CN105803055A - New target gene regional enrichment method based on multiple circulation extension connection - Google Patents
New target gene regional enrichment method based on multiple circulation extension connection Download PDFInfo
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Abstract
The invention provides a new target gene regional enrichment method based on multiple circulation extension connection. Experimental results show that the method can realize gene fragment enrichment of multiple target genes, and the number of the target gene fragments can be from tens to thousands. The enrichment product of the multiple target gene fragments can be used for sequencing analysis of various second-generation sequencing platforms after modification and purified quantification.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of target genetic region enrichment method extending based on Multiple Cycle and connecting.
Background technology
Although soon realizing by 1000 dollars of targets measuring human genome, but the totle drilling cost of genome research should include the cost of DNA sequencing, data management and data analysis (producing the data that can directly understand), this makes in big population level research and clinical practice, and the actual cost of genome research is difficult to reduce at short notice.In the recent period, a kind of new research method can to the specific region of disease and biological pathways, gene, even whole exon group (accounting for genomic 1%) being enriched with, then carry out the research of unbiased, this method is exactly target area enrichment high-flux sequence.
Target area enrichment high-flux sequence is to carry out probe design for interested one section or several sections of sequences, carries out catching enrichment by diverse ways, and further the sequence captured is carried out sequencing analysis.Owing to its probe design is flexible, overburden depth is high, it is more suitable for large sample amount disease sample analysis, or the results such as full-length genome, GWAS analysis or linkage analysis are verified；The site having been found that can not only be verified, the disease-susceptible humans site in candidate region can be found further simultaneously.The method carrying out high-flux sequence (NGS) after target gene is enriched with again has following advantage: 1) can reduce cost significantly, 2) the height order-checking degree of depth of target area ensure that sequencing result more accurately, 3) the shorter project turnaround time, 4) the clear and definite function of target area makes us that the analysis of result be more prone to.In view of these advantages, relative genome sequencing, bigger sample populations can be analyzed by target area enrichment associating high-flux sequence, this method can also have important using value in the clinical diagnosis of biomedical research and Mendel's disease, finally can also carry out Personalized medicine according to individual inherited characteristic.
Target area enrichment is carried out high-flux sequence, the enrichment of target region is the work primarily carried out, how a concrete research project selects most suitable enrichment method, need to consider the size of whole rich region, the number of sample and the need of factors such as (flux utilizing sequenator the most efficiently) that multiple samples are checked order simultaneously.Scientific research neutralizes the beneficiation technologies of some commercialization platforms use to be had a lot, but the reaction principle according to its core can be divided three classes: be based on the target area enrichment of pcr amplification, cyclisation and hybrid capture respectively.
" pcr amplification ": target area is made directly pcr amplification by multiple LA-PCRs (Long-rangePCR), the standard multiplex PCR of limited tuple or the substantial amounts of short-movie section of multiplexed PCR amplification of high tuple can also be selected, can also is that the multiplex PCR (IonAmpliSeqTMfromLifeTechnologies of innovation, GeneReadDNAseqSystemfromQiagen, TargetRichTMfromKailos), microlayer model PCR (RainDance), or the PCR (AccessArrayTMfromFluidigm) based on chip.The method of PCR-based is most suitable for the Small object region of 10-100kb scope, and this enrichment method typically requires and carries out the specific design of primers in target area and PCR reaction.The subject matter of PCR amplification method has: the sequence variations in PBR territory is easily caused amplicon and loses, and structure variation is found only by the reduction of order-checking Reads.
" cyclisation ": be also molecular inversion probes (Molecularinversionprobes, MIPs), padlock-probe (Gap-fillpadlockprobes) or selector probe (Selectorprobes) are filled in gap.Range intervals at 100-500kb, the single stranded DNA ring comprising target area sequence is formed by the mode (gap is filled and coupled reaction) of a kind of high specific, and then produce the structure comprising common DNA original paper, for target region interested carries out selective amplification, relatively representational method has Haloplex (Agilent) and MIPs.The subject matter of this method has: the sequence variations in PBR territory is easily caused amplicon and loses, and sensitivity and homogeneity are relatively low, and probe cost is of a relatively high.
" hybrid capture ": the nucleic acid in sample and be anchored on solid support or the DNA/RNA probe hybridization complementary with target area being directly stored in liquid, then passes through the mode that physics catches and isolates sequence interested.Capture range is from 500kb to whole full exon group, develop the commercial hybrids method of some classics, such as SureSelect (Agilent), Nextera (Illumina), TruSeq (Illumina), SeqCap (Nimble-Gen), IonTargetSeq (LifeTechnologies), these methods are to having better capture rate and cost efficiency on a large scale with pre-designed region.The subject matter of " hybrid capture method " has: the product quality and quantity of sample is had higher requirement, fundamentally cannot be used FFPE sample, and in practical operation, TruSeq and the SureSelect method of optimization can also be used for FFPE sample.
