CN102827830B

CN102827830B - Method for capturing nucleic acid fragment

Info

Publication number: CN102827830B
Application number: CN201210286589.7A
Authority: CN
Inventors: 盛司潼
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-08-14
Filing date: 2012-08-14
Publication date: 2015-04-15
Anticipated expiration: 2032-08-14
Also published as: CN102827830A

Abstract

The invention relates to the fields of gene engineering and molecular biology and provides a method for capturing a nucleic acid fragment. The method comprises the following steps: A, preparing a capture probe by using biotin labeled amplimers or joint elements with nucleic acid molecules containing a to-be-captured nucleic acid fragment as a raw material; and B, hybridizing the capture probe with a to-be-captured nucleic acid fragment library and capturing the to-be-captured nucleic acid fragment by using a solid phase carrier containing a streptavidin or avidin mark. The method for capturing the nucleic acid fragment in the invention can avoid occurrence of poor capture effects caused by poor homogeneity of the capture probe and enables cost for capturing of nucleic acid fragments to be reduced.

Description

A kind of method of capture nucleic acid fragment

Technical field

The present invention relates to genetically engineered and biology field, more particularly, relate to a kind of method of capture nucleic acid fragment.

Background technology

Along with appearance and the development of s-generation high throughput sequencing technologies, at present, scientists can carry out conventional de novo sequencing to the genome of a lot of living species, and checks order to the integral part in the genome of these species again.But, in the successful use procedure of the s-generation high throughput sequencing technologies of widespread use, there is a Main Bottleneck: namely how optionally to catch out by the nucleic acid fragment of the specific region become scattered about in genome, plastosome and/or other forms of DNA fast and effectively.Selectivity is caught and enrichment has a wide range of applications from the specific region of the genome of living species, plastosome and/or other forms of DNA.

Mainly caught the nucleic acid fragment of specific region in prior art by microarray technology, method is as follows: 1) on micro-array chip, directly generate the DNA fragmentation with object fragment complementation by chemosynthesis; 2) DNA fragmentation of the primer pair step 1) gained of band T7 promoter is utilized to carry out pcr amplification, then reverse transcription is carried out to PCR primer, transcribe in system and add with biotin labeled dUTP, generate with biotin labeled RNA fragment (capture probe); 3) mixed with biotin labeled RNA fragment by the genomic dna after fragmentation and hatch, target DNA fragment is combined with RNA, and extraction purification obtains target DNA fragment.In the method, biotin labeling with biotin labeled RNA fragment is introduced by the biotin labeled dUTP of band in process of reverse-transcription, biotin labeled quantity and position are random, the homogeneity of capture probe is bad, the biotin labeling on the part probe in the capture probe obtained may be caused too much and/or position is improper, affect the hybridization of itself and genomic dna, and then cause the capture effect of target DNA fragment not good; In addition, this programme needs the DNA fragmentation of chemosynthesis and object fragment complementation on chip in the process preparing capture probe, and production cost is high.

Therefore, need a kind of method of new capture nucleic acid fragment, can avoid because the generation of the homogeneity difference of capture probe and the not good phenomenon of the capture effect that causes, and reduce the cost of capture nucleic acid fragment.

Summary of the invention

The object of the present invention is to provide a kind of method of new capture nucleic acid fragment, be intended to solve the problem causing capture effect not good because of the homogeneity difference of capture probe in prior art.

In order to realize goal of the invention, the invention provides a kind of method of capture nucleic acid fragment, comprising the following steps:

A. to contain the nucleic acid molecule of nucleic acid fragment to be captured for raw material, biotin labeled amplimer or joint component is utilized to prepare biotin labeled capture probe;

B. make biotin labeled capture probe and nucleic acid fragment storehouse to be captured hybridize, utilize the solid phase carrier containing Streptavidin or avidin mark to catch described nucleic acid fragment to be captured.

Wherein, described step B can comprise the following steps:

B01. the biotin labeled capture probe in steps A is caught in utilization containing the solid phase carrier that Streptavidin or avidin mark, and must be fixed on the capture probe on solid phase carrier;

B02. make to be fixed on capture probe on solid phase carrier and nucleic acid fragment storehouse to be captured hybridize first time hybrid product, separation and purification first time hybrid product must containing the first product of solid phase carrier;

B03. make the product sex change that step B02 obtains, obtain and catch product for the first time.

Further, also can comprise the following steps after described step B03:

B04. utilize the first time being fixed on capture probe on solid phase carrier and step B03 gained obtained in step B01 to catch products thereof and obtain second time hybrid product, separation and purification second time hybrid product must containing the second product of solid phase carrier;

B05. make the product sex change that step B04 obtains, obtain second time and catch product.

In above-mentioned either a program, the hybridization in described step B is carried out in periodic vibration.

In above-mentioned either a program, the described nucleic acid molecule containing nucleic acid fragment to be captured is cloning vector storehouse.

Wherein, described steps A can have multiple implementation.

In one embodiment, described steps A can comprise the following steps:

A01. use Restriction Enzyme to be scaled off by the nucleic acid fragment enzyme to be captured in cloning vector storehouse, thus obtain DNA profiling;

A02. fragmentation DNA profiling, obtains DNA profiling fragmentation products;

A03.DNA template segments product is connected with biotin labeled joint component, obtains biotin labeled capture probe.

In another embodiment, described steps A can comprise the following steps:

A11. use Restriction Enzyme to be scaled off by the nucleic acid fragment enzyme to be captured in cloning vector storehouse, thus obtain DNA profiling;

A12. fragmentation DNA profiling, obtains DNA profiling fragmentation products;

A13.DNA template segments product is connected with joint component, must containing the DNA profiling fragment of joint;

A14. utilize biotin labeled amplimer to carry out pcr amplification to the product of steps A 13, obtain biotin labeled capture probe.

In another embodiment, described steps A comprises the following steps:

A21. fragmentation is containing the nucleic acid molecule of nucleic acid fragment to be captured, obtains fragmentation products;

A22. fragmentation products is connected with joint component, must containing the nucleic acid fragment of joint;

A23. utilize biotin labeled amplimer to carry out pcr amplification to the product of steps A 22, obtain biotin labeled capture probe.

In another embodiment, described steps A comprises the following steps:

A31. fragmentation is containing the nucleic acid molecule of nucleic acid fragment to be captured, obtains fragmentation products;

A32. fragmentation products is connected with biotin labeled joint component, obtains biotin labeled capture probe.

In such scheme, the joint component in described steps A 13 or A22 can be y splice.

In above-mentioned either a program, before described steps A, also can comprise step:

A00. be template with genomic dna, utilize the primer pair being used for specific amplification nucleic acid fragment to be captured to carry out pcr amplification, must containing the nucleic acid molecule of nucleic acid fragment to be captured.

