CN109182454A - A method of capture genome specific DNA fragments - Google Patents
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Abstract
The invention discloses a kind of methods of particular patch segment DNA in capture genome, comprising the following steps: (1) combines DNA to be detected and sgRNA/ gene editing albumen composition containing target site；It is cut into specific objective DNA；(2) sequencing library is constructed；(3) it is sequenced and determines specific region DNA sequence dna；The present invention is not necessarily to that the hybridization of conventional capture technique is used to combine, and can rapidly realize the enrichment of specific target site domain dna, avoids the disadvantages of conventional method hybridization enriching step is more, process is cumbersome, and testing result is also more accurate.
Description
Technical field
The present invention relates to a kind of capture genome specific DNA fragments, the method for subsequent DNA detection.
Background technique
The genome of people has 3,000,000,000 bases, that is, 3Gb, wherein containing code area, noncoding region and some regulations
Region.With the development of high throughput sequencing technologies, the cost for resurvey sequence to full-length genome is lower and lower, first base of the mankind
Because group takes 3,000,000,000 U.S. dollars, the full-length genome of a present people resurveys sequence in thousands of RMB, last decade sequencing cost
10,000 times are reduced, cost reduces very fast.?
Survey, polygenic disease detection, monogenic disease detection, be not each case require carry out genome sequencing, and only need into
The detection of row specific region, to quickly detect the textural anomaly whether specific region has DNA level.Occur currently on the market
The method of specific fragment sequencing, the method realized substantially have following three directions:
1, the core of PCR:PCR reaction is the combination by specific primer and target dna at appropriate temperatures, thus poly-
It is expanded in the presence of synthase.Using round pcr, the segment detected to needs is expanded, and is then carried out to amplified production
Sequencing detection.PCR method is simple and convenient, cheap, fast implements region enrichment.The technology is suitable for tens site richnesses
Collection, when needing detection site more, the primer for each PCR of adjusting and optimizing to react in an approximate temperature,
Carrying out reaction simultaneously will be extremely difficult, because each primer region is determined by specific site, and the G/C content in these regions is not
It is identical to the greatest extent, therefore it is not particularly suited for large-scale segment detection.
2, rna probe be enriched with: Agilent company of the U.S. issued by synthesis rna probe and genomic DNA hybridization to
The disposable technology for obtaining all gene extron subregions of full-length genome.The principle of this method is first to carry out genomic DNA
6~7 random fragmentation, filling-in plus A, link connector, amplification circulations, obtain the amplification library of a full-length genome small fragment, so
Library DNA is denaturalized afterwards and is carried out 4~16 hours of hybridization at the rna probe of single stranded DNA and the complementation of full exon region, RNA is visited
There is biotin labeling on needle, be combined by the magnetic-particle of marked by streptavidin, so that washing away is not targeting regions
DNA, the PCR amplification that remaining DNA carries out 10~16 circulations have obtained final library, which can be measured by high pass
Sequence is detected, and substantially in 50Mb or so 2G data volume or so is sequenced, so that it may right in general full exon+regulatory region
Exon and regulatory region have comprehensive detection.After the technology occurs, scientific research and clinical context are widely used, main at present
Supplier is Agilent and Roche Holding Ag.This method, can be disposably to whole exon 1s compared to PCR biggest advantage
Domain is enriched with, this is that Standard PCR, multiple PCR technique all cannot achieve.The technology can also be determined for specific region
System, flexibility are also good.But the technical operation is complicated compared to round pcr very much, time-consuming, needs 3~4 days, entire real
The process of testing has twice PCR process, although all ensureing PCR polymerase using high substantially now, is also not excluded for multiple PCR amplification
It can be artificially introduced base mispairing in the process.
3, DNA probe is enriched with: the technology that also useful double chain DNA probe and target area hybridization are enriched in the market.The technology
Realize the almost the same of route and rna probe, and genomic DNA is subjected to random fragmentation first, filling-in plus A, connect
6~7 head, amplification circulations, obtain the amplification library of a full-length genome small fragment, then library DNA are denaturalized into single stranded DNA
It is carried out 4~16 hours of hybridization with the DNA probe of full exon region complementation, has biotin labeling on probe, pass through strepto- parent
It is combined with the magnetic-particle of element label, so that washing away is not target zones domain dna, remaining DNA carries out 10~16 circulations
PCR amplification obtained final library, which can be detected by high-flux sequence, general full exon+regulation
In 50Mb or so, 2G data volume or so is sequenced substantially in region, so that it may have comprehensive detection to exon and regulatory region.
