the sample 12, and the base A to G of XRCC1 gene change causing Q399 R missense mutation; The base C to G of TP53 gene changes and causes P 72 R missense mutation in the sample 16; The base C to G of TP53 gene changes and causes P 72 R missense mutation in the sample 24, and the base A to G of XRCC1 gene change causing Q 399 R missense mutation; The base C to T in COX2 gene 3 ' regulation and control zone suddenlys change in the sample 27; All the other are all the wild type gene sequence.

Six, pcr amplification and detected through gel electrophoresis GSTM1 and GSTT1 gene

1. utilize the amplimer of GSTM1, GSTT1 and the internal control gene of design to carry out the amplification of GSTM1, GSTT1 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; If have the 480bp band in the running gel, then contain the GSTT1 gene, otherwise then lack.

Detected result shows: GSTM1, GSTT1 genetically deficient in the sample 2; GSTM1 genetically deficient in the sample 8; GSTM1, GSTT1 genetically deficient in the sample 12; GSTT1 genetically deficient in the sample 16; GSTM1 genetically deficient in the sample 24; All the other do not see GSTM1, GSTT1 genetically deficient.

Should be noted that present embodiment is one of them specific embodiments of the present invention,, use corresponding special primers and substitute, can be applied to detection method of the present invention equally not in order to restriction protection scope of the present invention.

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

Pass through conventional design; Make up the wild-type sequence (SEQID NO:79) and the plasmid of mutant nucleotide sequence (SEQ ID NO:80), the wild-type sequence (SEQ ID NO:81) in GSTP1 gene amplification zone and the plasmid of mutant sequence (SEQ ID NO:82) that contain CYP1A1 gene amplification zone respectively, and the wild-type sequence (SEQ ID NO:83) in TP53 gene amplification zone and the plasmid of mutant sequence (SEQ ID NO:84).

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 a last embodiment 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 of lung cancer tumor susceptibility gene of the present invention is limited to about 3%, 5% and more than detected result and practical situation basically identical.

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

Said lung cancer kit for detecting susceptibility genes; Can detect simultaneously a plurality of zones of lung cancer tumor susceptibility gene, accurately draw these regional sequence informations, comprise the variation situation in known mutations and unknown mutation site; Detection sensitivity is high, and can further detect a large amount of samples simultaneously.Detect the lung cancer tumor susceptibility gene sequence information that obtains through this test kit; Can combine the statistical study of follow-up further molecular biology test, clinical trial, clinical observation and integrated data; Set up certain disease model, realize the susceptibility of early stage assessment lung cancer.

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

Further, the said first sequence label base number is 3～20, more preferably between 4～10.Wherein, Said lung cancer tumor susceptibility gene to be measured has a plurality of; In the Auele Specific Primer corresponding, have at least a primer and target area part complementary, and 5 ' end of this primer have second sequence label with each target area; Be used for process, the target area amplified production of different testing samples is done corresponding mark in the amplification target area.Said second sequence label is preferably the nucleic acid molecule that has specific base sequence, and its base number is not limit, and the base number of said second sequence label is preferably 3～20, and more preferably 4～10.

Wherein, said lung cancer tumor susceptibility gene comprises at least a among APE1, CASP7, CASP8, CASP9, CHEK2, COX-2, CYP1A1, CYP2E1, ERCC1, ERCC2, ERCC6, Exo1, GSTP1, Hmlh1, IL-1 β, MDM2, MPO, MTHFR, NQO1, OGG1, P73, RASSF1, TERT, TGFB1, TP53, TP63, XPC and XRCC1 and GSTM1, the GSTT1.