Wherein, the described primer pair for specific amplification nucleic acid fragment to be captured 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, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

As from the foregoing, method of the present invention prepares biotin labeled capture probe by biotin labeled amplimer or joint component, the biotin labeled quantity of the capture probe of gained is all identical with position, homogeneity is good, then from nucleic acid fragment storehouse to be captured, treat capture nucleic acid fragment based on above-mentioned capture probe to catch, can avoid because the generation of the homogeneity difference of capture probe and the not good phenomenon of the capture effect that causes.In addition, method of the present invention because reduce the preparation cost of capture probe, and then reduces the cost of capture nucleic acid fragment.

Accompanying drawing explanation

Fig. 1 be in one embodiment of the invention capture probe catch the schematic flow sheet of nucleic acid fragment to be captured.

Fig. 2 be in another embodiment of the present invention capture probe catch the schematic flow sheet of nucleic acid fragment to be captured.

Fig. 3 is in the present invention one specific embodiment under different hybridization time condition, the accuracy rate figure that first time and second time are caught.

Embodiment

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

A method for capture nucleic acid fragment, comprises the following steps:

Method of the present invention prepares biotin labeled capture probe by biotin labeled amplimer or joint component, the biotin labeled quantity of the capture probe of gained is all identical with position, homogeneity is good, then from nucleic acid fragment storehouse to be captured, treat capture nucleic acid fragment based on above-mentioned biotin labeled capture probe to catch, can avoid because the generation of the homogeneity difference of capture probe and the not good phenomenon of the capture effect that causes.In addition, method of the present invention because reduce the preparation cost of capture probe, and then reduces the cost of capture nucleic acid fragment.

It should be noted that, described nucleic acid fragment to be captured can be one or more snippets sequence on genomic dna, Mitochondrial DNA, plasmid or RNA; The described nucleic acid molecule containing nucleic acid fragment to be captured can be cloning vector, cloning vector storehouse or pcr amplification product; One or more snippets sequence above-mentioned can be structured on cloning vector or cloning vector storehouse in advance; one or more snippets sequence above-mentioned also can be comprised in pcr amplification product, and this pcr amplification product utilizes corresponding PCR primer to obtain from genomic dna, Mitochondrial DNA, plasmid or RNA amplification.

Biotin labeled position on described biotin labeled amplimer or joint component and quantity are all determined.Described biotin labeled amplimer or joint component all obtain by the mode of synthetic.

Described biotin labeled joint component is nucleic acid molecule, for connecting, various structures can be adopted, include but not limited to flat end fitting, protruding terminus joint, y splice and the joint containing loop-stem structure, the joint of above-mentioned various structure is concrete open in Chinese patent CN201110222952.4, and here form by reference is all introduced.

Preferably, described biotin labeled joint component is biotin labeled y splice, and described biotin labeled y splice is for containing biotin labeled double chain acid molecule, and described biotin labeled y splice comprises crotch region and complementary district.

Wherein, mononucleotide protruding terminus can be contained in the complementary district of described biotin labeled y splice, and described mononucleotide is T, and described mononucleotide protruding terminus is positioned at 3 ' end of its place nucleic acid chains.This programme both can avoid joint from the appearance connecting phenomenon, can also be connected with the nucleic acid chains specificity containing A protruding terminus, improved joint efficiency.

Wherein, dideoxy nucleotide can be contained in the complementary district of described biotin labeled y splice, and described dideoxy nucleotide is positioned at 3 ' end of its place nucleic acid chains, and now 5 ' end of the complementary strand of dideoxy nucleotide place nucleic acid chains is containing phosphate group.This programme can avoid joint from the appearance connecting phenomenon.

Wherein, the complementary district of described biotin labeled y splice can contain without phosphoric acid Nucleotide, the described 5 ' end being positioned at its place nucleic acid chains without phosphoric acid Nucleotide.This programme can avoid joint from the appearance connecting phenomenon equally.

Wherein, the biotin labeling of described biotin labeled y splice is positioned at crotch region; Further, described biotin labeling is positioned at the end of y splice, preferred, and described biotin labeling is positioned at 5 ' end of its place nucleic acid chains.

Described amplimer, for amplification to prepare capture probe.3 ' end of described amplimer can with nucleic acid fragments complementary to be captured, 3 ' end of described amplimer also can be complementary with joint component.

Described nucleic acid fragment storehouse to be captured is the nucleic acid molecule set containing nucleic acid fragment to be captured, can be genomic dna, Mitochondrial DNA, plasmid or RNA, also can be the fragmentation products of genomic dna, Mitochondrial DNA, plasmid or RNA, can also be the connection product after the fragmentation products of genomic dna, Mitochondrial DNA, plasmid or RNA is connected with joint component; Joint component described herein is similarly nucleic acid molecule, for connecting the fragmentation products of genomic dna, Mitochondrial DNA, plasmid or RNA, various structures can be adopted, include but not limited to flat end fitting, protruding terminus joint, y splice and the joint containing loop-stem structure, the joint of above-mentioned various structure is concrete open in Chinese patent CN201110222952.4, and here form by reference is all introduced; But the sequence of joint component herein can not or complementary pairing identical with the sequence building the joint component used in capture probe process, like this after product is caught in acquisition, when utilization holds nucleic acid molecule that is identical or complementary pairing to carry out pcr amplification with 3 ' of joint component herein, can dilute the capture probe being mixed in and catching in product, the capture probe that minimizing is mixed into is to the interference of subsequent experimental.

The surface of described solid phase carrier is for being suitable for the material of Streptavidin or avidin mark, and this material includes but not limited to: slide, silicon chip, pottery, metal, metal oxide, plastics, rubber, nylon; Described solid phase carrier can be different shape, includes but not limited to: spherical solid phase carrier, hemispherical solid phase carrier, ellipsoid shape solid phase carrier, column solid phase carrier or erose solid phase carrier.

This programme is based on the existing nucleic acid molecule containing nucleic acid fragment to be captured, described nucleic acid fragment to be captured is caught from nucleic acid fragment storehouse to be captured, its essence is exactly enrich target fragment from nucleic acid fragment storehouse to be captured, and this target fragment is and the nucleic acid fragment containing the nucleic acid fragments complementary to be captured in the nucleic acid molecule of nucleic acid fragment to be captured.The sequence information of described nucleic acid fragment to be captured can be known, also can be unknown.

Wherein, described steps A has multiple implementation, below will be set forth further by multiple embodiment.

