CN107012221A - Enrichment system and its application based on blocker - Google Patents

Abstract

The invention discloses a kind of system of PCR-based and a small number of allele of enrichment and the method for mutation, it is included under conditions of one or more blocker is present and expands target region with a forward primer and a reverse primer.This method can detect nucleic acid mutation in high sensitivity, minimum detection limit (LOD) is up to 0.01% 0.001% in some cases, this method can be enriched with or detect various target nucleic acids interested, in terms of the alleviation or recurrence that can be used for after cancer early detection, operation or the chemicotherapy assessment of residual disease, staging and the analysis of molecules for prognosis, monitoring treatment result and cancer.

Description

Enrichment system and its application based on blocker

Technical field

The application is related to detection in Gene Mutation technical field, in particular to system and minority of a kind of PCR-based etc. Position gene and the enrichment method of mutation.

Background technology

Detect nucleic acid mutation or make a variation all extremely important to the various situations including disease detection and prognosis.It is related to and faces The protrusion concern of bed and diagnostic application is to detect clinically very important low-level mutation and a small number of equipotential bases Cause.At many aspects, it is extremely important to distinguish mutation, particularly true for following situations：From tissue biopsy and body fluid such as blood Slurry or serum carry out cancer early detection；To the assessment of residual after postoperative or radiotherapy, chemotherapy；Staging and it is used for The analysis of molecules of prognosis uses different therapeutic schemes according to different patients；To treatment results, cancer remission and recur into Row monitoring etc..The effective detection of the somatic mutation related to cancer depends greatly on selected technology and side Method.

Detection and identification oncogenic mutation and tumor suppressor gene mutation are mainly analyzed before cancer or cancerous tissue, phlegm, Urine, excrement and the circulation extracellular dna being discharged into blood.Sample generally includes wild type and mutant DNA, wild type DNA Often exceed the amount of the mutant DNA contribution.In many cases, wild type DNA substantially exceeds mutant DNA, makes detection and mirror The a small number of allele for determining extremely low concentration are extremely difficult.Thus need badly at this stage for be enriched with a small number of allele and mutation be System and method.Based on the necessity for the system and method for setting up the enrichment for being used for a small number of allele and mutation, the present invention is therefore .

The content of the invention

The application aims to provide a kind of system and method for being enriched with a small number of allele and mutation, to solve in the prior art The problem of.

To achieve these goals, according to the one side of the application there is provided a kind of nucleic acid molecules composition, for richness Collect the allelic mutation in the target nucleic acid sequence of target nucleic acid, above-mentioned a small number of allele and abrupt climatic change and mirror can be met The need for fixed minority allele and mutation.

In one aspect, the invention provides a kind of nucleic acid molecules composition, the target nucleic acid for being enriched in target nucleic acid The allelic mutation of sequence, including：(a) being capable of the forward primer and reverse primer and (b) of amplifying target nucleic acid sequence includes the First blocker of one section of sequence, this sequence meets following requirements (i) and the wild-type allele of target nucleic acid sequence matches Or complementary and (ii) can be extended by archaeal dna polymerase.This group of nucleic acid molecules may further include the second blocker, this blocker Match containing the complementary genes with wild-type allele or complementary second segment sequence.

In some embodiments, the first blocker or the second blocker not with any forward primer or reverse primer It is overlapping.In some other embodiment, the first blocker or the second blocker can include the nucleic acid of one or more modifications Or key.Can also have in end modified 3' nucleic acid or key in the first blocker or the second blocker.The nucleosides of described modification Acid or key can include-O- methyl the nucleic acid of PNA, LNA, 2 ', 2 '-O- alkyl nucleic acid, 2 '-fluorescence labeling nucleic acid, thiophosphate Key and their any combination.

In certain embodiments, target nucleic acid sequence covering coding EGFR T790M (EGFR Exon 20), EGFR L858R (EGFR exon 21)、EGFR exon 19del、BRAF V600E、BRAF V600K、BRAF V600D、BRAF V600G、 The region for any one mutation listed in BRAF V600A, BRAF V600R, or table 8 and 9.

Positive or reverse primer length can be between 10-50 nucleotides, than such as from about 15-30, about 16-28, about 17-26, about 18-24, or about 20-22 nucleotides.First blocker or the second blocker length can be approximately 5-100 Nucleotides, for example, about 10-50, about 15-30, about 16-28, about 17-26, about 18-24, or about 20-22 nucleotides.Excellent In the embodiment of choosing, in general, blocker is shorter than primer.

Present invention also offers including nucleic acid molecules composition as described above, one or more examinations for amplified reaction The kit of agent.The kit can include one or more selected from buffer, archaeal dna polymerase, RNase inhibitor, extension core The reagent of thuja acid (extension nucleotides) and probe composition.

Present invention also offers a kind of method for being used to be enriched with the allelic mutation of target nucleic acid sequence.This method includes carrying For a kind of nucleic acid molecules composition as described above；With forward primer and instead under conditions of the presence of the first or second blocker To primer amplifying target nucleic acid sequence.

The enrichment method can be used in detection target nucleic acid sequence whether there is allelic mutation.The method is included as above Described enrichment allelic mutation is used to prepare amplified production；It whether there is allelic mutation with detection amplified production. The detecting step can be carried out by gene sequencing or other technologies as known in the art.Target nucleic acid sequence can cover volume Code EGFR T790M, EGFR L858R, EGFR exon 19del, BRAF V600E, BRAF V600K, BRAF V600D, BRAF The region for any one mutation listed in V600G, BRAF V600A, BRAF V600R, or table 8 and 9.

Wherein table 8 is：

Table 9 is：

Described enrichment method can be used for assess with oncological patients or suspect with cancer (for example, lung cancer and Melanoma) subject.Such evaluation method, including：First biological sample is obtained from the subject；Then examined Survey to determine that one or more allelic mutations whether there is in above-mentioned biological sample.In an example, cancer is lung Gland cancer, such as non-small cell lung cancer.The biological sample can be serum, blood plasma, whole blood, saliva or phlegm.This method can also include Determined according to the presence of one or more above-mentioned allelic mutation or recommend a treatment course.This method can be with Including：In the presence of one or more of allelic mutations, the step of being administered in treatment course.

The details of one or more embodiments of the invention is illustrated in following embodiment description.Further feature, purpose And the advantage of the present invention will be shown by specification and/or claims.

The detailed description of the present invention

The present invention is based on or is based at least partially on one and is found surprisingly that, i.e., be mutated with specific nucleotide (such as wild Type thumping become) matching or complementation oligonucleotides blocker (the first blocker and/or the second blocker) can block or press down The amplification of specific nucleic acid mutation is made, so as to allow the enrichment of other mutation (for example, mutation variants).Therefore the present invention can be with A kind of method that system for being enriched with these jump reactions and the nucleic acid mutation for the target area of detection whether there is is provided.

Reaction system

In some embodiments, enrichment reaction system of the invention includes primer, and blocker and PCR amplifications are necessary Composition.As shown in Fig. 2A-C, system of the invention has：(i) the first blocker, this blocker can combine or hybridize to In same chain or sequence that forward primer is combined；(ii) second blocker and reverse primer can be combined or hybridized to conversely In chain and/or complementary series.Primer pair (i.e. one forward primer and a reverse primer), which is used to expand, contains hot spot mutation Region, and blocker is used for amplification (such as a kind of abundant allelic mutation such as wild type equipotential base for preventing nucleic acid mutation Cause).Blocker is the oligonucleotides complementary with nucleic acid mutation (such as wild-type allele), and its 3' end is designed to feeling emerging The mutation of interest is matched completely.For example, it anneals with wild-type allele completely, it can be extended by archaeal dna polymerase.Melting temperature with The length height correlation of oligonucleotides.By extending the length of blocker, blocker can bear higher reaction temperature, and keep And the combination of wild-type allele.On the other hand, under initial low reaction temperatures, blocker can also be with mutation equipotential base Because of annealing；However, because the mutating alkali yl of mutation allele is not matched with the 3' ends of blocker, extension can not be carried out.One Denier temperature is raised, and this causes blocker to be dissociated from mutant allele.Therefore, blocker is closely combined and held with wild type Easily it is denatured with mutation, primer is able to expand region interested in the reaction.Because blocker is annealed with wild type, primer extend Blockade zone can not be continued through, therefore mutation allele is by preferential amplification.