Further, the Auele Specific Primer of said APE1 is SEQ ID NO:1 and SEQ ID NO:2; The Auele Specific Primer of said CASP7 is SEQ ID NO:3 and SEQ ID NO:4; The Auele Specific Primer of said CASP8 is SEQ ID NO:5 and SEQ ID NO:6; The Auele Specific Primer of said CASP9 is SEQ ID NO:7 and SEQ ID NO:8; The Auele Specific Primer of said CHEK2 is SEQ ID NO:9 and SEQ ID NO:10; The Auele Specific Primer of said COX-2 is SEQ ID NO:11 and SEQ ID NO:12; The Auele Specific Primer of said CYP1A1 comprises at least one pair of among SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and the SEQ IDNO:16; The Auele Specific Primer of said CYP2E1 is SEQ ID NO:17 and SEQ IDNO:18; The Auele Specific Primer of said ERCC1 is SEQ ID NO:19 and SEQ ID NO:20; The Auele Specific Primer of said ERCC2 comprises 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 ERCC6 is SEQ ID NO:25 and SEQ ID NO:26; The Auele Specific Primer of said Exo1 is SEQ ID NO:27 and SEQ ID NO:28; The Auele Specific Primer of said GSTP1 is SEQ ID NO:29 and SEQ ID NO:30; The Auele Specific Primer of said Hmlh1 is SEQ ID NO:31 and SEQ ID NO:32; The Auele Specific Primer of said IL-1 β is SEQ IDNO:33 and SEQ ID NO:34; The Auele Specific Primer of said MDM2 is SEQ ID NO:35 and SEQ IDNO:36; The Auele Specific Primer of said MPO is SEQ ID NO:37 and SEQ ID NO:38; The Auele Specific Primer of said MTHFR is SEQ ID NO:39 and SEQ ID NO:40; The Auele Specific Primer of said NQO1 is SEQ ID NO:41 and SEQ ID NO:42; The Auele Specific Primer of said OGG1 is SEQ IDNO:43 and SEQ ID NO:44; The Auele Specific Primer of said P73 comprises at least one pair of among SEQ ID NO:45 and SEQ IDNO:46, SEQ ID NO:47 and the SEQ ID NO:48; The Auele Specific Primer of said RASSF1 is SEQ ID NO:49 and SEQ ID NO:50; The Auele Specific Primer of said TERT is SEQ IDNO:51 and SEQ ID NO:52; The Auele Specific Primer of said TGFB1 is SEQ ID NO:53 and SEQ IDNO:54; The Auele Specific Primer of said TP53 is SEQ ID NO:55 and SEQ ID NO:56; The Auele Specific Primer of said TP63 is SEQ ID NO:57 and SEQ ID NO:58; The Auele Specific Primer of said XPC is SEQ ID NO:59 and SEQ ID NO:60; The Auele Specific Primer of said XRCC 1 comprises at least one pair of among SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ IDNO:66, SEQ ID NO:67 and the SEQ ID NO:68; The Auele Specific Primer of said GSTM1 comprises at least one pair of among SEQ ID NO:69 and SEQ ID NO:70, SEQ ID NO:71 and the SEQ ID NO:72; The Auele Specific Primer of said GSTT1 is SEQ ID NO:73 and SEQ ID NO:74.

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 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 lung cancer tumor susceptibility gene, in other similar gene tests, also can use the method that the present invention sets forth.The method of detection of lung cancer tumor 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 sequence 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 disease model, can realize assessing in early days the susceptibility of lung 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. the method for a detection of lung cancer tumor susceptibility gene is characterized in that, said method comprising the steps of:

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

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

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

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

6. according to the method for each described detection of lung cancer tumor 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 corresponding mark is done in the order-checking library of different testing samples.

7. according to the method for each described detection of lung cancer tumor susceptibility gene of claim 1 to 5; It is characterized in that; In the steps A Auele Specific Primer corresponding, have at least a primer and target area part complementary, and 5 ' end of this primer have second sequence label with each target area; Be used for process, the target area amplified production of different testing samples is done corresponding mark in the amplification target area.

8. according to the method for each described detection of lung cancer tumor susceptibility gene of claim 1 to 5; It is characterized in that said lung cancer tumor susceptibility gene comprises at least one among APE1, CASP7, CASP8, CASP9, CHEK2, COX-2, CYP1A1, CYP2E1, ERCC1, ERCC2, ERCC6, Exo1, GSTP1, Hmlh1, IL-1 β, MDM2, MPO, MTHFR, NQO1, OGG1, P73, RASSF1, TERT, TGFB1, TP53, TP63, XPC and the XRCC1.

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

10. a lung cancer kit for detecting susceptibility genes is characterized in that, said test kit comprises:

11. lung cancer kit for detecting susceptibility genes 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. lung cancer kit for detecting susceptibility genes 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 corresponding mark is done in the order-checking library of different testing samples.

13. lung cancer kit for detecting susceptibility genes according to claim 10; It is characterized in that said lung cancer tumor susceptibility gene to be measured has in a plurality of, corresponding with each target area Auele Specific Primers; Have at least a primer and target area part complementary; And 5 ' end of this primer has second sequence label, is used for the process in the amplification target area, and the target area amplified production of different testing samples is done corresponding mark.

14. according to each described lung cancer kit for detecting susceptibility genes in the claim 10 to 13; It is characterized in that said lung cancer tumor susceptibility gene comprises at least one among APE1, CASP7, CASP8, CASP9, CHEK2, COX-2, CYP1A1, CYP2E1, ERCC1, ERCC2, ERCC6, Exo1, GSTP1, Hmlh1, IL-1 β, MDM2, MPO, MTHFR, NQO1, OGG1, P73, RASSF1, TERT, TGFB1, TP53, TP63, XPC and the XRCC1.

15. lung cancer kit for detecting susceptibility genes according to claim 14 is characterized in that, said lung cancer tumor susceptibility gene also comprises at least one among GSTM1, the GSTT1.