It is consistent with rna probe method compared to the advantage and disadvantage of round pcr.It is stable preferably with probe itself compared to rna probe, but
It is and is not so good as RNA in target patch segment DNA binding ability.
The direct amplification technique flux of PCR is lower, and in general genome capture all refers to the shape for hybridizing acquisition in a manner of probe
Formula.This technology has the shortcomings that several common: 1. is big for clinic development technical difficulty, complicated for operation, needs to carry out stringent
Subregion is unfavorable for the extensive development of the technology；2. being easy cross contamination between sample when sample size is big；3. probe
Method due to need 65 degree at a temperature of probe and target dna carry out prolonged hybridization combination, can just successfully obtain mesh
Standard film section, thus it is time-consuming permanent.
In recent years, the third generation gene editing system based on CRISPR/Cas obtains significant progress, and the system is originally
For the acquired immune system of bacterium, when invasion of the bacterium by external source, bacterium gets off specific fragment sequential recording, if
Next time is invaded by this external source again, and bacterium can start immune system immediately and quickly identify that external source is invaded, and cuts exogenous array, from
And guarantee inherently safe.This set CRISPR/Cas9 system this several years development it is very fast, be widely used in gene therapy,
For hereditary disease, malignant tumour, embryo's treatment, detection of nucleic acids.
Summary of the invention
In order to overcome the above problem in existing, the present invention provides a kind of sides of specific DNA fragments in capture genome
Method, the hybridization without conventional capture technique combine, and rapidly realize the enrichment of specific target site domain dna, avoid conventional method
Hybridize the disadvantages of enriching step is more, process is cumbersome, testing result is also more accurate.
A kind of capture genome specific DNA fragments method, which comprises the following steps:
(1) DNA to be checked is in conjunction with containing specific region sgRNA probe/gene editing albumen composition, warm bath cutting DNA；
(2) particular patch segment DNA constructs sequencing library；
(3) specific region DNA sequence dna is determined by being sequenced.
Further, the sgRNA probe is probe needed for gene editing albumen identifies specific fragment, probe sequence
Complementary with specific fragment, specific fragment is one or multiple targets, each specific fragment at least needs former and later two probes,
Midfeather distance is adjusted according to the testing requirements in later period.
Further, after sgRNA probe/gene editing albumen composition combination DNA, 37 DEG C warm bath 30 minutes, realize special
The cutting of stator segment DNA.
Further, the method also includes by cleaning, crossing the step of column removes nonspecific segment DNA.
Further, the method for constructing sequencing library is: the DNA cut down, by T4DNA polymerase,
One of Klenowexo-, T4DNA phosphokinase (T4DNA PNK), Taq polymerase or Klenow enzyme or a variety of filling-in and increasing
Add base A, 37 DEG C are reacted 20 minutes；Bacterial alkaline phosphatase (BAP) is added after 1 minute, 75 DEG C are denaturalized 5~10 minutes, to temperature
DNA ligase, DNA connector is added in degree after reducing, 20 DEG C connect 20 minutes；Albumen and buffering in purifying removal reaction system
Liquid constructs DNA sequencing library, is directly used in subsequent detection；If sequencing library concentration is too low, is expanded and be enriched with by PCR method.
The present invention uses identification and cutting power of the CRISPR/Cas family protein for specific sequence DNA for the first time, for
Particular patch segment DNA designs a plurality of~millions of probe simultaneously, and sgRNA probe and Cas albumen composition quickly identify particular patch
Segment DNA, 37 DEG C are incubated for 30 minutes, DNA fragmentation to be measured can be all cut down；The DNA cut down from genome, end
Filling-in plus A are carried out, DNA connector is then connected, the DNA library for constructing completion can be sequenced by sequenator.Sequenator can
Be with while synthesis while be sequenced for major technique representative two generation sequenators (illumina, Life), be also possible to PacBio,
Oxford Nanopore etc. is the third generation single-molecule sequencer of representative.
Typical probe is enriched in hybridization technique, and hybridization step needs 65 degree of 4~16 hours, is generally with overnight hybridization
It is more, it is time-consuming and laborious.This method uses the area that gene editing method removes identification DNA and needs are detected from DNA to be measured for the first time
Domain dna is cut down, and constructs sequencing library, so that multi-target DNA enrichment is realized for the first time, it is miscellaneous overnight without conventional probe
Friendship process can most complete specific fragment enrichment and sequencing library building, than conventional probe enrichment side in two hours fastly
Method will save for 90% time.