The present invention proposes the first embodiment, and the described nucleic acid molecule containing nucleic acid fragment to be captured is cloning vector storehouse, and described nucleic acid fragment to be captured has multiple and is constructed separately in multiple cloning vector, and described steps A comprises the following steps:

A02. fragmentation DNA profiling, obtains DNA profiling fragmentation products;

The mode that this programme is cut by enzyme, scales off nucleic acid fragment to be captured enzyme from cloning vector, and then is connected with biotin labeled joint component, obtain biotin labeled capture probe.Cloning vector in cloning vector storehouse in this programme can be converted in intestinal bacteria or yeast and carry out cultivation amplification respectively, catching of step B is carried out to prepare enough capture probes, and when carrier copies in intestinal bacteria or yeast, intestinal bacteria or saccharomycetic mechanism for correcting errors can be utilized, ensure that the high-fidelity of the nucleic acid fragment to be captured on cloning vector copies.This programme and realized treating capture nucleic acid fragment by PCR step amplification compared with, occur in nucleic acid fragment to be captured that the probability suddenlyd change is lower, thus ensure that the acquisition of high-quality biotin labeled capture probe, and then ensure that in follow-up step B the accuracy rate of the nucleic acid fragment to be captured of catching gained.

It should be noted that, described DNA profiling is the nucleic acid fragment to be captured that enzyme scales off from cloning vector storehouse.Restriction Enzyme restriction enzyme site is contained at nucleic acid fragment two ends to be captured in described cloning vector.The connection of the joint component of described DNA profiling fragmentation products and biomarker can only occur in the one or both ends of described DNA profiling fragmentation products.

The present invention also proposes the second embodiment, and the described nucleic acid molecule containing nucleic acid fragment to be captured is cloning vector storehouse, and described nucleic acid fragment to be captured has multiple and is constructed separately in multiple cloning vector, and described steps A comprises the following steps:

A12. fragmentation DNA profiling, obtains DNA profiling fragmentation products;

This programme is compared with the first embodiment, and the amount in required cloning vector storehouse is less, and realizes the amplification to the DNA profiling fragment containing joint by PCR step, thus obtains enough capture probes and carry out catching of step B.Because the speed that the speed of pcr amplification is cultivated compared with prokaryotic organism is faster, therefore, the efficiency that this programme prepares capture probe is higher.

It should be noted that, described DNA profiling is the nucleic acid fragment to be captured that enzyme scales off from cloning vector storehouse.Restriction Enzyme restriction enzyme site is contained at nucleic acid fragment two ends to be captured in described cloning vector.3 ' end of described biotin labeled amplimer is complementary with joint component.The connection of described DNA profiling fragmentation products and joint component occurs in the two ends of described DNA profiling fragmentation products.Described joint component is nucleic acid molecule, for connecting, various structures can be adopted, include but not limited to flat end fitting, protruding terminus joint, y splice and the joint containing loop-stem structure, the joint of above-mentioned various structure is concrete open in Chinese patent CN201110222952.4, and here form by reference is all introduced.

Preferably, described joint component is y splice, and described y splice is double chain acid molecule, and described y splice comprises crotch region and complementary district.

Wherein, mononucleotide protruding terminus can be contained in the complementary district of described y splice, and described mononucleotide is T, and described mononucleotide protruding terminus is positioned at 3 ' end of its place nucleic acid chains.This programme both can avoid joint from the appearance connecting phenomenon, can also be connected with the nucleic acid chains specificity containing A protruding terminus, improved joint efficiency.

Wherein, dideoxy nucleotide can be contained in the complementary district of described y splice, and described dideoxy nucleotide is positioned at 3 ' end of its place nucleic acid chains, and now 5 ' end of the complementary strand of dideoxy nucleotide place nucleic acid chains is containing phosphate group.This programme can avoid joint from the appearance connecting phenomenon.

Wherein, the complementary district of described y splice can contain without phosphoric acid Nucleotide, the described 5 ' end being positioned at its place nucleic acid chains without phosphoric acid Nucleotide.This programme can avoid joint from the appearance connecting phenomenon equally.

The present invention also proposes the 3rd embodiment, and described steps A comprises the following steps:

It should be noted that, the described nucleic acid molecule containing nucleic acid fragment to be captured is pcr amplification product, and described pcr amplification product utilizes the target area on corresponding PCR primer pair genomic dna, Mitochondrial DNA, plasmid or RNA to carry out acquisition of increasing.Described target area is made up of nucleic acid fragment to be captured.Described target area can have multiple.Described joint component is nucleic acid molecule, for connecting, various structures can be adopted, include but not limited to flat end fitting, protruding terminus joint, y splice and the joint containing loop-stem structure, the joint of above-mentioned various structure is concrete open in Chinese patent CN201110222952.4, and here form by reference is all introduced.The connection of described fragmentation products and joint component occurs in the two ends of described fragmentation products.3 ' end of described biotin labeled amplimer is complementary with joint component.

The present invention also proposes the 4th embodiment, and described steps A comprises the following steps:

It should be noted that, the described nucleic acid molecule containing nucleic acid fragment to be captured is pcr amplification product, and described pcr amplification product utilizes the target area on corresponding PCR primer pair genomic dna, Mitochondrial DNA, plasmid or RNA to carry out acquisition of increasing.Described target area is made up of nucleic acid fragment to be captured.Described target area can have multiple.Described fragmentation products is connected the one or both ends that can only occur in described DNA profiling fragmentation products with biotin labeled joint component.

3rd embodiment and the 4th embodiment respectively have advantage.In the step A of the 3rd embodiment, the consumption containing the nucleic acid molecule of nucleic acid fragment to be captured can be less, and lacked the step of a pcr amplification in the step A of the 4th embodiment, and efficiency is higher.

Preferably, also step can be comprised before described steps A:

It should be noted that, in this programme, described nucleic acid fragment to be captured has one at least, is arranged in genomic dna; The described primer pair for specific amplification nucleic acid fragment to be captured is corresponding with nucleic acid fragment to be captured, and each nucleic acid fragment to be captured all has the primer pair for specific amplification nucleic acid fragment to be captured described in a pair.This programme is particularly useful for the third and fourth embodiment.Preferred, described nucleic acid fragment to be captured is not more than 20kb, in case because template is long, and causes pcr amplification unsuccessful; Further, described nucleic acid fragment size to be captured is between 1kb to 4kb, and to ensure the efficient amplification of nucleic acid fragment to be captured, and the amplification condition of different nucleic acid fragment to be captured can be unified, and simplifies experimental design.

In a specific embodiment of the present invention, described nucleic acid fragment to be captured is the gene fragment relevant to mammary cancer, the described primer pair for specific amplification nucleic acid fragment to be captured comprises: 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, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, at least one pair of in SEQ ID NO:19 and SEQ ID NO:20.Above-mentioned each primer pair the gene fragment relevant to mammary cancer can carry out specific amplification, and amplification efficiency is also basically identical, and their amplified production is all between 1kb to 1.4kb.