In order to realize high flux, low cost, target region enrichment order-checking rapidly and efficiently, find out the change of the pathogenic mutation in this region, the new mutation of mendelian inheritance disease allele or exons coding information, fully develop sequence information and carry out medical diagnosis on disease and prevention for the mankind, realize personalized medical scheme and drug development, there is a need in the art for low cost, high efficiency target gene beneficiation technologies that exploitation makes new advances.
Summary of the invention
It is an object of the invention to provide a kind of target genetic region enrichment method extending based on Multiple Cycle and connecting.
A first aspect of the present invention, it is provided that the enrichment method of a kind of nucleic acid fragment, described method includes step:
(1) reaction system is provided
Described reaction system at least includes: sample to be tested, n probe groups, nucleic acid polymerase and nucleic acid ligase；
Described n >=2, comprise the first probe and the second probe respectively in each probe groups；
Described first probe and described second probe hybridize (described specific hybrid refers to complementary at least partly or complete complementary) respectively with the 3 ' of same target nucleic acid fragment ends and 5 ' end；
Described first probe can not by 5' extreme direction Exonucleolytic enzymatic degradation；
Described second probe can not by 3' extreme direction Exonucleolytic enzymatic degradation；
Described first probe includes and the Part I of target nucleic acid fragment 3' end hybridization and the Part II corresponding with subsequent PCR amplification primer sequence (the described corresponding reverse complementary sequence referring to described Part II with pcr amplification primer can specific hybrid)；
Described second probe includes the Part I with the hybridization of target nucleic acid fragment 5 ' end and the Part II with subsequent PCR amplification primer sequence specific hybrid；
When described first probe and described second probe are with same target nucleic acid fragment specific hybrid, the distance of the 3' end of described first probe and the 5' end of described second probe at least 1 nucleotide in interval；
(2) hybridization and extension connect
Described first probe and described second probe in high-temperature denatured, annealing process with the target nucleic acid fragment specific hybrid of described sample to be tested, under described nucleic acid polymerase effect, described first probe carries out DNA extension along described target nucleic acid fragment, blocked by it when extending to the 5' end of described second probe, it is thus achieved that the first probe extended DNA chain；And under the effect of described nucleic acid ligase, described first probe extended DNA chain 3' end is connected with described second probe 5' end, thus formed containing the reactant mixture connecting product；With
Optionally repeat above-mentioned " degeneration-annealing-extension-connection ", thus increasing the quantity connecting product described in described reactant mixture；
(3) digestion
Described reactant mixture to step (2), carries out digestion process with exonuclease, thus obtaining the reactant mixture through digestion, containing not digested described connection product in described reactant mixture；
(4) enrichment
With the described not digested described connection product of step (3) for template, carrying out pcr amplification, thus obtaining pcr amplification product, being the nucleic acid fragment of enrichment.
In another preference, also including undigested described connection product is purified in step (4), thus obtaining purified described connection product, and the connection product of this purification being used as template.
In another preference, after step (4), further comprise the steps of: and described pcr amplification product is fabricated to nucleic acid fragment library.
In another preference, described first probe can not by 5' extreme direction Exonucleolytic enzymatic degradation, it is possible to by 3' extreme direction Exonucleolytic enzymatic degradation.
In another preference, the 5' end of described first probe is with the blocking group preventing Exonucleolytic enzymatic degradation.
In another preference, described second probe can not by 3' extreme direction Exonucleolytic enzymatic degradation, it is possible to by 5' extreme direction Exonucleolytic enzymatic degradation.
In another preference, the 3' end of described second probe is with the blocking group preventing Exonucleolytic enzymatic degradation.
In another preference, described second probe can not by 3' extreme direction Exonucleolytic enzymatic degradation, it is possible to by 5' extreme direction Exonucleolytic enzymatic degradation.
In another preference, described nucleic acid polymerase is high-temperature heat-resistance nucleic acid polymerase, it is preferable that described nucleic acid polymerase is selected from lower group: Hemo(NEB), AMPLITAQDNAPOLYMERASE (AMPLITAQDNA polymerase), STOFFELFRAGMENT (LIFETECHNOLOGIES)；HotStartFlexDNAPolymerase(NEB)。
In another preference, described nucleic acid polymerase is the polymerase that there is no 5' to 3' exonuclease activity.
In another preference, described nucleic acid ligase is high-temperature heat-resistance nucleic acid ligase, it is preferable that described nucleic acid ligase is selected from lower group: TaqDNALigase (NEB)；AMPLIGASE(EPICENTRE)；9°NTMDNALigase(NEB)。
In another preference, the Tm value expanding described second probe of same target nucleic acid fragment is higher than the Tm value of described first probe.
In another preference, the Tm value of described second probe exceeds the Tm value 3 DEG C-10 DEG C of described first probe, it is preferable that the Tm value of described second probe exceeds the Tm value 4 DEG C-6 DEG C of described first probe, such as 5 DEG C.
In another preference, the Tm value of each first probe in each described probe groups is 59 DEG C-68 DEG C.