For example as shown in Figure 2 A, blocker and wild-type allele annealing, the blocker of extension prevent outer primer PCR is expanded.In Fig. 2 B, each blocker has mispairing so the combination to mutation allele can not be kept at 3 ' ends, institute With can be by external primer amplification.In fig. 2 c, non-specific extension only result in the allele of whole specific template at this The amplification failure of circulation, as a result non-false positive PCR primer is formed.

System disclosed herein is better than ApoE gene (AS-PCR), also referred to as amplification retardance mutantion line System (amplification mutation refractory system, ARMS, with reference to Newton C, Graham A, Heptinstall L,et al.Analysis of any point mutation in DNA.The amplification refractory mutation system(ARMS).Nucleic acids….1989；17(7):2503-2516.http:// Nar.oxfordjournals.org/content/17/7/2503.short.Accessed March 18,2015), this is one Plant and find correct technology by attempting to be enriched with hot spot mutation.In AS-PCR methods, the 3' ends of primer are designed to Mutation interested is matched with completely, and allows specific mutation amplification (Figure 1A and 1B).Such as Qiagen companies The Cobas EGFR systems of Therascreen EGFR and Roche use this technology.However, the non-spy of allele-specific The inherent defect of different in nature extension primer (Fig. 1 C) reduces sensitivity, causes the area between rare somatic mutation and wild type It is unreliable to divide.Therascreen LOD is 0.5%-7.02% (with reference to therascreen EGFR RGQ PCR Kit.https://www.qiagen.com/us/products/catalog/assay-technologies/complete- Assay-kits/personaliz ed-healthcare/therascreen-egfr-rgq-pcr-kit-na), Cobas's LOD is 5% (referenceEGFR Mutation Test.http://molecular.roche.com/assays/ Pages/cobasEGFRMutationTest.aspx)。

Blocker

As described above, blocker (herein referred to as " blocking agent ") and the desired specific nucleic acid suppressed Complementation, such as substantial amounts of allelic mutation (such as wild-type allele).Such blocker can be designed to short single-stranded Oligomer, this oligomer is no more than 100 nucleotides, more preferably 50 nucleotides or less, still more preferably 30 cores Thuja acid is smaller, most preferably 20 nucleotides or smaller, and minimum lower limit is about 5 nucleotides.

Blocker can be modified in some cases with certain methods as known in the art to protect it from by 3' or 5 ' 5 prime excision enzyme activities are degraded.Blocker can carry out one or more modifications to prevent the drop of 3' or 5' exonuclease activities Solution.Such modification includes but is not limited to the modification of 2'-O- methyl ribonucleotides, phosphorothioate backbone modification, two thio phosphorus Acid esters backbone modifications, phosphate backbone modification, methyl acid phosphate backbone modification, the modification of 3' terminal phosphateizations and the substitution of 3' ends alkyl. In some embodiments, because there is one or more modification without by 3 ' and/or 5' exonuclease activities in blocker Effect.

The 3' ends of blocker are designed to be matched with the mutation of specific nucleic acid interested completely.As shown in following example, One blocker is annealed with wild-type allele completely, can be extended by archaeal dna polymerase.

The melting temperature (Tm) of blocker and its length height correlation.The Tm values scope of blocker can be from 40 DEG C to 70 DEG C, such as 40 DEG C to 70 DEG C, 41 DEG C to 69 DEG C, 42 DEG C to 68 DEG C, 43 DEG C to 67 DEG C, 44 DEG C to 66 DEG C, or about 53 DEG C to about 56 DEG C, or any scope therebetween.In other embodiments, the Tm values of blocker can compare under the conditions of PCR cycle in amplification Annealing/elongating temperature is high about 3 DEG C to 6 DEG C.

In certain embodiments, blocker is not degraded during PCR is expanded.According to the present invention, blocker can be can Extension is inextensible.In certain embodiments, blocker can include an inextensible blocking unit in its 3' end Point.In certain embodiments, blocker can also be in its 3' end, and 5' ends and/or optional position therebetween include it Its part (including but is not limited to extra inextensible blocking part, quencher moieties, fluorescing fractions etc.).In other embodiment In, blocker may extend away, and not include any inextensible blocking part in its 3' end.In this case, block Thing extends during PCR.By extending the length of blocker, blocker bears higher reaction temperature, and keep with it is wild Type allele is matched.

Primer

According to the present invention, a forward primer and/or a reverse primer are designed to mutual with various suitable positions Mend (complete or partial), these positions correspond to the mutation of one or more nucleic acid interested.For example, in some cases and mesh When marking area hybridization, 3 ' ends of forward primer or reverse primer can distance objective region one or more nucleic acid mutation 0, 5,10,15,20,25,30,35,40,45,50,55,60,80,100,250,500,1000,2000 or more nucleotides. In some embodiments, when being hybridized to target region, 3 ' ends of forward primer or reverse primer can be with one in hybridising region Or multiple nucleic acid mutations are remote at a distance of less than 30 nucleotides.Primer can be about 10-50bp oligonucleotides, e.g., from about 15- 30, about 16-28, about 17-26, in about 18-24 or about 20-22, or any scope therebetween.

In some cases, primer and blocker may all or part of targets of overlapping and competitive hybridization forward or backwards Region.Such as primer and blocker may have 0,5,10,15, or more nucleotides it is overlapping.In certain embodiments, primer and Blocker is not overlapping or the not all or part of target area of competitive hybridization.

Primer discussed above and/or blocker may include the base of one or more modifications or different from naturally occurring The base of base (i.e. adenine, cytimidine, guanine, thymine and uracil).In certain embodiments, the alkali through modification Base is remained able to effectively with containing adenine, guanine, cytimidine, the nucleic acid unit hybridization of uracil or thymidine. In some embodiments, the base through modification can increase matching unmatched target nucleic acid sequence between Tm values difference and/or Unmatched joint efficiency is reduced, therefore not only increases the specificity of detection, selectivity is also improved.

Modified base is to make it different from naturally occurring base by adding or removing one or more functions group, Difference is embodied in the connection of heterocyclic ring structure (that is, carbon is replaced by hetero atom, or opposite replacement) and/or one or more bases Arm configuration.In certain embodiments, naturally occurring base, all tautomerism shapes of base and base analogue through modification Formula can also be included in the primer and blocker of the present invention.

Some examples of modified base may include, for example, the 7- deazapurines of general base analog analog and its derivative Thing and pyrazolopyrimidine and their derivative (are incited somebody to action see, for example, WO90/14353 and US20100285478, and by quoting The entire content of its these patent is incorporated herein).The example of such base analogue includes, for example, guanine is similar Thing 6- amino -1H- pyrazolos [3,4-d] pyrimidine -4 (5H) -one (PPG), Adenine derivatives 4- amino -1H- pyrazolos [3,4- D] pyrimidine (PPA) and (the 7H)-diketone (PPX) of xanthine analog 1H- pyrazolos [4,4-d] pyrimidine -4 (5H) -6.These bases Analog, when there is oligonucleotides in some embodiments of the invention, can strengthen the differentiation for hybridizing and improving mispairing.

In addition, in certain embodiments, sugar or sugar analogue through modification can be in one or more oligonucleotides Exist in nucleotides subunit.It is sugar-modified to include, but not limited on the 2' that substituent is attached to sugar, 3' and/or 4 ' carbon atom, The sugar of different epimeric forms, in the change of the α of glycosidic bond or the difference of beta comfiguration and other anomers.Sugar moieties bag Include, but be not limited to, pentose, deoxypentose, hexose, deoxyhexamethylose, ribose, deoxyribose, glucose, arabinose, five rings furan Mutter sugar, xylose, lyxose and cyclopenta.