Typical probe catching method is hybridized by the excess probe of overlapping and a DNA mixing library, combination
DNA in target area is finally eluted, and not can control the original length of eluted dna, thus when late detection just
It needs just obtain to be detected the DNA progress overall length sequencing eluted with DNA sequencing method as long as possible
The DNA variation situation in region.And the present invention can customize out since specific cleavage site has been determined and meet subsequent detection requirement
DNA length, so as to substantially reduce cost and the time of DNA sequencing, precisely orientation carries out depth detection for target area.
The present invention has following technical characterstic:
(1) quickly: multiple, full-length genome exon is captured through the invention, can be terminated within 2 hours, and
Routine PCR reaction experiment is consistent, is considerably faster than conventional probe catching method.
(2) without hybridization: using CRIPSR/Cas9 system identification target fragment and cutting for innovation cuts down building
Sequencing library, whole flow process, which is in the industry cycle initiated, can carry out target area enrichment without hybridization.
(3) favorable expandability: the identification region of CRIPSR/Cas9 is 20 bases, without the requirement of special identification region, and
And multidigit point it is different with the G/C content in each region when working at the same time and caused by Standard PCR TM temperature difference problem at this
System is not present, therefore design difficulty is not high within the scope of full-length genome, without adjusting primer location repeatedly as conventional multiplex PCR
Multiple capture can be realized with concentration.
(4) plasticity is strong: it can be determined due to cutting the DNA length captured with designed, designed, it can be according to subsequent
The adjustment of detection platform difference, for example specific site can be placed on to the centre of different length.And conventional catching method is due to passing through
Probe and random library are hybridized, and the position of the indefinite specific site in the segment captured and capture dna
Length.
(5) specificity is good: conventional probe catching method, due to having magnanimity overlapping covering probe, that is, overlapped probes
It is put into reaction system, therefore has many non-specific adsorptions, causes generally to have 40~50% number in final sequencing result
According to the DNA for nontarget area.And the present invention is not due to needing probe magnanimity overlay area, it is only necessary to specific fragment left and right ends
Two probes, therefore non-specific rich region can greatly reduce, without the sequencing data of magnanimity.
(6) DNA long fragment direct Sequencing: previous all high-flux sequence methods are all based on the principle of shotgun substantially,
DNA to be measured is smashed first, constructs random sequencing library, the data obtained, which is then sequenced, will carry out splicing analysis.Work as target patch
As soon as section regional DNA is the change of whole segment DNA, the method for shotgun is not suitable for detecting this kind of disease, such as fragile X synthesis
Sign, Erb's atrophy (DMD) etc., and this method can directly cut the DNA before and after region to be measured, use is novel
The instrument platforms such as long segment sequenator PacBio, Oxford Nanopore directly carry out long segment sequencing, can directly detect disease
Lesion domain, to quickly patient be allowed to be diagnosed.
Detailed description of the invention
Fig. 1 is flow chart of the present invention.
Fig. 2 fast enriching EGFR gene exon region schematic illustration.
Fig. 3 is fast enriching EGFR gene exon region result schematic diagram.
Fig. 4 is enrichment FMR1 repetitive sequence region for detecting fragile X mental retardation schematic diagram.
Specific embodiment
It is better understood the present invention by means of following specific embodiments, however, these specific embodiments are only used for
It illustrates the present invention, is not necessarily to be construed as limitation of the present invention.
Flow chart as shown in Figure 1, capture genome specific DNA fragments method of the invention, main includes including following
Step:
(1) DNA to be checked is in conjunction with containing specific fragment sgRNA probe/gene editing albumen composition, warm bath cutting DNA；
(2) particular patch segment DNA constructs sequencing library；
(3) specific fragment DNA sequence dna is determined by being sequenced.
Embodiment 1:
The present embodiment is used to illustrate the application that the present invention is used for fast enriching EGFR gene exon region.
It is fast enriching EGFR gene exon region schematic illustration shown in Fig. 2.For tetra- exon designs of EGFR
Four pairs of probes, Cas Protein cleavage goes out entire exon, after library construction, is detected using high-flux sequence instrument,
To find the gene mutation situation on exon.