In another preference, the Tm value of each second probe in each described probe groups is 68 DEG C-75 DEG C.
In another preference, by carrying out the modification of anti-exonuclease at the 3' end of the 5' end of described first probe and/or described second probe in described method, with realize described first probe can not can not by 3' end Exonucleolytic enzymatic degradation by 5' end Exonucleolytic enzymatic degradation and/or described second probe.
In another preference, described modification includes but not limited to: Phosphorothioates modifies, 5-PropynepdC modifies, pdU modifies, and 2'-Fluorobases modifies, and 2'-O-methylbases modifies, 2'-5'linkedbases modifies, LNAbases modifies, and Chimericlinkage modifies, and 3'InverteddT modifies.
In another preference, the 5' end of described second probe is phosphorylated modification.
In another preference, in described step (2), amplification cycles number is 2-100 time, it is preferred to 3-80 time, more preferably 4-20 time, such as 16 times.
In another preference, in described step (3), add multiple nucleic acids excision enzyme and digest, it is preferable that be simultaneously introduced 5' extreme direction exonuclease and 3' extreme direction exonuclease.
In another preference, one or more in lower group of described exonuclease: T5Exonuclease, T7Exonuclease, LambdaExonuclease, ExonucleaseT, ExonucleaseI, ExonucleaseV, ExonucleaseIII.
In another preference, described n (the kind number of probe groups) is 2-100000, it is preferred to 3-5000, more preferably 10-500, it is most preferred that for 20-200.Preferably, the probe groups for same target (purpose) nucleic acid fragment is called one (individual) probe groups by the present invention, for instance, when n is 2, then two kinds of probe groups are respectively directed to target nucleic acid fragment two kinds different.
In another preference, in described sample, the total amount of target nucleic acid fragment is 1-2000ng, it is preferred to 200-500ng.
In another preference, extending distinguished sequence (i.e. target nucleic acid sequence) length connecting product in described step (2) is 30-5000bp, it is preferred to 100-1000bp, more preferably 150-310bp.
In another preference, with sequence label in described PCR primer, described sequence label length is 1-100bp, it is preferred to 5-10bp.The product that connects of different samples can expand by the PCR primer with difference sequence label, and the amplified production of so different samples may be mixed together, and according to this sequence label, sequencing sequence can be sorted out in follow-up sequencing data.
In another preference, the length of the Part I of the first described probe is 30-50bp (being preferably 35-45bp, more preferably 44bp), and/or the length of Part II is 18-30bp.
In another preference, the length of the Part I of the second described probe is 30-50bp (being preferably 35-45bp, more preferably 42bp), and/or the length of Part II is 21-36bp.
In another preference, the Part II of the first probe of each probe groups is identical or essentially identical.
In another preference, the Part II of the second probe of each probe groups is identical or essentially identical.
In another preference, the length of described PCR primer is 42-58bp.
In another preference, only with a kind of PCR primer pair in step (4).In another preference, described sample is the sample of nucleic acid being derived from animal, plant or microorganism, it is preferred to DNA sample or RNA reverse transcription product cDNA sample.
In another preference, described sample is for being derived from the sample of nucleic acid of animal (being preferably mammal, more preferably people), it is preferred to DNA sample or RNA reverse transcription product cDNA sample.
In another preference, described sample to be tested only comprises a kind of sample or described sample to be tested comprise the multiple detection sample (as being taken respectively from the sample of multiple patient or the sample of multiple not homologue) coming from different objects.
In another preference, in described step (4), the primer used in described pcr amplification includes forward primer and reverse primer, described forward primer comprises can with the sequence of the reverse complementary sequence specific hybrid of the described Part II sequence of described first probe, and described reverse primer comprises the sequence of the described Part II specific hybrid with described second probe.
In another preference, in described step (4), described forward primer and/or in described reverse primer containing and the compatible universal sequence of high flux chip order-checking platform.
In another preference, in described step (4), described forward primer and/or in described reverse primer containing sequence label, adopt different sequence labels for different samples.
In another preference, the described Part II sequence of described first probe is:
5’CACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3’(SEQIDNO.:1)。
In another preference, the described Part II sequence of described second probe is:
5’AGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTTCGCA3’(SEQIDNO.:2)。
In another preference, in described step (4), described forward primer sequence is:
5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACAC3’(SEQIDNO.:3)。
In another preference, in described step (4), described reverse primer sequences is:
5 ' CAAGCAGAAGACGGCATACGAGAT [X] GTGACTGGAGTTCAGACGTGTGCT3 ', wherein [X] is sequence label；Preferably, [X] length is 1bp-100bp, it is preferred to 5bp-10bp, such as 8bp.
A second aspect of the present invention, it is provided that a kind of method for nucleic acid sequencing, described method includes step: use the method described in first aspect present invention, purpose nucleic acid fragment is enriched with.