For example, the modification of lock nucleic acid (LNA) type is usually directed to the change of the pentose to ribose and deoxyribonucleotide, this changes Become constraint, or the N-type conformation seen in " locking " Aform DNA sugar.In certain embodiments, this locking can pass through 2'- O, 4'-C- methene key are 1,2:Realized in the different furanoses of 5,6- diisopropylidenes-alpha-D-.In other embodiments, this Plant to change and can be used for synthesis locking phosphoramidite monomer.(for example, with reference to Wengel J., Ace.Chem.Res., 32: 301-310(1998),U.S.Pat.No.7,060,809；Obika,et al.,Tetrahedron Lett 39:5401-5405 (1998)；Singh,et al.,Chem Commun 4:455-456(1998)；Koshkin,et al.,Tetrahedron 54:3607-3630 (1998), the content of each open source literature is integrally incorporated herein by quoting).

In some preferred embodiments, the base through modification includes 8- azepine -7- denitrogenations-DA (PPA), 8- azepines -7- Denitrogenation-DG (PPG), the false different cytidine (2'-Deoxypseudoisocytidine) of 2 '-deoxidation, the fluoro- 2' BrdUs (FDU) of 5-, Locked nucleic acid (LNA), or 2'-O, 4'-C- ethene bridging nucleic acid (ENA) base).The alkali through modification that can be used in the present invention Other examples of base are described in United States Patent (USP) 7517978, and the disclosure of the patent application is by quoting its entirety It is incorporated herein.

Many bases through modification, include such as LNA, ppA, ppG, 5- fluorine dU (FDU), commercially available, and can use In oligonucleotide synthesis method as known in the art.In certain embodiments, the primer and blocker of modification can be used Standard is chemically synthesized.For example, in certain embodiments, the part of modification or base can be by using following several The support that the nucleosides of mode (a) modification is synthesized as DNA, the nucleosides of (b) modification is as phosphoramidite, examination when (c) DNA is synthesized Agent (such as the convertible acid amides handled when mixing DNA sequence dna by benzylamine), or modified after the synthesis of (d).

Due to the flexibility of nucleotide structure, the base-pair of part mispairing may partly form Watson-Crick (Watson- Crick) combine.This feature to block efficiency reduction, because it can also extend in the allele of mutation.Locked nucleic acid (LNA) and some other nucleic acid analogs have more rigid structure can be used for mitigate the problem.The blocker used can be with Contain one or two LNA or close to the position of mutation interested.

In certain embodiments, when primer or blocker are synthesized, modified base is placed on 3' ends.In certain embodiments, repair Decorations base is placed between primer or 3 ' the 1-6 nucleotides in end of blocker, for example, away from primer or the end of blocker 3 ' 2,3,4 or 5 Individual nucleotides.In some preferred embodiments, primer or blocker are synthesized so that modified base is placed on 3'- least significant ends.

Key can also be present in primer and blocker disclosed in the present invention between modification.The key so modified includes, but not It is limited to, peptide, phosphoric acid, di-phosphate ester, alkyl phosphate, alkane phosphonate ester, thio, thiophosphate, phosphorodithioate, methyl Phosphonate ester, phosphoramidate, substituted phosphoramidate and the base of analog, key is some further between sugar and/or nucleotides Improvement, this be to they in oligonucleotides as blocker and/or primer using related, for those skilled in the art Member is apparent.

The 5 prime excision enzyme activity of the 3 ' of archaeal dna polymerase → 5 ' can remove base mismatch, therefore difference from 3 ' ends of oligonucleotides Also it is digested in the base of blocker.Phosphorothioate bond is than phosphodiester bond to 5 prime excision enzyme activity more indigestibility；Therefore can It is used in 3 ' ends of blocker.

Method

Said system can make for recognizing in the various methods for being enriched with and/or quantifying the mutation of sample allelic With.

Method disclosed in the present invention be included in the presence of one or more blocker with a forward primer and One reverse primer expands target region.Blocker is including one and the target to be enriched with the case of no nucleic acid mutation The sequence of regional complementarity.This method can further comprise the amplification for detecting target region.The method of the invention can be with high sensitivity Ground detects nucleic acid mutation, and minimum detection limit (LOD) is up to 0.01%-0.001% in some cases.

As shown in Figure 2 A, when blocker is annealed in mutation (such as wild-type mutant), the blocker extends and prevented PCR is expanded, and thus prevents the extension of the forward primer or reverse primer of a distal end.In certain embodiments, forward or backwards Primer can apart from the hybridising region 0 of blocker, 5,10,15,20,25,30,35,40,45,50,55,60,80,100,250, 500,1000,2000 nucleotides or farther.In certain embodiments, blocker has sufficiently high Tm, it is impossible to be replicated Positive or reverse primer is replaced.In certain embodiments, the enzyme used in amplified reaction does not have strand-displacement activity.

A variety of archaeal dna polymerases can be used in the present invention.In some cases, target area is expanded with the inventive method Used enzyme includes but is not limited to：High-fidelity DNA polymerase and the repair enzyme with 3 ' end 5 prime excision enzyme activities.Available for this hair Enzyme in bright methods described is exemplified below:Including but not limited to, (Pfu Turbo thermal startings DNA gathers Pfu thermal startings archaeal dna polymerase Synthase), Phusion^TMThermal starting high-fidelity DNA polymerase, Phusion Hot^TMStart II high-fidelity DNA polymerases, Phire^TM Thermal starting archaeal dna polymerase, Phire^TMThermal starting II archaeal dna polymerases, KOD thermal starting archaeal dna polymerases, Q5 high-fidelity thermal startings DNA Polymerase, AmpliTaq, Phusion HS II, Deep Vent and Kapa high-fidelity DNA polymerases.

Conventional method for amplifying nucleic acid sequence is well known in the art.The present invention can use it is any this The method existed in the prior art of sample.In certain embodiments, the method that can use digital pcr is expanded, such as Vogelstein and Kinzler("Digital PCR,"PNAS,96:Described by 9236-9241 (1999).This method It is included in the dilution sample to be expanded before the amplification of target area.Dilution includes being diluted to traditional conventional plate, porous plate, nanometer Hole, and it is diluted to micro- pad or as droplet.(with reference to Beer N R, et al., " On-chip, real-time, single- copy polymerase chain reaction in picoliter droplets,"Anal.Chem.79(22):8471- 8475(2007)；Vogelstein and Kinzler,"Digital PCR,"PNAS,96:9236-9241(1999)；With Pohl and Shih,"Principle and applications of digital PCR,"Expert Review of Molecular Diagnostics,4(1):41-47(2004)).When being used in combination with digital pcr, the present invention can be significantly Improve the sensitivity of digital pcr.This is due to that the invention provides significantly inhibited when detecting that Genetic conditions include rare hereditary The related ambient noise of wild type (such as at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or about 100%).By the method for the present invention due to can be in a numeral It is more easy to be combined with target area in the single volume of PCR reactions and substantially increases targeting sensitivity.

When being changed with the rare heredity of PCR (for example, digital pcr) detections, the overwhelming majority in given reactant mixture is all It is wild-type sequence, and seldom includes rare genetic event.The method of the invention can effectively suppress wild type, such as such as this It is more than 1 described in text：10000.In some instances, each digital pcr includes 10000 wild type molecules, but because Amplification of the method for the invention to the suppression of wild-type amplification without suppressing single rare target, still can detect 1 Rare mutation.