Above-mentioned first embodiment and the nucleic acid molecule containing nucleic acid fragment to be captured described in the second embodiment are cloning vector storehouse, because in cloning vector storehouse except nucleic acid fragment to be captured, also containing other structural nucleic acid sequence, if cut step without Restriction Enzyme enzyme nucleic acid fragment to be captured is cut out, directly carry out the words of fragmentation step, can cause in the capture probe prepared containing the probe for catching other the structural nucleic acid sequence in cloning vector storehouse, cause non-targeted to be caught, it is not high to catch accuracy rate.In addition, described cloning vector storehouse refers to the storehouse that the cloning vector using nucleic acid fragment to be captured as Insert Fragment forms, described cloning vector includes but not limited to bacterial artificial chromosome (Bacterial Artificial Chromosome, BAC), yeast artificial chromosome (Yeast Artificial Chromosome, YAC), P1 derives from artificial chromosome (P1 Derived Artificial Chromosome, and artificial mammalian chromosome (Mammal Artificial Chromosome, MAC) PAC).The size of the Insert Fragment in above-mentioned cloning vector, can between 50bp to 200kb without particular requirement.Preferably, described Insert Fragment, namely nucleic acid fragment size to be captured is between 1kb to 30kb.Because cloning vector can copy in such as intestinal bacteria or yeast, not by the impact of cloning vector molecular size, and fidelity of reproduction is high, and the amplification that PCR reacts for long segment (being such as greater than the nucleic acid molecule of 5kb or 10kb or 20kb) has difficulties.So, when needs are to comparatively large regions (such as 100kb, even 4000kb) nucleic acid fragment when catching, this nucleic acid fragment can be split into multiple nucleic acid fragment to be captured and build in cloning vector respectively, described multiple nucleic acid fragment to be captured can form this complete nucleic acid fragment compared with large regions after superimposing.Described multiple nucleic acid fragment to be captured can be more than or equal to 1kb or 5kb or 10kb or 20kb.

In 3rd embodiment and the 4th embodiment, because the described nucleic acid molecule containing nucleic acid fragment to be captured is pcr amplification product, and described pcr amplification product utilizes the target area on corresponding PCR primer pair genomic dna, Mitochondrial DNA, plasmid or RNA to carry out increasing acquisition; So the 3rd embodiment and the nucleic acid molecule containing nucleic acid fragment to be captured described in the 4th embodiment directly can carry out fragmentation step, and without the need to increasing the step removed by non-nucleic acid fragment to be captured before fragmentation.

In above-mentioned four embodiments, all containing fragmentation step, it is more consistent and be suitable for the capture probe of catching that object is nucleic acid fragment to be captured to make size.

Preferably, after the fragmentation step in above-mentioned four embodiments, also can comprise and fragment products is carried out gel electrophoresis, and then cut the step of fragmentation products of the specific size of glue purification.Further, the size of the fragmentation products of the specific size of purifying is between 100bp to 600bp; Further, the size of the fragmentation products of the specific size of purifying is between 200bp to 500bp; Further, the size of the fragmentation products of the specific size of purifying is between 300bp to 400bp.

In addition, end-filling, dephosphorylation or phosphorylation can be comprised toward contact, add the process such as A tail after the fragmentation step in above-mentioned four embodiments, and be connected with joint component more after being like this.Described end-filling, dephosphorylation, phosphorylation and add the process of A tail and be prior art, do not repeat them here.

It should be noted that the implementation of described steps A includes but not limited to above-mentioned four embodiments.Such as, when nucleic acid fragment to be captured is not more than 20kb, when being particularly less than or equal to 5kb, also replace the step that restriction enzyme cuts, by separating containing the nucleic acid fragment to be captured in the nucleic acid molecule of nucleic acid fragment to be captured described in first embodiment and the second embodiment by the mode of pcr amplification.In addition, when nucleic acid fragment to be captured is less than or equal to 500bp, and when the sequence at its two ends is end to end known, can, without the need to fragmentation step, biotin labeled primer or joint component be directly utilized to carry out the preparation of capture probe with reference to above-mentioned four embodiments.

Wherein, described step B has multiple implementation, below will be set forth further by multiple embodiment.

The present invention proposes the 5th embodiment, and described step B comprises the following steps:

Because in the capture probe of steps A gained of the present invention, the probe of the positive-sense strand complementation of nucleic acid fragment existing and to be captured, also the probe with the antisense strand complementation of nucleic acid fragment to be captured is had, so, in the crossover process of step B, the phenomenon of complementary pairing between part probe in the capture probe of steps A gained, may be there is.As shown in Figure 1, first, biotin labeled capture probe and nucleic acid fragment storehouse to be captured are hybridized, recycling catches biotin labeled capture probe containing the solid phase carrier of Streptavidin or avidin mark, then obtain through denaturing step and catch product, may complementary pairing be there is between biotin labeled capture probe in this scheme in crossover process, and containing Streptavidin or avidin mark solid phase carrier when catching biotin labeled capture probe, might not be combined with all biotin labelings, so catching in product of this scheme gained may be mixed with capture probe, and the subsequent applications of the capture probe meeting noise capture product that these are mixed with, include but not limited to sequential analysis etc.

As shown in Figure 2, the basic procedure of the 5th embodiment is: be first fixed on solid phase carrier by capture probe, then utilize the solid phase carrier being fixed with capture probe to catch nucleic acid fragment to be captured in nucleic acid fragment storehouse to be captured, and then obtained by denaturing step and catch product.In the crossover process of the 5th embodiment, same contingent complementary pairing between biotin labeled capture probe, but can avoid catching in product is mixed with capture probe, it is higher to catch accuracy rate, and the ratio of namely catching in product shared by nucleic acid fragment to be captured is higher.

It should be noted that, described first time hybrid product be fixed on capture probe on solid phase carrier and nucleic acid fragment storehouse to be captured complete hybridization after whole reaction system; Described the first product containing solid phase carrier refers to the product containing solid phase carrier be purified into from first time hybrid product; Described first time catches product and refers to through denaturing step, from the nucleic acid molecule be not fixed on solid phase carrier that the first product containing solid phase carrier discharges.

Catch accuracy rate to improve further, the present invention proposes the 6th embodiment, and described step B comprises the following steps:

B03. make the product sex change that step B02 obtains, obtain and catch product for the first time;

This programme is in fact utilize the capture probe on solid phase carrier of being fixed on obtained in step B01 in the 5th embodiment to catch product to the first time in the 5th embodiment and carry out second time and catch.This programme can improve further catches accuracy rate.

It should be noted that, described second time hybrid product be obtain in step B01 be fixed on capture probe on solid phase carrier and first time catch product complete hybridization after whole reaction system; Described the second product containing solid phase carrier refers to the product containing solid phase carrier be purified into from second time hybrid product; Described second time is caught product and is referred to through denaturing step, from the nucleic acid molecule be not fixed on solid phase carrier that the second product containing solid phase carrier discharges.

The amount of catching product in order to the first time ensureing in described step B04 is enough carried out second time and is caught, and can obtain after first time catches product in step B03, before the hybridization in step B04, catch product carry out pcr amplification to first time.

It should be noted that the implementation of described step B includes but not limited to above-mentioned two embodiments, such as, scheme shown in Fig. 1 can realize object of the present invention equally.