In another preference, described method for nucleic acid sequencing use high flux chip order-checking platform to through using the purpose nucleic acid fragment of the enrichment of the method described in first aspect present invention carry out unimolecule amplification order-checking or be made directly single-molecule sequencing.
In another preference, described method further comprises the steps of: and sequencing data is analyzed, and the sample of sequencing sequence is sorted out, and reads gene mutation site and/or calculates each genetic fragment copy number.
A third aspect of the present invention, it is provided that a kind of test kit, described test kit is for the enrichment of nucleic acid fragment, and described test kit includes: corresponding to one or more probe groups of sample to be tested nucleotide sequence, nucleic acid polymerase and nucleic acid ligase；
Probe groups comprises the first probe and the second probe,
Described first probe and described second probe hybridize (described specific hybrid refers to complementary at least partly or complete complementary) respectively with the 3 ' of same target nucleic acid fragment ends and 5 ' end；
Described first probe can not by 5' extreme direction Exonucleolytic enzymatic degradation；
Described second probe can not by 3' extreme direction Exonucleolytic enzymatic degradation；
Described first probe includes and the Part I of target nucleic acid fragment 3' end hybridization and the Part II corresponding with subsequent PCR amplification primer sequence (the described corresponding reverse complementary sequence referring to described Part II with pcr amplification primer can specific hybrid)；
Described second probe includes and the Part I of target nucleic acid fragment 5 ' end hybridization and the Part II corresponding with subsequent PCR amplification primer sequence (described corresponding refer to that described Part II and pcr amplification primer can specific hybrids)；
When described first probe and described second probe are with same target nucleic acid fragment specific hybrid, the distance of the 3' end of described first probe and the 5' end of described second probe at least 1 nucleotide in interval.
In another preference, described test kit also includes PCR primer, described PCR primer includes forward primer and reverse primer, described forward primer comprises can with the sequence of the reverse complementary sequence specific hybrid of the described Part II of described first probe, and described reverse primer comprises the sequence of the described Part II specific hybrid with described second probe.
In another preference, described forward primer and/or in described reverse primer containing and the compatible universal sequence of high flux chip order-checking platform.
In another preference, described forward primer and/or in described reverse primer containing sequence label, adopt different sequence labels for different samples.
In another preference, described test kit also includes the PCR reagent of routine.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus constituting new or preferred technical scheme.As space is limited, tired no longer one by one state at this.
Accompanying drawing explanation
Fig. 1 shows the operating process of invention.
Fig. 2 shows 3 clinical samples genes of interest fragment copy number detected values.
Detailed description of the invention
The present inventor is by extensive and deep research, obtain a kind of target genetic region enrichment new technique extending based on Multiple Cycle and connecting, test result indicate that, this technology can realize the genetic fragment enrichment of multipurpose gene, and genes of interest segments can be tens of to tens thousand of.The enriched product of multipurpose genetic fragment can pass through modification and purification quantitatively afterwards for the sequencing analysis of various high flux chips order-checking platform such as secondary order-checking platform.
Specifically, the present invention connects the ultimate principle heat-staple archaeal dna polymerase of employing and DNA ligase based on extending, carry out multi cycle and extend coupled reaction, adopt extension primer and the linking probe of anti-exonuclease enzyme modification, non-specific PCR primer and residual primers probe can be removed by the digestion of multiple nucleic acids excision enzyme, then adopt the universal primer matched with secondary order-checking platform to carry out amplification purification and obtain sequencing library.On this basis, the present invention is completed.The method of the present invention is special and efficient to catching of target sequence, and the sequencing data of its amplified production can be also used for target gene fragment copy number analysis, thus detecting while realizing genes of interest fragment point mutation and copy number.
In one preferred embodiment of the invention, the step of described method following (as shown in Figure 1):
nullA () designs two specificity DNA probing needles for target nucleic acid fragment，One is that 5 ' ends extend primed probe，Another 3 ' end extends retardance probe，5 ' end probe first half sequences are the universal sequences that subsequent PCR amplification primer is consistent，And latter half is and the distinguished sequence of target nucleic acid fragment hybridization，5 ' ends of 3 ' end probes carry out phosphorylation modification，First half is and the distinguished sequence of target nucleic acid fragment hybridization，Latter half is the universal sequence that subsequent PCR amplification primer is consistent，5 ' the several bases of end of 5 ' end probes carry out protection and modify from Exonucleolytic enzymatic degradation，3 ' the several bases of end of 3 ' end probes carry out protection and modify from Exonucleolytic enzymatic degradation，Several base distances are had between the two probe，Probe with hybridize with template DNA after first extend under the polymerase effect not having 5 '-> 3 ' 5 prime excision enzyme activities two probe gaps filled，Then it is attached under ligase effect；In order to increase the amount connecting product, it is possible to use high-temperature heat-resistance polymerase and high-temperature heat-resistance ligase carry out denaturation renaturation-extension connection and repeatedly circulate；
B () coupled reaction product combines common digestion process with various exonucleases, purification after the strand of all disconnected products or double-stranded DNA being removed；
C () utilizes the PCR primer pair that check order with subsequent high pass amount chip platform amplimer or sequencing primer match to be connected product a pair to carry out amplification and obtain the applicable subsequent high pass amount chip being enriched multiple genes of interest fragment and check order the sequencing library of platform.Under normal circumstances, PCR primer also has the hop count the sequence label to dozens of bases longs, the product that connects of different samples can expand by the PCR primer with difference sequence label, the amplified production of so different samples may be mixed together, and can be referred in different sample by sequencing sequence according to this sequence label in follow-up sequencing data；
D () linking probe amplified production utilization high flux chip of future generation order-checking platform carries out unimolecule amplification order-checking or direct single-molecule sequencing；
E sequencing data is analyzed by (), the sample realizing sequencing sequence is sorted out, gene mutation site reads and each genetic fragment copy number calculates: be first grouped on corresponding sample according to sequence label by the sequence that order-checking obtains, then utilize corresponding software that each sequence carries out with reference gene group sequence mate and read diversity sequence difference and obtain mutational site, add up the sequencing sequence number of each connection product, by referring to after the correction of genetic fragment again with the copy number of this corrected value this genetic fragment of comparing calculation of normal sample.