The method of the invention further comprises the expansion that target area is detected using detection method general in this area Increase.For example, detection can be obtained by the melting curve of amplified production by mass spectrography or by the way that amplified production is sequenced.Root The number made a variation according to amplified production amplifying nucleic acid is different with type, and amplified production shows different melting curves.For determining to melt The method of solution curve has been fully described and generally well-known in the art, for the method using mass spectrum and nucleic acid sequencing also all Generally use in the art (referring to Sambrook and Russell, Molecular Cloning:A Laboratory Manual,(3^rdEd.) (2001) and Plum, Optical Methods, Current Protocols in Nucleic Acid Chemistry, 2001-2011) and Current Protocols in Nucleic Acid Chemistry, 2001- 2011,specifically Liquid Chromatography-Mass Spectrometry Analysis of DNA Polymerase Reaction Products).Method for nucleic acid sequencing is also known to conventional and those skilled in the art.Appoint What sequence measurement can be all used in conjunction with the present invention (referring to Current Protocols in Molecular Biology, 1995-2010)。

The method of the present invention is also included by comparing with target area with the presence or absence of the associated known amplification of nucleic acid mutation The amount of the amplified production of level detects the amplification situation of target area.For the side for the amount for detecting amplification or measure amplified production Method is well-known in the art, and any such method can be used (to be write referring to Sambrook and Russell《Molecule Cloning experimentation room handbook (the 3rd edition)》(2001) and written by Gallager et al.《Current experimental program：Indispensable experiment Room technology》(2008))).

The variance that the method for the present invention is detected includes mutation in the target area, missing and/or insertion.Missing Single nucleotide acid base including removing target area.The missing that can be detected includes 1 base deletion, 2,3,4,5 or more Many (such as hundreds of or thousands of) nucleotide bases.Mutation may include but be not limited to replace (such as transversion and conversion), abasic site, friendship The site of connection, and chemical modification or modification base.The mutation that can be detected is included in 1 mutation in target area, 2,3,4,5 or more nucleotide base mutation.Insertion includes one nucleotides of addition to target region.Can detect may include 1 Individual base insertion, 2,3,4,5 or more (such as hundreds of or thousands of) nucleotide bases are inserted into target area.In some implementations In scheme, missing, mutation and/or insertion are detected by the method for the present invention.

System and methods described herein can minimize false positive, and false positive is AS-PCR maximum weakness.This Research method is not the method with the DNA of elongation mutagenesis, but using blocking oligonucleotide to be annealed on wild type DNA and hinder Its method expanded of breaking causes the mutant DNA by preferential amplification (Fig. 2A and 2B).Accidental allele non-specificity is blocked The extension of thing has little to no effect (Fig. 2 C) to the result of enrichment.The remaining amplification that wild type is not blocked can be in follow-up NGS Sequencing is identified.

As disclosed herein, system and method for the present invention can effectively block the wild type more than 99.9% to expand Increase, cause the ratio of mutation allele to be significantly increased to 1/7 from 1/10000, i.e., from about 0.01% to about 14%.Rare false sun Property be not that the intrinsic non-specific extension of allele specific oligonucleotide is introduced, but only by the nucleotides of archaeal dna polymerase Incorporation mistake is triggered.False positive can further be reduced using the exo+ polymerase with 3' to 5' 5 prime excision enzyme activities, entered One step reduces rare wrong report.

Purposes and application

Method disclosed herein can be used for being enriched with or detect various target nucleic acids interested.Target nucleic acid can be double-strand A part for nucleic acid or single-chain nucleic acid.

The source of workable nucleic acid samples, including but not limited to human cell such as blood circulation, the cell of culture and swollen Oncocyte.The cell of the tissue of other mammals, blood and culture is also the available source of template nucleic acid.In addition, it is viral, bite Thalline, bacterium, fungi and other microorganisms can also be the sources of foranalysis of nucleic acids.The DNA can be genome or matter Grain, bacteriophage, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or other clones for carrier.RNA can be straight Connect and separated from relevant cell, can also be by expanding or being obtained by in-vitro transcription from suitable RNA promoters in vitro. The present invention can be used for the mutation for detecting genomic DNA, the either mankind, animal or other organisms.The inventive method is being lost There is special purposes in the analysis of transmissibility or acquired disease or illness；One of which specific use be used for genetic disease and The detection of cancer.

In some embodiments, template sequence or nucleic acid samples can be genomic DNAs.In other embodiments, mould Plate sequence or nucleic acid samples can be cDNA.In other embodiments, as being analyzed gene expression simultaneously by RT-PCR In the case of, template sequence or nucleic acid samples can be RNA.DNA or RNA template sequences or nucleic acid samples can be from any kind of groups It is extracted in knitting, for example, formalin fixes the tumor specimen of FFPE.

In some embodiments, target nucleic acid may reside in the cell of subject, such as mammal (for example The mankind), plant, fungi (such as yeast), protozoan, bacterium or virus.For example, target nucleic acid may reside in and interested have In the genome of body (for example on chromosome) or in the nucleic acid beyond chromosome.In some embodiments, target nucleic acid Can be RNA, such as mRNA.In some other embodiment, target nucleic acid can be DNA (such as double-stranded DNA).

In a particular embodiment, target nucleic acid can be specifically present in organism interested, i.e., target nucleic acid does not have Have and find or can not be found in the organism similar to interested organism in other organisms.In some embodiment party In case, target nucleic acid can be viral nucleic acid.For example, viral nucleic acid can be in human immunodeficiency virus (HIV), influenza virus (such as influenza A virus, influenza B virus, or influenza hepatitis viruse), or in dengue fever virus.Target nucleic acid may have In bacterium.In some embodiments, target nucleic acid can be a protozoan nucleic acid.In some embodiments, target nucleic acid It is the nucleic acid of fungi (such as yeast).

In some preferred embodiments, target nucleic acid can be the nucleic acid of mammal (such as mankind).For example, lactation Animal nucleic acid can be found in circulating tumor cell, epithelial cell or fibroblast.In other examples, target nucleic acid be Circulation in the nucleic acid freely circulated in the blood of subject, such as acellular nucleic acid (cfDNA) or the blood of cancer patient is swollen Knurl DNA (ctDNA)., can be with order to better profit from the limited acellular nucleic acid (cfDNA) obtained from patients blood plasma Use the PCR response procedures that multiplex PCR and one are general.As shown in the examples below, as a result show, all three tests Mutantional hotspot be successfully enriched with altogether.Multiple assay allows to detect a series of driving gene mutation simultaneously, and this satisfies lung cancer liquid The demand of body biopsy.

In order to reach this target of the turnaround time of 5 days that CAP is recommended, library preparation process has been optimized to 4 hours Below.The cycle estimator of whole liquid biopsy is 4 days, including sample stream, sequencing, data analysis and result are explained.

In an example, target nucleic acid chain is a chain for containing specific variation, such as SNP (single Nucleotide polymorphism, SNP) or genetic mutation.The example of such mutation includes point mutation, transposition or reversion.

In some embodiments, composition, method and/or the kit disclosed herein in the present invention can be in diagnosis During be used to detect cell in the circulatory system.In one embodiment, composition, method and/or the examination in the present invention Agent box can be used for early-stage cancer to detect the DNA of tumor cell in blood in diagnosing.In some embodiments, the group in the present invention Compound, method and/or kit can be used for the detection and checking of cancer or the related hereditary variation of disease or somatic mutation. In some embodiments, composition, method and/or kit in the present invention can be used for SNP Genotypings --- four-, three-with The SNP of two-allele.In other embodiments, composition, method and/or the kit in the present invention can be used for Recognize one or more nucleotides inserteds or deletion mutation.In some embodiments, the present invention in composition, method and/ Or kit can be used for cell line in the parting of DNA in the hybrid dna sample that QC human identifications test, cell contamination analyte detection QC, the gene expression analysis of allele, Viral typing/rare pathogen detection carries out abrupt climatic change from the sample of mixing, Detection circulating tumor cell and/or pre-natal diagnosis in blood.