In above-mentioned any embodiment, the hybridization in described step B can be carried out in periodic vibration.This programme can allow capture probe fully contact with the storehouse molecule in nucleic acid fragment storehouse to be captured, improves the capture rate that capture probe treats the nucleic acid molecule containing nucleic acid fragment to be captured in capture nucleic acid fragment library.This programme is particularly useful for the 5th and the 6th embodiment.

In the 5th and the 6th embodiment, capture probe is fixed on solid phase carrier to carry out hybridization with the nucleic acid molecule containing nucleic acid fragment to be captured; When the density of the liquid in the density and hybridization system of described solid phase carrier is inconsistent, sedimentation or floating can be there is in solid phase carrier, and then the capture probe causing being fixed on solid phase carrier and containing nucleic acid fragment to be captured nucleic acid molecule between contact insufficient, too high in the density being fixed on the capture probe on solid phase carrier at the bottom of hybridization system or top, the probability making capture probe that complementary pairing occur each other increases, and reduces capture rate.Therefore, in the 5th and the 6th embodiment, the hybridization in described step B is made to be carry out in periodic vibration, the capture rate that capture probe is treated capture nucleic acid fragment library or caught the nucleic acid molecule containing nucleic acid fragment to be captured in product for the first time can be improved, reduce the probability that complementary pairing occurs between capture probe.

It should be noted that, the implementation of described periodic vibration has multiple, includes but not limited to: on gyroscope, carry out hybridization, or by certain time interval, piping and druming or concussion hybridization system.

Below will be described in further detail the present invention by the first specific embodiment, this specific embodiment utilizes capture probe from the genomic dna of breast cancer patients blood sample, catch the relevant gene fragment of mammary cancer.

One, the preparation of capture probe.

1. with normal people's genomic dna for template, the gene fragment that amplification mammary cancer is relevant.

BRCA1-F1(SEQ ID NO:1) and BRCA1-R1SEQ ID NO:2 the gene fragment amplification primer pair that mammary cancer is relevant is as follows:, BRCA1-F2(SEQ ID NO:3) and BRCA1-R2(SEQ ID NO:4), BRCA1-F3(SEQ ID NO:5) and BRCA1-R3(SEQ ID NO:6), BRCA1-F4(SEQ ID NO:7) and BRCA1-R4(SEQ ID NO:8), BRCA1-F5(SEQ ID NO:9) and BRCA1-R5(SEQ ID NO:10), BRCA1-F6(SEQ ID NO:11) and BRCA1-R6(SEQ ID NO:12), BRCA2-F1(SEQ ID NO:13) and BRCA2-R1(SEQ ID NO:14), BRCA2-F2(SEQ ID NO:15) and BRCA2-R2(SEQ ID NO:16), BRCA2-F3(SEQ ID NO:17) and BRCA2-R3(SEQ ID NO:18), BRCA2-F4(SEQ ID NO:19) and BRCA2-R4(SEQ ID NO:20).

With above-mentioned 10 pairs of amplimers for PCR primer, be configured by following PCR reaction system respectively: 5 × LongAmp Taq Reaction Buffer, 10 μ L; The each 2.5mM of dNTP(), 1.5 μ L; Upstream primer (10 μMs), 2 μ L; Downstream primer (10 μMs), 2 μ L; Genomic dna, 300ng, LongAmp Taq DNA polymerase(NEB, E5200S, 2.5U/ μ L), 2 μ L; ddH ₂o, adds to 50 μ L.

PCR reaction conditions is as follows:

95℃，4min；

95 DEG C, 30min; 58 DEG C, 30s; 72 DEG C, 1.5min; Repeat 25 circulations;

72℃，10min。

Utilize PCR cleaning agents box, clean respectively to amplified production, remove the primer and dNTP that do not increase, column purification also uses TE(10mM Tris-HCl, 1mM EDTA, pH7.5) reclaim, obtain amplified production.

2. the gene fragment that fragmentation mammary cancer is relevant.

The amplified production concentration of the column purification recovery of determination step 1 gained take TE as solution, by the amplified production waiting the column purification of mass mixing step 1 gained to reclaim, and the mixed solution 600 μ L of configuration 100ng/ μ L, then ultrasonic fragmentation.Reaction conditions is as follows: under the power condition of 430 to 440W, ultrasonic 5s, suspends 5s, repeats 40 times.After ultrasonic end, the product after ultrasonic is run the PAGE glue of 8%, reclaim the fragment of 200 to 500bp size, obtain fragmentation products one.

3. dephosphorylation.

Dephosphorylation process being carried out to the fragmentation products one of step 2 gained, to prevent in follow-up ligation process, occurring from connecting between fragmentation products one, and then the capture reaction of interfere with subsequent.Reaction system is as follows: fragmentation products one (50ng/ μ L), 40 μ L; 10 × AP Buffer, 10 μ L; Fast AP(Fermentas, EF0651,1U/ μ L), 10 μ L; ddH ₂o, adds to 100 μ L.After 37 DEG C of digestion 30min, column purification also reclaims with TE, obtains dephosphorylation product.

4. end reparation.

End reparation is carried out to the dephosphorylation product of step 3 gained.Reaction system is as follows: dephosphorylation product (30ng/ μ L), 60 μ L; 5 × T4 DNA Polymerase Buffer, 20 μ L; T4 DNA Polymerase(Fermentas, EP0061,5U/ μ L), 1 μ L; The each 2.5mM of dNTP(), 2 μ L; ddH ₂o, adds to 100 μ L.12 DEG C of reaction 30min, column purification also reclaims with TE, obtains end and repairs product one.

5. be connected with joint component one.

The end of step 4 gained is repaired product one be connected with joint component one (SEQ ID NO:21 and SEQ ID NO:22), wherein 3 ' the terminal nucleotide C of SEQ ID NO:21 is dideoxy nucleotide, 5 ' the terminal nucleotide G of SEQ ID NO:22 is phosphorylated, containing phosphate group.Reaction system is as follows: end repairs product one (20ng/ μ L), 40 μ L; Joint component one (150ng/ μ L), 4 μ L; 10 × T4 Ligase Buffer, 10 μ L; 10mM ATP, 10 μ L; 4 × PEG 6000(30%), 25 μ L; T4 Ligase(Fermentas, EL0013,30U/ μ L), 1 μ L; ddH ₂o, adds to 100 μ L.16 DEG C of reaction 2h, column purification also reclaims with TE, must connect product one.

6. primer is replaced.