The modification of the anti-exonuclease of the present invention includes but not limited to Types Below: Phosphorothioates, 5-PropynepdC, pdU, 2'-Fluorobases, 2'-O-methylbases, 2'-5'linkedbases, LNAbases, Chimericlinkage, 3'InverteddT.
The exonuclease of the present invention includes but not limited to Types Below: T5Exonuclease, T7Exonuclease, LambdaExonuclease, ExonucleaseT, ExonucleaseI, ExonucleaseV, ExonucleaseIII.
The present invention has a major advantage in that:
(1) method of the present invention can realize the enrichment of multipurpose genetic fragment, and genetic fragment number can be tens of to thousands of, even tens thousand of.
(2) this multipurpose genetic fragment enrichment method is simple to operate quickly, it is possible to realize the purpose fragment enrichment of hundreds of samples within a few hours.
(3) relatively conventional extension interconnection technique, this technology adopts high temperature conjunction enzyme and polymerase, and simultaneously completes 1 reaction system, it is possible to repeatedly circulate, it is achieved the efficiently concentrating of low concentration DNA profiling multipurpose genetic fragment.
(4) by introducing anti-exonuclease enzyme modification at extension primer 5 ' end and retardance probe 3 ' end, extension connection product is carried out digestion and can remove non-specific extension products and residual primers, probe and template nucleic acid by the mixture utilizing various exonuclease, promotes the content of following amplification product specific fragment.
(5) in the product being enriched with by this technology there is certain corresponding relation with the relative scale of these fragments of primary template in the relative scale of different fragments content, therefore, the sequencing data of these products can also by referring to the copy number information obtaining target fragment after the dual correction of fragment and sample for reference except can providing point mutation information.
Below in conjunction with specific embodiment, the further detailed old present invention.Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted detailed conditions in the following example, generally conventionally condition such as Sambrook et al., molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to manufacturer it is proposed that condition.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.Experiment material used in following example and reagent all can obtain from commercially available channel.
Embodiment 1
Each exon for MVK, MVD, PMVK, FDPS4 gene devises 42 pairs of probes, and distinguished sequence amplification length is 183bp-280bp, devises 8 pairs of probes also for 8 reference gene fragments simultaneously, and distinguished sequence amplification length is 185bp-283bp.Utilize these probes to adopting the technology of the present invention that genes of interest fragment and reference gene fragment are expanded in 1 reaction system simultaneously.3 clinical samples and 1 normal person's sample adopt the universal primer pair containing different sequence labels when extending the pcr amplification after connecting, the amplified production of different samples first mixes, purification quantitatively adopts the secondary sequenator of MiSeq of illumina company of the U.S. to check order afterwards, sequencing data first sorts according to different sequence labels, the sequencing data of each sample utilizes Burrows-WheelerAligner (BWA) software to carry out carrying out matching with Radix Ginseng photograph genome then carrying out sequencing data statistics, utilize the copy number that this statistical data carries out genes of interest fragment to estimate simultaneously.
(1) specific experiment step
1, probe design
According to primer3 primer-design software
(http://bioinfo.ut.ee/primer3-0.4.0/primer3/) ultimate principle, for MVK, MVD, all exons of PMVK, FDPS4 gene devise 42 pairs of probes, and distinguished sequence amplification length is 183bp-280bp, devising 8 pairs of probes also for 8 reference gene fragments, distinguished sequence amplification length is 185bp-283bp simultaneously.null5 ' extension probes (the first probe) are held universal sequence (Part II) to form plus 3 ' terminal specific sequences (Part I) by 5 '，Its 5 ' end universal sequence starts the phosphide key thioester bond between 4 bases and replaces (Phosphorothioates modification)，5 ' end universal sequences are 5 ' CACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3 ' (SEQIDNO.:1),3 ' retardances probe (the second probe) are held universal sequence (Part II) to form by 5 ' terminal specific sequences (Part I) plus 3 ',Its 5 ' end carries out phosphorylation modification，And 3 ' hold the phosphide key thioester bond between last 4 bases to replace，3 ' end universal sequences are 5 ' AGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTTCGCA3 ' (SEQIDNO.:2).The Tm value of 5 ' extension probes distinguished sequences is 59 DEG C-68 DEG C, and the Tm value of 3 ' retardance probe distinguished sequences is 68 DEG C-75 DEG C, the Tm value generally bigger than 5 ' extension probes more than 5 DEG C of 3 ' retardance probes of same amplified fragments.Rich segment and probe distinguished sequence information are in Table 1.