The relevant purposes of cancer

System and method disclosed by the invention in following field it is particularly useful：(1) from tissue biopsy and such as blood plasma or Cancer early detection done in the body fluid of serum etc；(2) operation or chemicotherapy after residual disease assessment；(3) disease By stages, for the analysis of molecules of prognosis or for individual patient personalized customization therapeutic scheme；(4) monitoring treatment result and cancer Alleviation or recurrence.

2015, american cancer death toll was estimated as 589430 people (with reference to Siegel RL, Miller KD, Jemal A.Cancer statistics,2015.CA Cancer J Clin.2015；65(1):5-29.doi:10.3322/ caac.21254).In all cancers, non-small cell lung cancer (NSCLC) is the first cause of cancer mortality, accounts for 22.8%. Combined chemotherapy based on platinum class can moderately improve patients with advanced NSCLC 9% i.e. 12 compared to independent supportive treatment The life cycle of individual month is (with reference to Spiro SG, Rudd RM, Souhami RL, et al.Chemotherapy versus supportive care in advanced non-small cell lung cancer:improved survival without detriment to quality of life.Thorax.2004；59(10):828-836.doi:10.1136/ thx.2003.020164).By contrast, targeted therapy combines oncogene parting in Treatment decsion formulation process, treatment Effect improves a lot.For example, Gefitinib, one kind can targeting epidermal growth factor receptor (EGFR) small molecule tyrosine Kinase inhibitor (TKI) is (with reference to Paez JG, Ja PA, Tracy S, et al.EGFR Mutations in Lung Cancer:Correlation with Clinical Response to Gefitinib Therapy.2004；304 (June):1497-1501), compared to 20% reaction rate of typical chemotherapy, its reaction rate may be up to 75%；Compared to The survival period of patient was up to (reference in 28.2 months in 10.3 months middle position survival rates of typical chemotherapy, Gefitinib scheme Barr Kumarakulasinghe N,Zanwijk N Van,Soo R a.Molecular targeted therapy in the treatment of advanced stage non-small cell lung cancer(NSCLC) .Respirology.February 2015.doi:10.1111/resp.12490).However, targeted therapy does not benefit institute Some Patients with Non-small-cell Lung.The high efficiency of targeted therapy depends on operable carcinogenic driving gene mutation in patient body to deposit .These mutation are initiations or maintain the molecule abnormality of neoplastic process can be by the medicament that changes for each genome Neutralize.If during target tumor, Gefitinib in the Patients with Non-small-cell Lung of wild type EGFR gene only There is very little 0-6.6% response speed, but have in the patient for thering is EGFR to be mutated the response speed of unusual 75% (with reference to such as Douillard J-Y, Shepherd F a, Hirsh V, et al.Molecular predictors of outcome with gefitinib and docetaxel in previously treated non-small-cell lung cancer:data from the randomized phase III INTEREST trial.J Clin Oncol.2010；28(5):744-752.doi:10.1200/JCO.2009.24.3030 with Hirsch FR, Varella- Garcia M,Bunn P a.,et al.Molecular Predictors of Outcome With Gefitinib in a Phase III Placebo-Controlled Study in Advanced Non-Small-Cell Lung Cancer.J Clin Oncol.2006；24(31):5034-5042.doi:10.1200/JCO.2006.06.3958 with Maruyama R, Nishiwaki Y,Tamura T,et al.Phase III study,V-15-32,of gefitinib versus docetaxel in previously treated Japanese patients with non-small-cell lung cancer.J Clin Oncol.2008；26(26):4244-4252.doi:10.1200/JCO.2007.15.0185 and Mok T,Wu Y.Gefitinib or carboplatin–paclitaxel in pulmonary adenocarcinoma.N Engl J Med.2009Sep 3；361(10):947-57 and http://scholar.google.com/scholarHl=en＆ BtnG=Search＆q=intitle:new+england+journal#2.Accessed April 13,2015).Therefore, target Mutant test don't fail to be driven before to treatment.

Tissue biopsy is always the main method of mutation identification.However, this method is to the patient's of non-small cell lung cancer State of an illness detection is unsatisfactory.About 75% non-small cell lung cancer case is in late period in diagnosis, but tissue solid is lived Examine the inherent defect between detection tumour and in terms of the heterogeneity of intra-tumor and often lead to drug resistance.In fact, tumour is heterogeneous Property presence be during effective cancer treatment method is developed using targeted therapy a significant challenge (reference Yancovitz M,Litterman A,Yoon J,et al.Intra-and inter-tumor heterogeneity of BRAF(V600E))mutations in primary and metastatic melanoma.PLoS One.2012；7(1): e29336.doi:10.1371/journal.pone.0029336).Liquid biopsy has complete as an attractive method The potentiality that face capture genome changes, so that targeted therapy is effectively realized.In addition, liquid biopsy is easily repeated, this causes It is used into tumour monitoring change to be possibly realized, thus available for direction of medication usage during treating.Liquid biopsy is also potential to be used for It is postoperative or treatment after may cause recur minimal residual disease measurement (refer to Diehl F, Schmidt K, Choti M a, et al.Circulating mutant DNA to assess tumor dynamics.Nat Med.2008；14(9):985- 990.doi:10.1038/nm.1789).In addition, it can also serve as the early stage sign of detection recurrence in follow-up nursing process An important kit.However, due to the nucleic acid (ctDNA) of tumour cell and the quantity pole of tumour cell in the circulatory system Low, along with even more serious by detecting brought human error, liquid biopsy is not yet in the routine work of doctor.

System and method disclosed in this invention can be as a kind of reliable, quick measure advanced Non-small cell lung The liquid biopsy method of patient.The obstacle for developing such a reliable and quick detection method is dual, be there is no so far gram The difficult platform of following two aspect is taken：

First difficulty is sensitivity.In patient with advanced cancer, Tumour DNA (ctDNA's) deposits in the circulatory system Amount may be very low.Newman et al. scopes that late Patients with Non-small-cell Lung blood plasma is observed be 0.04% to 3.2% (with reference to Newman AM, Bratman S V, To J, et al.An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.Nat Med.2014； 20(5):548-554.doi:10.1038/nm.3519).This conventional technology of quantitative PCR (qPCR) can reach about 1% Monitoring lower-cut (LOD), the big multipotency of next generation's sequencing (NGS) reaches 1-2%LOD.Digital pcr, which in this respect show, to be most hopeful Progress, its LOD as little as 0.01% is (with reference to Detection of rare mutations in blood samples by droplet digital PCR.http://www.bio-rad.com/webroot/web/pdf/lsr/literature/ Bulletin_6317.pdf)。

Second difficulty is the ability of multiple assay.The quantity of operable mutation and the volume of blood sample having found have Limit causes substance is determined to be unsuitable for liquid biopsy.Therefore, a key function of functional liquid biopsy is single from one The ability of the mutation of the multiple association drivings of plasma dna sample parallel detection.At present, NGS is can to provide enough multi-functionals Unique ripe platform.

Liquid biopsy disclosed herein is absorbed in the operable Cancer-causing mutation of identification with the selection of guiding treatment scheme.About 75% non-small cell lung cancer is metastatic in diagnosis or in late period (with reference to Reade C, Ganti A.EGFR targeted therapy in non-small cell lung cancer:potential role of cetuximab.Biol targets Ther.2009:215-224.http://www.ncbi.nlm.nih.gov/pmc/ Articles/PMC2726075/.Accessed April 13,2015), targeted therapy can be used to such patient.At this It is heterogeneous to there is higher level in the tumour in a little patients.The main purpose of the inventive method is in an xenograft tumor cell mass The middle operable mutation of test, and monitoring tumour closely is to the reaction for the treatment of and the rising of molecule drug resistance.After after treatment The patient in continuous nursing period also can significantly be benefited from liquid biopsy.Studies have found that, molecular testing can compare radiological examination The early several months finds the possibility of palindromia (with reference to Misale S, Yaeger R, Hobor S, et al.Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer.Nature.2012；486(7404):532-536.doi:10.1038/nature11156 and Diaz L a, Williams RT,Wu J,et al.The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.Nature.2012；486(7404):537- 540.doi:10.1038/nature11219)。

As described in embodiment below, system and method disclosed herein can be successfully used to the richness of hot spot mutation Collection and detection include those codings EGFR T790M, EGFR L858R, EGFR exon 19Del, BRAF V600E, BRAF V600K, BRAF V600D, BRAF V600G, BRAF V600A and BRAF V600R.