Because containing dideoxy nucleotide on joint component one, so the chain connected in product one can contain an otch, in order to remove this otch, use one to hold the primer with identical sequence to replace with 3 ' of the chain containing dideoxy nucleotide in joint component one, the 3 ' end of this replacement primer (SEQ ID NO:23) is not containing dideoxy nucleotide.Reaction system is as follows: connect product one (15ng/ μ L), 40 μ L; 5 × Buffer, 20 μ L; Replace primer (10 μMs), 10 μ L; The each 2.5mM of dNTP(), 1 μ L; 10mM ATP, 5 μ L; ddH ₂o, adds to 100 μ L.First place 5min at 60 DEG C, be then cooled to 37 DEG C, then add 6 μ L Taq DNA Polymerase(GenScript, 5U/ μ L) and 1 μ L T4 DNA Ligase(Fermentas, EL0013,30U/ μ L), in 37 DEG C of reaction 30min, then column purification reclaims, and obtains primer and replaces product.

7.PCR increases.

Utilize the identical Primer-F1(SEQ ID NO:24 of 5 ' end portion of 3 ' end and displacement primer) and 3 ' to hold and the PCR primer Primer-R1(SEQ ID NO:25 of joint component complementation) product is replaced to step gained primer carry out pcr amplification and obtain the gene fragment capture probe that mammary cancer is correlated with.Wherein, the 5 ' end of Primer-F1 contains two biotin labeling.Reaction system is as follows: primer replaces product (10ng/ μ L), 50 μ L; Primer-F1(10 μM), 100 μ L; Primer-R1(10 μM), 100 μ L; 10 × Ex Taq Buffer, 100 μ L; Ex Taq(TaKaRa, 5U/ μ L), 8 μ L; The each 2.5mM of dNTP(), 80 μ L; ddH ₂o adds to 1000 μ L.

Reaction conditions is as follows:

94℃，2min；

94 DEG C, 15s; 58 DEG C, 10s; 72 DEG C, 30s; Repeat 15 circulations;

72℃，5 min。

Utilize PCR cleaning agents box, clean amplified production, remove the primer and dNTP that do not increase, column purification also uses TE(10mM Tris-HCl, 1mM EDTA, pH7.5) reclaim, obtain the gene fragment capture probe that mammary cancer is relevant.

Two, the gene fragment capture probe that mammary cancer is relevant is fixed on magnetic bead.

The magnetic bead (Dynabeads M-280 Streptavidin) of the gene fragment capture probe that the mammary cancer of getting 1 μ g is correlated with and 30 μ L combines, and ambient temperatare puts 30min, flicks tube wall every 2min.Use magnet adsorption magnetic bead, remove supernatant, add 50 μ L, the NaOH of 0.1M, sex change 5min under room temperature, repeats once, finally dissolves magnetic bead with 25 μ L TE.Because in the step 7 of step one, only have a PCR primer to contain biotin labeling, and the magnetic bead in this step is excessive, so can think that the capture probe be fixed on magnetic bead is about 0.5 μ g in this step.

Three, the preparation in nucleic acid fragment storehouse to be captured.

1. with the genomic dna of a breast cancer patients (Shenzhen BJ Univ Hospital patient) for starting material, carry out fragmentation process.Measure breast cancer patients Whole Blood Genomic DNA concentration, and be configured to 100ng/ μ L with TE, go 600 μ L to carry out ultrasonic fragmentation.Reaction conditions is as follows: under the power condition of 430 to 440W, ultrasonic 5s, suspends 5s, repeats 40 times.After ultrasonic end, the product after ultrasonic is run the PAGE glue of 8%, reclaim the fragment of 300 to 400bp size, obtain fragmentation products two.

2. phosphorylation and end reparation.

Carry out phosphorylation to the fragmentation products two of step 1 gained, reaction system is as follows: fragmentation products two, 1 μ g; 10 × T4 Ligase Buffer, 10 μ L; The each 2.5mM of dNTP(), 1.5 μ L; T4 DNA Polymerase(Fermentas, EP0061,5U/ μ L), 1 μ L; Klenow DNA Polymerase(Fermentas, 3 ' to 5 ' exo minus, EP0421,5U/ μ L), 0.1 μ L; T4 PNK(Fermentas, EK0031,10U/ μ L), 0.5 μ L, 10mM ATP, 1.5 μ L; ddH ₂o, adds to 100 μ L.20 DEG C of reaction 20min, column purification also reclaims with TE, obtains end and repairs product two.

3. be connected with joint component two.

Step 2 gained end is repaired product two be connected with joint component two (SEQ ID NO:26 and SEQ ID NO:27), reaction system is as follows: end repairs product two, 700ng; Joint component two, 700ng; 10 × T4 Ligase Buffer, 20 μ L; 10mM ATP, 25 μ L; 4 × PEG 6000(30%), 50 μ L; T4 Ligase(Fermentas, EL0013,30U/ μ L), 10 μ L; ddH ₂o, adds to 200 μ L.16 DEG C of reaction 2h, column purification also reclaims with TE, must connect product two.

4.PCR increases.

Utilize the primer that has an identical sequence with the nucleic acid chains (SEQ ID NO:26) in joint component two as the upstream primer of pcr amplification and downstream primer, product two is connected to step 3 gained and to increase to obtain nucleic acid fragment storehouse to be captured.Reaction system is as follows: connect product two, 100ng; 10 × Ex Taq Buffer, 5 μ L; Ex Taq(TaKaRa, 5U/ μ L), 0.5 μ L; The each 2.5mM of dNTP(), 4 μ L; PCR primer (SEQ ID NO:26,100 μMs), 0.5 μ L; ddH ₂o adds to 50 μ L.

Reaction conditions is as follows:

94℃，2min；

94 DEG C, 15s; 58 DEG C, 10s; 72 DEG C, 30s; Repeat 15 circulations;

72℃，5 min。

Utilize PCR cleaning agents box, clean amplified production, remove the primer and dNTP that do not increase, column purification also uses ddH ₂o reclaims, and obtains nucleic acid fragment storehouse to be captured.

Four, hybridize and catch.

1. first time hybrid capture.

Get the nucleic acid fragment storehouse 2.5 μ g to be captured of step 3 gained, be dissolved in the ddH of 5 μ L ₂in O, 95 DEG C of sex change 5min, then ice bath 2min, then be placed in 65 DEG C of process 15min; Then the capture probe be fixed on magnetic bead of 25 μ L step 2 gained is added, 30 μ L 2 × hybridization buffers (pH 8.0,10 × SSPE, 10 × Denhardt ' s, 10 mM EDTA, 0.2% SDS); Be placed on the gyroscope of 65 DEG C of baking ovens, rotate hybridization 36h.

2. cleaning and sex change.

Product after magnetic bead adsorption step 1 hybridization, abandons supernatant; Then 500 μ L cleaning buffer solutions (1.5mM Trisodium Citrate, 150mM sodium-chlor, 0.1%SDS) are used to clean three times; Then 50 μ L 0.1M sodium-hydroxide treatment 5min are added; Magnetic bead adsorbs, and stays supernatant; In supernatant, add the acetic acid of 3 μ L 20%, with in and supernatant in sodium hydroxide, obtain first time catch product.

3.PCR increases.