2, denaturation renaturation-extension connects repeatedly circular response
1) 20 μ l reaction systems are configured, wherein containing 5mM (NH4)2SO4,10mMKCl2,5.0mMMgCl2, 10mMNAD, 6%Glycerol, 60mMTricine (pH8.7), 0.2mMdNTP mixed liquor, 0.001 μM of each 5 ' extension probes, 0.0015 μM of each 3 ' retardance probe, 0.3 μ lHemoKlenTaqTM(NEB), 40UTaqDNAligase (NEB) and 200ng genomic DNA
2) reaction system mixed liquor runs by following PCR program: 98 DEG C of 2min；(95℃20s,60℃2hr)×6；4 DEG C of insulations
3, extend connection product exonuclease digestion and process
Above-mentioned reaction adds 1 μ lLambdaExonuclease (5U/ μ l, NEB), 1 μ lExonucleaseI (5U/ μ l, NEB) and 0.5 μ lExonucleaseIII (100U/ μ l, NEB), then 37 DEG C of 2h；75℃15min.
Enzymic digestion product PCR purification kit (QIAGEN) carried out column purification
4, connect product pcr amplification
1) pcr amplification primer pair is a forward universal primer
(5 ' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACAC3 ', SEQIDNO.:3) and sample specific reverse primer (5 ' CAAGCAGAAGACGGCATACGAGAT [n1n2n3n4n5n6n7n8] GTGACTGGAGTTCAGACGTGTGCT3 ', SEQIDNO.:104), n here1n2n3n4n5n6n7n8For sequence label, the sequence label that 4 samples are corresponding is TGGAAGGA, CGCCTTCA, TAGAAATC and CATTCTGC.
2) PCR reaction system is 20 μ l, Qi ZhonghanReactionBuffer(NEB),2.5mMMgCl2, 0.3mMdNTP mixed liquor, 0.3 μM of every pair of primer, 0.4UQ5DNA polymerase (NEB) and the above-mentioned extension of 10 μ l connect purified product.
3) reaction system mixed liquor runs by following PCR program: 98 DEG C of 30s；(98℃10s,65℃30s,72℃1min)x30；72℃5min；4 DEG C of insulations.
5, after being mixed by the pcr amplification product of above-mentioned 4 samples, isolate the fragment between 200bp-500bp with rubber tapping after 2% agarose gel electrophoresis, it is quantitative that fragment products adopts RT-qPCR to carry out molecular number.
6, on the library after quantitatively, the secondary sequenator of MiSeq of illumina company of the U.S. checks order.
7, data analysis: sequencing data is carried out sorting according to different sequence labels and obtains the sequencing data of each sample；Sequencing data Burrows-WheelerAligner (BWA) program carries out matching with human genome canonical sequence, adds up each sample always order-checking amount, the order-checking degree of depth of each purpose and reference fragment and the bioaccumulation efficiency of each sample；The order-checking degree of depth of each purpose fragment of clinical samples is respectively divided by 8 order-checking degree of depth with reference to fragment and obtains 8 ratios, each ratio is respectively divided by the corresponding ratio of normal sample again and is multiplied by 2 again, thus obtain 8 numerical value, take its median and be this sample copy number detected value in target fragment.
(2) experimental data
1) the sequencing data statistics of 4 sample 50 fragments
The order-checking degree of depth of each fragment of 3 patient's sample (P1, P2, P3) and 1 normal sample (C1) is in Table 2, and the statistical result of sequencing data is in Table 3.From statistical data, 4 sample standard deviations realize effective enrichment of 50 genetic fragments: its bioaccumulation efficiency all reaches more than 80%, and average effective reading 400 × more than, the order-checking degree of depth arrives 2 × above fragment respectively 49,49,49,48, and 10 × above fragment is 47.
2) sample copy number detected value
Utilize the copy number that order-checking depth data carries out each fragment to calculate, 4 fragment eMVKE01, eMVDE02, eFDPSE01b and eFDPSE11 due to the order-checking degree of depth generally less than 20 × therefore reject when carrying out copy number and calculating.Three patient's sample (P1, P2 and P3) residue 38 genetic fragments copy number detected value see Fig. 2, as can be seen from the figure P1 has at least lacked exon 2 to exon 5 section on mvk gene, and P2 and P3 has lacked exons 1 respectively to exon 3 section and exon 5 to exon 8 section on FDPS gene.Through the checking of RT-PCR experiment, these deletion mutation results are accurately.