EGFR T790M mutation are one frequent acquired prominent in tyrosinase inhibitor (TKI) targeted therapy patient Become, the mutation causes the threonine of EGF-R ELISA 790 to be replaced by methionine.Residue after this mutation is added The binding site of affinity and competition binding inhibitor to ATP is (with reference to Yun C-H, Mengwasser KE, Toms A V, et al.The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.Proc Natl Acad Sci U S A.2008；105(6):2070-2075.doi:10.1073/ pnas.0709662105).Cancer cell less than 5% is obtained after the mutation, and patient will be shown to TKI therapeutic sensitivities Reduction is (with reference to Lindeman NI, Cagle PT, Beasley MB, et al.Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors:guideline from the College of American Pathologists,International Association for the Study of Lung Cancer,and Association for Molecular Patho.Arch Pathol Lab Med.2013；137(6):828-860.doi:10.5858/arpa.2012-0720-OA). Therefore, it is necessary to supervision T790M appearance closely.T790M detection can assist a physician makes switching medicine during treating To decision (reference Watanabe S, Tanaka J, the Ota T, et of the third generation EGFR TKI decision or pause medication al.Clinical responses to EGFR-tyrosine kinase inhibitor retreatment in non- small cell lung cancer patients who benefited from prior effective gefitinib therapy:a retrospective analysis.BMC Cancer.2011；11(1):1.doi:10.1186/1471- 2407-11-1)。

EGFR L858R are carcinogenic driving genes, account for all EGFR activation lung cancer 43% (refer to Mitsudomi T, Yatabe Y.Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer.FEBS J.2010；277(2):301-308.doi:10.1111/j.1742- 4658.2009.07448.x).Mixed, made with wild type DNA with different ratios containing the EGFR L858R cell line dnas being mutated It can be enriched with method and system (table 3) disclosed above.Similar to T790M result, 863 times of enrichment is used for examining It is more than sufficient to survey 0.01% allelic mutation.

Except above-mentioned point mutation, other variances or mutation also by the method and system of the present invention carry out enrichment and/ Or amplification.The example of adaptable variance is listed in following list 8 and 9.

System and method disclosed herein can provide optimal liquid biopsy product and mode.Compared on silicon chip by The all told enhancing liquid biopsy that single biopsy sample takes whole Illumina companies HiSeq (refers to Sullivan M.Guardant Health takes another $ 50M for " liquid biopsy " cancer test.2015 and http://venturebeat.com/2015/02/03/guardant-health-takes-another-50m-for- Ground-breaking-liquid-biop sy-test/), determination method disclosed herein reduces depositing for external not mutated DNA The more sensitive detection of Cancer-causing mutation can allowed；All told enhancing liquid relative to Illumina companies HiSeq is lived Inspection, the present invention can handle 10,000 sample with identical HiSeq sequencing capacity.

Composition and kit

The present invention includes composition or reactant mixture containing above-mentioned primer and blocker.Said composition can be further Comprising selected from nucleic acid polymerase, the nucleotides of extension and one or more reagents of detection reagent.

The detection reagent can include nucleotide probe, for example, molecular beacon probe or be marked with fluorogen and quencher Mark fluorescent probe --- negative and positive probe.For example, with reference to U.S. Patent number 5925517,6103476,6150097,6270967, Described in 6326145 and 7799522.Except above-mentioned reagent, said composition may also comprise, one or more salt, such as sodium chloride, Magnesium chloride, potassium chloride, magnesium sulfate；Buffer, such as Tris buffer solutions, N- (2- ethoxys) piperazine-N'-2- ethane sulfonic acids (HEPES), 2- (N- morpholines) ethyl sulfonic acid (MES), MES sodium salts, 3- (N- morpholines) propane sulfonic acid (MOPS), N- tri- (methylol) Methyl -3- amino propane sulfonic acids (TAPS)；Solubilizer；Detergent, such as non-ionic detergent, such as Tween-20；Nuclease Inhibitor and the like.

The present invention further comprises kit and the target nucleic acid sequence for being expanded, being enriched with and/or for detection Diagnostic system.Therefore, one or more reactive components for method disclosed herein can be in the richness of target nucleic acid chain The form of the kit used in collection and detection is provided.Such kit is provided in one or more containers or in matrix The proper content of one or more reactive components of (by electrostatic interaction or Covalent bonding together).

One or more target nucleic acid sequences are expanded or are enriched with or are sequenced using method described herein (for example for NGS or Sanger sequencings) kit, it may include it is following：Forward primer, reverse primer, one or more blocker, nucleic acid Polymerase, the nucleotides and detection probe of extension.The example of the other components of kit includes but is not limited to, it is one or more not Same polymerase, for polymerisation (in 1X or the form of concentration) buffer solution and one or more dyestuffs or for detecting polymerization The fluorescence molecule of product, one or more of which primer is used for specific amplification correspondence nucleic acid or target nucleic acid, and one or more are visited Pin is used for specific bond in control nucleic acid or target nucleic acid.The kit can also include with one or more of lower component：Branch Hold, terminate, changing or digestion reagent, bleeding agent and the device for detecting a detection probe.

The reactive component used in amplification and/or detection method can be provided in a variety of manners.For example, component (example Such as enzyme, triphosphopyridine nucleotide, blocker and/or primer) aqueous solution can be suspended in or as freeze-drying or freezing Powder, ball or pearl.In the latter case, when component regenerates, forming complete mixture is used to use in the assay.

The need for kit or system can be sufficient at least one described measure comprising its consumption.It is described herein Any combinations of component, can also include instructing the form that its component is used.In some applications, one or more reacted constituents It can be packed with individually surveying measured personal consumption in advance, typical case's packaging is including the use of disposable pipe or equivalent Container.In this case, the sample of test target nucleic acid can be added to the amplification individually managed and directly carried out.In examination The amount of the component provided in agent box can be any appropriate amount, and can depend on demand of the target market to the product. It is determined that the general standard of appropriate amount can be found in following books：For example, Sambrook and Russell writes《Molecular cloning Laboratory manual (the 3rd edition)》(2001) CSH Press；Frederick M.Ausube,《Molecular biology side Method》, John Wiley＆Sons publishing houses (2003).

The kit of the present invention may include any amount of useful other reagent of method for implementing the present invention or Material.Such material includes but is not limited to：Cell cracking agent (including buffer solution), the chelating agent of bivalent cation or suppression The reagent of other nucleic acid, the normally functioning comparison DNA of other compositions for ensuring the multienzyme complex and reaction, DNA fragments Reagent (including buffer solution), amplification reaction reagent (including caching), liquid and cleaning solution.The present invention can be provided at any temperature Kit.For example, for containing protein component or during the complex stock being present in liquid, being preferably maintained in less than 0 DEG C, Preferably equal to or lower than -20 DEG C, or it is otherwise in frozen state.

The container of kit can be any conventional container, and it can keep provided form, for example, microcentrifugation Pipe, ampoule bottle, or detection means, such as liquid flow device, kit, lateral flow, or other similar devices.The kit can With including target nucleic acid mark the or unlabelled nucleic acid probe for detection.In some embodiments, kit can be with Including indicating any method described herein in the component to be used, for example, using the thick of no nucleic acid extraction and/or purifying The method of matrix.For such kit and the typical packaging material of system include solid matrix (such as glass, plastics, paper, Paper tinsel, particulate etc.), under various settings (such as in bottle, micro titer plate well, microarray etc.), fixed reacted constituent or inspection Probing pin.