Catch product to the first time of step 2 gained and carry out pcr amplification, reaction system is as follows: first time catches product, 100ng; 10 × Ex Taq Buffer, 5 μ L; Ex Taq(TaKaRa, 5U/ μ L), 0.5 μ L; The each 2.5mM of dNTP(), 4 μ L; PCR primer (SEQ ID NO:26,100 μMs), 0.5 μ L; ddH ₂o adds to 50 μ L.

Reaction conditions is as follows:

94℃，2min；

94 DEG C, 15s; 58 DEG C, 10s; 72 DEG C, 30s; Repeat 25 circulations;

72℃，5 min。

Utilize PCR cleaning agents box, clean amplified production, remove the primer and dNTP that do not increase, column purification also uses ddH ₂o reclaims, and must be used for the nucleic acid fragment storehouse to be captured that second time is caught.

4. second time hybrid capture.

With reference to the step of above-mentioned steps 1 and 2, utilize capture probe on the magnetic bead nucleic acid fragment storehouse to be captured of catching for second time to step 3 gained that is fixed on of step 2 gained to carry out second time and catch, obtain and catch product for the second time.

Five, order-checking inspection capture effect.

Product is caught to the first time obtained in step 4 and second time is caught product and carried out high-flux sequence respectively.Result shows, and first time catches product and catching product all can cover the relevant gene fragment of mammary cancer (region that 10 pairs of amplimers in step one can amplify) completely for the second time, and namely their coverage all reaches 100%; In addition product is caught for the first time also basically identical with the uniformity of catching product for the second time.

In addition is caught product the first time obtained in step 4 and second time is caught product and increased respectively, amplification condition can step 3 in refer step four, and then PCR is clean reclaims amplified production, and will reclaim the product and pGEM that obtain -T Easy Vector(Promega, Cat# A1360) connect, product conversion will be connected again in competence bacillus coli DH 5 alpha (TIANGEN Biotech (Beijing) Co., Ltd. CB101-01), and intestinal bacteria DH 5 α after transforming is applied on the LB flat board of ammonia benzyl resistance, carry out blue hickie screening.Using the hickie on flat board as template, primers designed (SEQ ID NO:26), as the upstream primer of bacterium colony PCR and downstream primer, carries out bacterium colony PCR checking.Each random picking 100 positive colonies carry out Sanger order-checking, and the gene fragment that sequencing result is relevant to published above-mentioned 10 mammary cancer on NCBI are compared.Result shows, and by catching for the first time in 100 positive colonies that product obtains, has in the comparison of 88 positive colony energy, by catching for the second time in 100 positive colonies that product obtains, has in the comparison of 97 positive colony energy; The accuracy rate of namely catching for the first time is 88%, and the accuracy rate that second time is caught is 97%.

Should be noted that, the present invention typically applies and includes but not limited to that the gene fragment that above-mentioned mammary cancer is relevant is caught, in other similar gene fragments, plasmid fragments, Mitochondrial DNA or RNA fragment are caught, such as human leucocyte antigen (human leukocyte antigen, HLA), in the catching of gene fragment or DMD gene (x linked recessive inherited disease gene) fragment, also method set forth in the present invention can be applied.Described HLA is the major histocompatibility complex of the mankind, and HLA gene is positioned at 6p21.3 on the mankind's No. 6 the short arm of a chromosome, and total length is 4000Kb.Described DMD gene is positioned on mankind's X chromosome galianconism Xp21, and total length is 2500kb.

In addition, the present invention also respectively for whether rotate on gyroscope in the temperature in crossover process, hybridization time, crossover process and hybridization time capture probe and the factor such as ratio in nucleic acid fragment storehouse to be captured carried out a series of contrast experiment.

Result shows, when the temperature in crossover process is respectively 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, when other reaction conditionss are identical with the first specific embodiment, along with the rising of temperature, the amount that first time and second time catch product constantly declines, and the accuracy rate that first time and second time are caught constantly rises; Wherein 65 DEG C time catch accuracy rate and 70 DEG C time to catch accuracy rate basically identical, but the first time when amount that first time when 70 DEG C and second time catch product is 65 DEG C respectively and second time catch 73% and 76% of the amount of product.

When hybridization time is respectively 12h, 18h, 24h, 36h, 48h, 72h, when other reaction conditionss are identical with the first specific embodiment, along with the prolongation of time, the amount that first time and second time catch product constantly rises; First time and the second time accuracy rate then (see figure 3) on a declining curve of catching, wherein, when hybridization time is 18h, first time and the second time accuracy rate of catching respectively comparatively 36h time improve 8% and 3%; The coverage that first time and second time are caught is 93% when 12h, is 100% at 18h and more than 18h.

When hybridization in crossover process leaves standstill to carry out, when other reaction conditionss are identical with the first specific embodiment, the accuracy rate that first time and second time are caught is 66% and 72% respectively, far below the accuracy rate that gyroscope is hybridized.The accuracy rate that the accuracy rate that gyroscope is caught for the first time is just caught than standing second time is high.

When amount and the ratio of the amount in nucleic acid fragment storehouse to be captured of capture probe, to be respectively 2:1,1:1,1:2,1:5(identical with the first specific embodiment), 1:10 time, wherein the amount of capture probe is fixed as 0.5 μ g, along with the increase in nucleic acid fragment storehouse to be captured, gained catches the amount of product for the first time in the trend risen with second time, the coverage of catching with second time for the first time, accuracy rate are basically identical.Therefore can according to practical situation, the ratio of amount to the amount in the amount of capture probe and nucleic acid fragment storehouse to be captured of the genomic dna of gained carries out suitable adjustment per sample.

To sum up, best hybridization condition is: 65 DEG C of gyroscopes hybridize 18h.In actual experiment process, it is generally acknowledged that the accuracy rate of catching is higher than 85%, coverage reaches 100% can think acquisition success.Therefore, catching method of the present invention in fact only needs once to catch and can reach this area to the general requirement of catching, and this can simplify experimental procedure greatly, improves conventional efficient, reduces experimental cost.Certainly, carry out catching for twice better experiment effect can be provided.In addition, general all between 36h to 72h at the hybridization carrying out capture probe and nucleic acid fragment storehouse to be captured in prior art, (periodically hybridization is shaken) in preferred version of the present invention, hybridization only needs 18h can meet or exceed the hybridization effect of prior art, greatly reduce the time of hybrid capture, this can improve conventional efficient greatly, reduces the time cost of experiment.

Following the present invention has also been two contrast experiments, for setting forth experiment effect of the present invention further.

One, contrast experiment one.

Contrast experiment one is mainly by step 7 step 7 in step one ' replace, other reaction conditionss and the first specific embodiment are consistent.