Table 1 is enriched with genetic fragment and probe distinguished sequence information thereof
A, the mRNA respectively MVK (NM_000431.2), PMVK (NM_006556.3), MVD (NM_002461.1) and FDPS (NM_002004.2) of the statistics correspondence of this gene location.
The order-checking depth data of table 50 genetic fragments of 24 samples
The sequencing data statistics of table 50 genetic fragments of 34 samples
Discuss:
The present invention is that the method carrying out pcr amplification to extend connection product for template after a kind of utilization repeatedly circulation extends coupled reaction carries out the simple and quick enrichment of genes of interest fragment, what reaction system adopted is that thermally-stabilised ligase and polymerase complete denaturation renaturation-extensions multiple circulations of connection to realize an individual system, and period (n) can be 2-50.
5 ' ends of 5 ' extension probes (the first probe) have carried out nuclease-resistant modification; 3 ' ends of 3 ' retardances probe (the second probe) have been also carried out nuclease-resistant modification so that the sequence two ends only accurately extending the product connected all obtain nuclease-resistant protection.Extend the connection multiple exonuclease of product and carry out digestion removal non-specific amplification product, non-specific extension products, residual probe and genomic DNA.Extension connects the product utilization universal primer containing different sequence labels and carries out the library of the applicable next generation's order-checking platform of amplification foundation, utilizes the library of the universal primer structure containing different sequence labels may be mixed together simultaneously and carries out next generation's order-checking.This technology is applicable to the Enrichment Amplification of multiple gene fragment, and the genetic fragment number expanded together can be tens of, hundreds of or thousands of, even tens thousand of, also can comprise some reference gene fragments except genes of interest fragment, and reference gene segment number is to be 0-999.
This technology is by the use of hot resistant DNA polymerase and heat-resistant dna ligase, and probe is carried out nuclease-resistant modification, greatly improves the bioaccumulation efficiency of purpose fragment.
The sequencing data of this technology Enrichment Amplification product can pass through to analyze the copy number obtaining genes of interest fragment, analysis method is add up each purpose and the order-checking degree of depth with reference to fragment, the order-checking degree of depth of each purpose fragment of clinical samples is respectively divided by each order-checking degree of depth m ratio of acquisition with reference to fragment, and (m is reference gene fragment, reference gene can be any genetic fragment beyond this fragment), the median ratio of corresponding ratio or all samples that each ratio is respectively divided by normal sample again is multiplied by normal sample again at the copy number of this purpose fragment or overwhelming majority sample copy number in this fragment, thus obtain m numerical value, take its median as this sample copy number detected value in target fragment.
The enrichment method of the nucleic acid fragment of the present invention, compares multiplexed PCR amplification and can realize the amplification (～10-10 of more multiplicity in single reaction3Plex), use high temperature conjunction enzyme and polymerase to carry out the repeatedly circulation that denaturation renaturation-extension connects, it is ensured that connecting the concordance extending efficiency, fidelity is better, and low concentration DNA sample is had better adaptability simultaneously.And compare and cyclisation catching method needs to use longer inversion probes (～70-300nt), the probe shorter (～50-60nt) that the enrichment method of the nucleic acid fragment of the present invention uses, and introduce anti-exonuclease enzyme modification at extension primer 5' end and retardance probe 3' end, not only it is substantially reduced synthesis difficulty and the cost of probe, avoid the phenomenon of hybridization in the long easy generation ring of probe simultaneously, and the digestions of amplified production is also improved the simple specificity relying on clip size to select to be purified recovery.The selection of rich region is mainly had greater flexibility by the advantage that the enrichment method relative hybridization of the nucleic acid fragment of the present invention is caught in moderate fluxes level, it is independent of species and genome area distribution, compare its specificity of probe hybridization better simultaneously, complex sequence there is preferred solution, it is possible to more targeted arbitrary target region is carried out is enriched with order-checking.