In described system, except containing reagent constituents, the instrument for determining can also be included, such as from mark Remember the photometer of probe in detecting signal.

The mode of the system using kit or the present invention is described in detail in specification such as written instruction or video recording demonstration, optionally by trying Agent box is provided.Further, the present invention there is provided herein the method using any combinations thing or kit, also carry herein Implementation for any method or measure and/or any device for implementing any measure or method for kit are supplied Use.

Optionally, the system of kit or the present invention also include software, to accelerate the generation of data, analysis and/or storage, And it is easy to the access to database.The software include logical order, instruction set, or can data collection, storage and/or The suitable computer program used in analysis.The comparison and association analysis of data can use the system of kit or the present invention Subsidiary software.

In all above methods, reagent and system provide various based on blood, subject's (warp of the assessment of Noninvasive Its allow) morbid state diagnostic tool.These reagents and system diagnostics method of testing, it can be plus other screening examinations Test, the use of such as chest X-ray or CT scan, improve the accuracy of diagnosis and/or directly additional test.In other sides Face, the present invention described herein can be used for the routine assessments of the Prognosis scoveillance of disease and the risk of recurrence, based on monitoring Can be in response to specific therapy.The present invention described herein can be also used for being present in sample after operation consent and treatment and examine The evaluation of the change of disconnected feature, and determine to reflect the presence of tumour and the gene expression profile available for the probability for assessing recurrence.

After such scheme, the present invention has the advantages that following prominent and effect compared with prior art：The present invention Morbid state can be characterized from a minimal invasive procedures, i.e., by blood sampling without separating cancer cell.The way of currently available technology Tissue sample, typically tumor sample are depended on from the classification of gene expression profile cancer.In the situation of very small tumour Under, biopsy is problematic, if tumour does not occur or invisible, the sample for finding it is impossible.The technology of the present invention side Tumour need not be purified or separated when analyzing tumor sample in case；And its extra advantage exists when blood sample It is easily prepared in the material, and the quite stable in follow-up analysis；When sample to be analyzed is mRNA, this stability seems It is particularly important.

Brief description of the drawings

The Figure of description for constituting the part of the application is used for providing further understanding of the present application, and the application's shows Meaning property embodiment and its illustrate be used for explain the application, do not constitute the improper restriction to the application.In the accompanying drawings：

Fig. 1 is shown illustrates ApoE gene (AS-PCR) with the specific primer of mutation.Wherein, Tu1AZhong Oligonucleotides matches mutant nucleotide sequence and allows to allow extension and PCR to expand (not shown) with reverse primer completely.It is few in Figure 1B Nucleotides has in 3' ends mismatches and mismatches wild-type sequence, so as to prevent oligonucleotides to extend and expand.Fig. 1 C In have the false positive PCR primer of sequence that non-specific extension occurs and cause the allele comprising mutation.

Fig. 2A, 2B and 2C are the system principle schematic diagrames of the present invention.A blocker and wild-type allele in Fig. 2A The blocker of annealing and extension has blocked the PCR of outer primer to expand.Each blocker is mismatched at 3' ends in Fig. 2 B, discord mutation Allele annealing, so as to allow external primer amplification.Non-specific extension only causes the equipotential of whole specific template in Fig. 2 C The amplification failure in this circulation of gene, non-false positive PCR primer is formed.

Embodiment

It is noted that described further below is all exemplary, it is intended to provide further instruction to the application.Unless another Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.

It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.

It should be noted that term " first " in the description and claims of this application and above-mentioned accompanying drawing, " Two " etc. be for distinguishing similar object, without for describing specific order or precedence.It should be appreciated that so using Data can exchange in the appropriate case, so that presently filed embodiment described herein for example can be with except herein Order beyond those of diagram or description is implemented.In addition, term " comprising " and " having " and their any deformation, it is intended that Be to cover it is non-exclusive include, for example, containing process, method, system, product or the equipment of series of steps or unit not Be necessarily limited to those steps or the unit clearly listed, but may include not list clearly or for these processes, side The intrinsic other steps of method, product or equipment or unit.

As background technology is introduced, in the prior art in many cases, wild type DNA substantially exceeds mutant DNA, Make detection and a small number of allele of identification extremely low concentration extremely difficult, be used for richness the invention provides a kind of nucleic acid molecules composition Collect the allelic mutation in the target nucleic acid sequence of target nucleic acid, including：(a) be capable of amplifying target nucleic acid sequence forward primer and Reverse primer and (b) include the first blocker of first paragraph sequence, and this sequence is met in following requirements (i) and target nucleic acid sequence Wild-type allele matches or complementary and (ii) can be extended by archaeal dna polymerase.By being mutated with specific nucleotide (such as open country Raw type thumping becomes) the oligonucleotides blocker of matching or complementation can block or suppress the amplification of specific nucleic acid mutation, from And allow the enrichment of other mutation (for example, mutation variants), it whether there is with the nucleic acid mutation of the target area of detection.

Definition

" nucleic acid " refers to DNA molecular (for example, cDNA or genomic DNA), RNA molecule (for example, mRNA), or DNA or RNA Analog.DNA or RNA analogs can be synthesized from nucleotide analog.The nucleic acid molecules can be single-stranded or double-stranded, but excellent Choosing is double-stranded DNA.

As used herein, term " target nucleic acid " or " target " refer to the nucleic acid containing target nucleic acid sequence.Target nucleic acid can be with It is single-stranded or double-stranded, and often DNA, RNA, DNA or RNA or the derivative of combinations thereof." target nucleic acid sequence ", " target nucleic acid sequence " or " target area " refers to include the particular sequence of a part for all or single-chain nucleic acid sequence.Target nucleic acid Sequence can be that in nucleic acid-templated scope, it can be any type of single-stranded or double-stranded nucleic acid.Template can be purifying or The nucleic acid of separation, or can be purified with right and wrong or non-isolated.

Term " allele " typically refers to be pointed in the same position of pair of homologous chromosome, controls relativity One pair of genes.For example, on homologue.Allele can refer to wherein individual cells or organism or in multiple cells or In the difference of organism identical physical location (" allelelic mutation ") same physical location is found that on homologue Between different DNA sequence dna.In some cases, the mononucleotide that allele can correspond in specific physics site is poor It is different.(single or multiple) insertions of nucleotides are may correspond in other embodiments and allele or are lacked.

As used herein, term " Rare allele variation " is in target polynucleotide compared to replacement allelic variation The lower allelic mutation of storage in chain.Rare allele variation be also referred to as " minorAllele mutation " and/or " mutation allele mutation ".For example, Rare allele variation can be found less than 1/10,1/100,1/ in a frequency 1000,1/10000,1/100000,1/1000000,1/10000000,1/100000000 or 1/1000000000 compared to another Individual allelic variation is for given a SNP or gene.In other words, rare allele variation can be, 1,10, Have in 100,1000 microlitres of samples or reactant volume less than 2,3,4,5,6,7,8,9,10,15,20,25,50,75,100, 250,500,750,1000,2500,5000,7500,10000,25000,50000,75000,100000,250000,500000, 750000 or 1000000 copies.

As used herein, term " abundant allelic variation " can refer to present in sample than substituting allele change Different higher levels of variation.Abundant allelic variation is also referred to as one " major allele variation " and/or " wild type Allelic variation ".For example, the frequency of abundant allelic variation may be greater than 10 ×, 100 ×, 1000 ×, 10000 ×, 100000 ×, 1000000 ×, 10000000 ×, 100000000 × or 1000000000 × compare another allele Variation is for given a SNP or gene.In other words, abundant allelic variation can be, for example, 1,10,100, 1000 microlitres of samples or reactant volume have more than 2,3,4,5,6,7,8,9,10,15,20,25,50,75,100,250 again, 500,750,1000,2500,5000,7500,10000,25000,50000,75000,100000,250000,500000, 750000 or 1000000 parts copies.