Step 7 ': utilize the identical Primer-F1(SEQ ID NO:24 of 5 ' end portion of 3 ' end and displacement primer) and 3 ' to hold and the PCR primer Primer-R1(SEQ ID NO:25 of joint component complementation) product is replaced to step gained primer carry out pcr amplification and obtain the gene fragment capture probe that mammary cancer is correlated with.Wherein, Primer-F1 and Primer-R1 be not all containing biotin labeling.Reaction system is as follows: primer replaces product (10ng/ μ L), 50 μ L; Primer-F1(10 μM), 100 μ L; Primer-R1(10 μM), 100 μ L; 10 × Ex Taq Buffer, 100 μ L; Ex Taq(TaKaRa, 5U/ μ L), 8 μ L; The each 2.5mM of dNTP(), 80 μ L; DUTP(10mM), 4 μ L; ddH ₂o adds to 1000 μ L.

Reaction conditions is as follows:

94℃，2min；

94 DEG C, 15s; 58 DEG C, 10s; 72 DEG C, 30s; Repeat 15 circulations;

72℃，5 min。

Utilize PCR cleaning agents box, amplified production cleaned, remove do not increase primer, dNTP, dUTP, column purification and use TE(10mM Tris-HCl, 1mM EDTA, pH7.5) reclaim control group mammary cancer be correlated with gene fragment capture probe.

Result shows, compared with the first specific embodiment, the amount that the first time of contrast experiment one gained and second time catch product only has 73% and 77% of the first specific embodiment respectively, the accuracy rate of catching that first time and second time are caught is respectively 86% and 94%, and the coverage that first time and second time are caught is respectively 87%, 86%.Wherein first time and second time catch the amount of product may be in the magnetic capture process after completing steps 7 ' far below the reason of the first specific embodiment, there is the situation that multiple magnetic bead is combined with same capture probe, the capture probe of ' capture probe of gained is not completely by magnetic capture, and the step 7 ' gained that causes step 7 may further not caused the obvious decline of the coverage that first time of this contrast experiment and second time are caught completely by magnetic capture.And the combination of multiple magnetic bead and same capture probe of the specificity hybridization of capture nucleic acid fragment library is treated in to(for) capture probe does not have too much influence, thus the first time of this contrast experiment and second time catch catch accuracy rate and the first specific embodiment is basically identical.

Two, contrast experiment two.

Contrast experiment two is mainly by step 2 in the first specific embodiment: capture probe by this step of the magnetic capture of marked by streptavidin be placed in simultaneously step 4 first time hybrid capture and second time hybrid capture after.

Result shows, and the first time of contrast experiment two gained is basically identical with the amount and the first specific embodiment of catching product for the second time, is 101% and 99% of the first specific embodiment respectively, far above contrast experiment one; The accuracy rate of catching that first time and second time are caught is respectively 87% and 95%, substantially identical with contrast experiment one with the first specific embodiment; The coverage that first time catches with second time is identical with the first specific embodiment, is 100%, far above contrast experiment one.

The high-flux sequence result of contrast experiment two is compared with the first specific embodiment, catch in the relevant gene fragment of mammary cancer, there is more heterozygous mutant, its reason may be there occurs complementary pairing between capture probe in crossover process, and containing the magnetic bead of marked by streptavidin when catching biotin labeled capture probe, be not combined with all biotin labelings, and then cause catching in product of gained to be mixed with capture probe, thus make follow-up high-flux sequence result contain the sequence of capture probe, and the inconsistent place between the sequence of capture probe and nucleic acid fragment storehouse to be captured, heterozygous mutant has been shown as in sequencing result, and nucleic acid fragment in fact to be captured may be homozygous mutation or wild-type in these inconsistent places.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

SEQUENCE LISTING

<110> Sheng department Tong

The method of a <120> capture nucleic acid fragment

<130>

<160> 27

<170> PatentIn version 3.3

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Claims

1. a method for capture nucleic acid fragment, is characterized in that, comprises the following steps:

(1). take genomic dna as template, utilize the primer pair being used for specific amplification nucleic acid fragment to be captured to carry out pcr amplification, must containing the nucleic acid molecule of nucleic acid fragment to be captured;

(2). with the nucleic acid molecule containing nucleic acid fragment to be captured for raw material, utilize biotin labeled amplimer or biotin labeled joint component to prepare biotin labeled capture probe;

(3). biotin labeled capture probe and nucleic acid fragment storehouse to be captured are hybridized;

Described step (3) comprises the following steps:

1.. the solid phase carrier utilizing avidin to mark catches the biotin labeled capture probe in step (2), must be fixed on the capture probe on solid phase carrier;

2.. make to be fixed on capture probe on solid phase carrier and nucleic acid fragment storehouse to be captured hybridize first time hybrid product, separation and purification first time hybrid product must containing the first product of solid phase carrier;

3.. make the product sex change that in step (3), 2. step obtains, obtain and catch product for the first time;

Described hybridization is carried out in periodic vibration;

The described primer pair for specific amplification nucleic acid fragment to be captured is 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, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, and SEQ ID NO:19 and SEQ ID NO:20,

The time of described hybridization is 18h, and the temperature of hybridization is 65 DEG C, and the ratio of the amount of capture probe and the amount in nucleic acid fragment storehouse to be captured is respectively 2:1,1:1,1:2,1:5 or 1:10.

2. the method for capture nucleic acid fragment according to claim 1, is characterized in that, further comprising the steps of after the middle step of described step (3) is 3.:

4.. 3. the first time of gained catches products thereof and obtains second time hybrid product for be fixed on capture probe on solid phase carrier and the step in step (3) that obtain in utilizing in the step (3) step 1., and separation and purification second time hybrid product must contain the second product of solid phase carrier;

5.. make the product sex change that in step (3), 4. step obtains, obtain second time and catch product.

3. the method for capture nucleic acid fragment according to claim 1, is characterized in that, described step (2) comprises the following steps:

1.. fragmentation, containing the nucleic acid molecule of nucleic acid fragment to be captured, obtains fragmentation products;

2.. fragmentation products is connected with joint component, must containing the nucleic acid fragment of joint;

3.. utilize biotin labeled amplimer to carry out pcr amplification to step product 2. in step (2), obtain biotin labeled capture probe.

4. the method for capture nucleic acid fragment according to claim 1, is characterized in that, described step (2) comprises the following steps:

2.. fragmentation products is connected with biotin labeled joint component, obtains biotin labeled capture probe.

5. the method for the capture nucleic acid fragment according to claim 3 or 4, is characterized in that, described joint component is y splice; Described y splice is double chain acid molecule, and described y splice comprises crotch region and complementary district; Mononucleotide protruding terminus is contained and without phosphoric acid Nucleotide in the complementary district of described y splice; Described mononucleotide is T, and described mononucleotide protruding terminus is positioned at 3 ' end of its place nucleic acid chains; Described 5 ' the end being positioned at its place nucleic acid chains without phosphoric acid Nucleotide.

6. the method for capture nucleic acid fragment according to claim 1, is characterized in that, the middle step of described step (3) 1. solid phase carrier is the solid phase carrier of marked by streptavidin.