Step is mainly as follows:
1. extracting genomic DNA from human peripheral leucocytes, 200ul whole blood and the mixing of 200ul lysate；
2. taking 5ul total amount is 2ug genomic DNA and 20ul EGFR complementary probe sgRNA/Cas9 albumen composition, add
25ul Cas9 buffer (Tris-HCl pH 8.0), 37 DEG C warm bath 30 minutes；
3. being added Streptavidin MagneSphere 5ul (Thermo Fisher), mixed at room temperature 5 minutes；
4. separating magnetic bead with magnetic frame, supernatant is removed, magnetic bead is rinsed twice with PBS, is separated with magnetic frame, and abandon supernatant；
5. 3N NaOH 40ul is added magnetic bead and mixes 5 minutes, is separated with magnetic frame, take supernatant；
6. in supernatant be added 40ul 0.1N acetic acid in and supernatant；
7. addition 2 units/ul end-filling enzyme (T4DNA polymerase or the dNTPs with Klenow exo-), 490uM,
The Tris buffer of 55mM forms, and 37 DEG C are reacted 20 minutes；
8. directly in test tube be added 1ul BAP, 37 DEG C 1 minute, 75 DEG C be denaturalized 5 minutes；
9. DNA connection reagent is directly added in test tube, reagent is connected by 100 units/ul DNA ligase, 3.5mM's
The Tris buffer of ATP, 55mM, dithiothreitol (DTT) and DNA the connector composition of 30mM.20 DEG C react 20 minutes, after reaction
It directly can directly upper high-flux sequence instrument detection by silicagel column or magnetic-particle purifying.
It is fast enriching EGFR gene exon region result schematic diagram shown in Fig. 3.EGFR 18,19,20,21 4 outer
Aobvious son, is directly cut down by four pairs of probes, then constructs sequencing library, and since initial position is clear, sequencing data amount is wanted
Ask not high, as shown in figure 3, lower section is that sequencing gained sequence and the template DNA base that there is any discrepancy are indicated with letter.
| Location | sgRNA targeting sequence | |
| sgRNA1 | Intron18 | ccagatcactgggcagcatg |
| sgRNA2 | Intron19 | ggtccatggctctgaacctc |
| sgRNA3 | intron19 | ggtccatgtgcccctccttc |
| sgRNA4 | intron20 | ggagccaggatcctcacatg |
| sgRNA5 | intron20 | catgaacatgaccctgaatt |
| sgRNA6 | intron21 | gccagcattttcctgacacc |
| sgRNA7 | intron21 | gagttaactttttccaacag |
| sgRNA8 | intron22 | agtttgtactgaggccaagc |
Embodiment 2:
The present embodiment is used to illustrate the application that the present invention is used for fast enriching full-length genome exon region.
1. extracting genomic DNA from human peripheral leucocytes, 200ul whole blood and the mixing of 200ul lysate；
2. taking 5ul total amount is 2ug genomic DNA and 20ul outer complementary probe sgRNA/Cas9 albumen composition entirely, add
25ul Cas9 buffer (Tris-HCl pH 8.0), 37 DEG C warm bath 30 minutes；
3. being added Streptavidin MagneSphere 5ul (Thermo Fisher), mixed at room temperature 5 minutes；
4. separating magnetic bead with magnetic frame, supernatant is removed, magnetic bead is rinsed twice with PBS, is separated with magnetic frame, and abandon supernatant；
5. 3N NaOH 40ul is added magnetic bead and mixes 5 minutes, is separated with magnetic frame, take supernatant；
6. in supernatant be added 40ul 0.1N acetic acid in and supernatant；
7. addition 2 units/ul end-filling enzyme (T4DNA polymerase or the dNTPs with Klenow exo-), 490uM,
The Tris buffer of 55mM forms, and 37 DEG C are reacted 20 minutes；
8. directly in test tube be added 1ul BAP, 37 DEG C 1 minute, 75 DEG C be denaturalized 5 minutes；
9. DNA connection reagent is directly added in test tube, reagent is connected by 100 units/ul DNA ligase, 3.5mM's
The Tris buffer of ATP, 55mM, dithiothreitol (DTT) and DNA the connector composition of 30mM.20 DEG C react 20 minutes, after reaction
It directly can directly upper high-flux sequence instrument detection by silicagel column or magnetic-particle purifying.
Embodiment 3:
The present embodiment is for illustrating the present invention for fast enriching FMR1 gene repeat sequence region, for detecting fragile X
Syndrome.Fig. 4 is enrichment FMR1 repetitive sequence region for detecting fragile X mental retardation schematic diagram.It is crisp causing by Cas albumen
Property X syndrome leading portion repetitive sequence region cut down, by long segment three generations sequencing (Oxford Nanopore or
PacBio direct Sequencing) is carried out, can accurately detect whether fragile X mental retardation.Conventional PCR method is due to the region G/C content
It is too high, it is difficult to carry out direct augmentation detection.