The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (10)
1. the enrichment method of a nucleic acid fragment, it is characterised in that described method includes step:
(1) reaction system is provided
Described reaction system at least includes: sample to be tested, n probe groups, nucleic acid polymerase and nucleic acid ligase；
Described n >=2, comprise the first probe and the second probe respectively in each probe groups；
Described first probe and described second probe hybridize (described specific hybrid refers to complementary at least partly or complete complementary) respectively with the 3 ' of same target nucleic acid fragment ends and 5 ' end；
Described first probe can not by 5' extreme direction Exonucleolytic enzymatic degradation；
Described second probe can not by 3' extreme direction Exonucleolytic enzymatic degradation；
Described first probe includes and the Part I of target nucleic acid fragment 3' end hybridization and the Part II corresponding with subsequent PCR amplification primer sequence (the described corresponding reverse complementary sequence referring to described Part II with pcr amplification primer can specific hybrid)；
Described second probe includes the Part I with the hybridization of target nucleic acid fragment 5 ' end and the Part II with subsequent PCR amplification primer sequence specific hybrid；
When described first probe and described second probe are with same target nucleic acid fragment specific hybrid, the distance of the 3' end of described first probe and the 5' end of described second probe at least 1 nucleotide in interval；
(2) hybridization and extension connect
Described first probe and described second probe in high-temperature denatured, annealing process with the target nucleic acid fragment specific hybrid of described sample to be tested, under described nucleic acid polymerase effect, described first probe carries out DNA extension along described target nucleic acid fragment, blocked by it when extending to the 5' end of described second probe, it is thus achieved that the first probe extended DNA chain；And under the effect of described nucleic acid ligase, described first probe extended DNA chain 3' end is connected with described second probe 5' end, thus formed containing the reactant mixture connecting product；With
Optionally repeat above-mentioned " degeneration-annealing-extension-connection ", thus increasing the quantity connecting product described in described reactant mixture；
(3) digestion
Described reactant mixture to step (2), carries out digestion process with exonuclease, thus obtaining the reactant mixture through digestion, containing not digested described connection product in described reactant mixture；
(4) enrichment
With the described not digested described connection product of step (3) for template, carrying out pcr amplification, thus obtaining pcr amplification product, being the nucleic acid fragment of enrichment.
2. the method for claim 1, it is characterised in that described first probe can not by 5' extreme direction Exonucleolytic enzymatic degradation, it is possible to by 3' extreme direction Exonucleolytic enzymatic degradation；And/or
Described second probe can not by 3' extreme direction Exonucleolytic enzymatic degradation, it is possible to by 5' extreme direction Exonucleolytic enzymatic degradation.
3. the method for claim 1, it is characterised in that described nucleic acid polymerase is high-temperature heat-resistance nucleic acid polymerase, it is preferable that described nucleic acid polymerase is selected from lower group: Hemo(NEB), AMPLITAQDNAPOLYMERASE (AMPLITAQDNA polymerase), STOFFELFRAGMENT (LIFETECHNOLOGIES)；HotStartFlexDNAPolymerase(NEB)。
4. the method for claim 1, it is characterised in that for the Tm value of described second probe of same target nucleic acid fragment higher than the Tm value of described first probe.
5. the method for claim 1, it is characterised in that described n (the kind number of probe groups) is 2-100000, it is preferred to 3-5000, more preferably 10-500, it is most preferred that for 20-200.
6. the method for claim 1, it is characterised in that described nucleic acid polymerase is high-temperature heat-resistance nucleic acid polymerase；And/or described nucleic acid ligase is high-temperature heat-resistance nucleic acid ligase.
7. the method for claim 1, it is characterised in that the 5' end of described second probe is phosphorylated modification.
8. the method for claim 1, it is characterised in that with sequence label in described PCR primer, described sequence label length is 1-100bp, it is preferred to 5-10bp；
Preferably, in described step (4), the primer used in described pcr amplification includes forward primer and reverse primer, described forward primer comprises can with the sequence of the reverse complementary sequence specific hybrid of the described Part II sequence of described first probe, and described reverse primer comprises the sequence of the described Part II specific hybrid with described second probe.
9. a method for nucleic acid sequencing, it is characterised in that described method includes step: use the method described in any one of claim 1-8, purpose nucleic acid fragment is enriched with.
10. a test kit, it is characterised in that described test kit is for the enrichment of nucleic acid fragment, and described test kit includes: corresponding to one or more probe groups of sample to be tested nucleotide sequence, nucleic acid polymerase and nucleic acid ligase；
Probe groups comprises the first probe and the second probe,
Described first probe and described second probe hybridize (described specific hybrid refers to complementary at least partly or complete complementary) respectively with the 3 ' of same target nucleic acid fragment ends and 5 ' end；
Described first probe can not by 5' extreme direction Exonucleolytic enzymatic degradation；
Described second probe can not by 3' extreme direction Exonucleolytic enzymatic degradation；
Described first probe includes and the Part I of target nucleic acid fragment 3' end hybridization and the Part II corresponding with subsequent PCR amplification primer sequence (the described corresponding reverse complementary sequence referring to described Part II with pcr amplification primer can specific hybrid)；
Described second probe includes and the Part I of target nucleic acid fragment 5 ' end hybridization and the Part II corresponding with subsequent PCR amplification primer sequence (described corresponding refer to that described Part II and pcr amplification primer can specific hybrids)；
When described first probe and described second probe are with same target nucleic acid fragment specific hybrid, the distance of the 3' end of described first probe and the 5' end of described second probe at least 1 nucleotide in interval.
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| WO2019144582A1 (en) * | 2018-01-26 | 2019-08-01 | 厦门艾德生物医药科技股份有限公司 | Probe and method for high-throughput sequencing targeted capture target region used for detecting gene mutations as well as known and unknown gene fusion types |
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