1. extracting genomic DNA from human peripheral leucocytes, 200ul whole blood and the mixing of 200ul lysate；
2. taking 5ul total amount is 2ug genomic DNA and 20ul outer complementary probe sgRNA/Cas9 albumen composition entirely, add
25ul Cas9 buffer (Tris-HCl pH 8.0), 37 DEG C warm bath 30 minutes；
3. being added Streptavidin MagneSphere 5ul (Thermo Fisher), mixed at room temperature 5 minutes；
4. separating magnetic bead with magnetic frame, supernatant is removed, magnetic bead is rinsed twice with PBS, is separated with magnetic frame, and abandon supernatant；
5. 3N NaOH 40ul is added magnetic bead and mixes 5 minutes, is separated with magnetic frame, take supernatant；
6. in supernatant be added 40ul 0.1N acetic acid in and supernatant；
7. the dNTPs, 55mM of 2 units/ul end-filling enzyme (T4DNA polymerase or and Klenowexo-), 490uM is added
Tris buffer composition, 37 DEG C react 20 minutes；
8. directly in test tube be added 1ul BAP, 37 DEG C 1 minute, 75 DEG C be denaturalized 5 minutes；
9. DNA connection reagent is directly added in test tube, reagent is connected by 100 units/ul DNA ligase, 3.5mM's
The Tris buffer of ATP, 55mM, dithiothreitol (DTT) and DNA the connector composition of 30mM.20 DEG C react 20 minutes, after reaction
Long segment survey directly can be carried out by Oxford Nanopore or PacBio sequenator by silicagel column or magnetic-particle purifying
Sequence.
Sequence table
<110>Hangzhou Kai Si Medical Devices Co., Ltd.
<120>a kind of method for capturing genome specific DNA fragments
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ccagatcact gggcagcatg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggtccatggc tctgaacctc 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggtccatgtg cccctccttc 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggagccagga tcctcacatg 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
catgaacatg accctgaatt 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gccagcattt tcctgacacc 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gagttaactt tttccaacag 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
agtttgtact gaggccaagc 20
Claims (5)
1. a kind of method for capturing genome specific DNA fragments, which comprises the following steps:
(1) DNA to be checked is in conjunction with containing specific fragment sgRNA probe/gene editing albumen composition, warm bath cutting DNA；
(2) particular patch segment DNA constructs sequencing library；
(3) specific fragment DNA sequence dna is determined by being sequenced.
2. the method as described in claim 1, which is characterized in that the sgRNA probe is that the identification of gene editing albumen is specific
Probe needed for segment, probe sequence is complementary with specific fragment, and specific fragment is one or multiple targets, each particular patch
Section at least needs former and later two probes, and midfeather distance is adjusted according to the testing requirements in later period.
3. method according to claim 2, which is characterized in that after sgRNA probe/gene editing albumen composition combination DNA,
37 DEG C warm bath 30 minutes, realize particular patch segment DNA cutting.
4. the method as described in claim 1, which is characterized in that the method also includes nonspecific by cleaning, crossing column removal
The step of piece segment DNA.
5. the method as described in claim 1, which is characterized in that the method for constructing sequencing library is: the DNA cut down leads to
It crosses in T4 archaeal dna polymerase, Klenowexo-, T4 DNA phosphokinase (T4 DNA PNK), Taq polymerase or Klenow enzyme
One or more filling-in and increase base A, 37 DEG C are reacted 20 minutes；Bacterial alkaline phosphatase (BAP) is added after 1 minute, 75 DEG C
DNA ligase, DNA connector is added in denaturation 5~10 minutes after temperature reduction, and 20 DEG C connect 20 minutes；Purifying removal reactant
Albumen and buffer in system construct DNA sequencing library, are directly used in subsequent detection；If sequencing library concentration is too low, pass through
PCR method amplification enrichment.
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| CN113667714A (en) * | 2020-05-15 | 2021-11-19 | 武汉华大医学检验所有限公司 | Target area capturing method, kit and sequencing method |
| CN113667714B (en) * | 2020-05-15 | 2024-07-09 | 武汉华大医学检验所有限公司 | Target area capturing method, kit and sequencing method |
| CN111676289A (en) * | 2020-06-19 | 2020-09-18 | 安徽微分基因科技有限公司 | Method for detecting EGFR gene 19 exon E746-A750 mutant gene |
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