Term " amplification " as used herein and its variation include any for producing multiple copies or at least many nucleosides The method of the complementary strand of a part for acid, the polynucleotides are commonly known as " template ".The template polynucleotide can be single Chain or double-strand.The colony of one amplified production to solid plate, is referred to as " amplicon ".The polynucleotides of amplicon can be single Chain or double-strand, or both mixtures.Under normal circumstances, template will include target nucleic acid sequence, by the amplicon of gained By including essentially identical sequence, or the polynucleotides being substantially complementary with target nucleic acid sequence.In some embodiments, it is specific The polynucleotides of amplicon be substantially the same, it is or substantially complimentary to one another；A given expansion in certain embodiments The polynucleotides increased in son can have nucleotide sequence different from each other.Amplification can be carried out with linear or exponential manner, and And a repetition to solid plate and continuous duplication can be related to form two or more amplified productions.Some are typical Amplified reaction is related to the synthesis of the nucleic acid based on template that is continuous and repeating, so as to produce multiple partial nucleotide sequences for including template Row subchain, and to a certain extent share template nucleotide sequence homogeneity or with template or complementation.In some embodiments, Each nucleic acid synthesis is referred to alternatively as once " circulating " for amplification, including creates free 3' ends (for example, passing through otch double-strand A DNA chain), resulting primer and primer extension procedures；Optionally, additional denaturing step may also be included in which Template is partially or completely to be denatured.In some embodiments, the repetition for the single loop that a wheel amplification includes amplification gives Number of times.For example, can include 5,10,15,20,25,30,35,40,50,75,100 or more times of a wheel amplification are specific The repetition of circulation.In an exemplary embodiment, amplification includes wherein specific polynucleotide template progress nucleic acid synthesis Two any reactions for continuously circulating.Synthesis can include template-dependent nucleic acid synthesis.

" blocker " (also referred to as " oligonucleotides blocker ", " blocker ", " blocker probe " or " widow's blocking Thing ") this word refers to a chain of nucleic acid or can hybridize to a constraint by primer (forward primer or reverse primer), The oligonucleotides of the DNA of the identical specific allelic variation of composition on opposite or complementary strand, the few nucleosides can be located at Acid can reduce or prevent the amplification of the specific allelic variation.Blocker can be designed to be can combine closely it is wild Type allele (for example, abundant allelic variation), to suppress wild-type allele amplification, and allows amplification in phase With or the chain of mutation allele (for example, rare allelic variation) on opposite chain on extended by primer and Occur.

Term " primer " or " primer tasteless nucleotide " refer to single-chain nucleic acid or can hybridize with template nucleic acid and serve as extension Starting point, an oligonucleotides of nucleic acid synthesis is carried out according to the composition of template nucleic acid.Term " extension nucleotides " is to refer to Be impregnated in any nucleotides of extension products during expanding, i.e. DNA, RNA or its DNA or RNA derivative such as DNA or RNA, It may include a label.

" modified base " one word used herein is generally referred to as in nucleic acid and naturally occurring base or base Learn the different any modification of key.This modification includes the chemical constitution of base, or in nucleic acid base chemical bond, or nucleic acid backbone Change in structure (referring to embodiment).

Term " detection probe " refers to have forms steady with the abundant complementation of its target nucleic acid sequence, under strict hybridization conditions Fixed probe:One section of oligonucleotides of target nucleic acid sequence hybrid product.The oligomer that probe is generally synthetic, it may include and mesh The base of the sequence complementation beyond region is marked, these bases are not influenceed under strict hybridization conditions between probe and target nucleic acid Hybridization.One can not be homopolymer (such as Poly-A or Poly-T), promoter sequence, limit with the sequence of complementary target Property endonuclease recognition sequence processed, or assign the sequence (such as catalytic site or hairpin structure) of desired two grades or tertiary structure, Or the marked region for helping to detect and/or expanding." stabilization " or " detection is stable " refers to the temperature ratio of a reactant mixture The melting temperature (Tm) of contained double-strandednucleic acid is low 2 DEG C in mixture, more preferably lower than Tm at least 5 DEG C, even more preferably compares Tm It is low at least 10 DEG C.

Term " hybridization " or " annealing " refer to that the nucleotide chain of all or part of complementation can be under given conditions with parallel Or preferably antiparallel formation one stable duplex structure or region, two chains of composition nucleotides are connected by hydrogen bond Together.Although hydrogen bond is generally between adenine and thymine or uracil (A and T or U), formation or cytimidine and bird are fast Formed between purine (C and G), other base-pairs can form hydrogen bond.

Term " preferential hybridization (Preferentially hybridize) " refers in stringent hybridization condition, nucleic acid or few core Its target nucleic acid sequence can be hybridized under thuja acid (such as primer, blocker, or probe) and forms stable crossbred, for example, is indicated There is at least one sequence or organism interested in the sample.Nucleic acid and the specific hybridization of target nucleic acid, i.e., it is special to foot Can be there is (or in the absence of) and detected exactly by enough degree in target nucleic acid sequence.Preferential hybridization is generally referred to as in sample The difference of at least 10 times of hybridization signal between target nucleic acid sequence and non-target nucleic acid sequences.

Term " strict hybridization conditions " or " strict condition " refer to that a nucleic acid or oligomer specific hybrid arrive it Expected target nucleic acid sequence, rather than another sequence condition.Strict condition may be according to some well-known factors Change, such as G/C content and sequence length, and (such as standard side can be predicted or determined by the common method of molecular biology Method is determined).

Term " substantially homologous " or " corresponding to substantially " refer to that one section of sequence in probe, nucleic acid or oligonucleotides has at least 10,20,30,40,50,100,150,200,300,400, or 500 continuous bases, these bases have at least 80% (preferably extremely Lack 85%, 90%, 95%, 96%, 97%, 98% and 99% and most preferably 100%) continuous with the equal length of reference sequences Base is identical.Homology between sequence can be expressed as the base mismatch number in every group of at least ten base being compared.

Term " being substantially complementary " refers to an oligonucleotides comprising one section at least 10,20,30,40,50,100,150, 200,300,400, or 500 continuous bases, these bases have at least 80% (preferably at least 85%, 90%, 95%, 96%, 97%, 98% and 99% and most preferably 100%) identical with the continuous base of the equal length of reference sequences.It is same between sequence Source property can be expressed as the base mismatch number in every group of at least ten base being compared.

Term as used herein " subject " refers to any organism with genome, it is preferable that animal living, Such as mammal, this is always diagnosis, treatment, the object of observation or experiment.The example of subject can be the mankind, and domestic animal moves Thing (beef cattle and milk cow, sheep, poultry, pig etc.), or companion animals (dog, cat, horse etc.).

Term " biological sample " refers to from organism (such as patient) or from body constituents (such as cell) The sample of acquisition.The sample can be any biological tissue, cell or liquid.The sample can be " clinical sample ", this It is the sample come from subject, the sample of such as human patientses or veterinary subject.Such sample, includes but is not limited to：Saliva Liquid, phlegm, blood, haemocyte (such as leucocyte), amniotic fluid, blood plasma, seminal fluid, marrow and tissue or aspiration biopsy sample, urine, abdomen Film liquid and liquor pleurae, or the cell therefrom obtained.Biological sample can also include histotomy, such as be used for the ice of histology purpose Freeze section.Biological sample can also be referred to as " Patient Sample A ".Biological sample can also include the egg for substantially purifying or separating In vain, film preparation, or cell culture.

As used herein, when being used to refer to any a set of constituent, the term of " contact " and its mutation include following Described any process, is thus mixed into same mixture by the composition of contact and (is for example added to identical compartment or molten Liquid), it is not necessarily to the actual physics contact between cited component.Cited component can be in any order or any Combine (or sub-portfolio) contact, and can include, one of them or some cited by component then removed from mixture, It can be selected in before adding other listed compositions and remove.For example, " A contacts B and C ", including any and all following several feelings Condition：(I) A is mixed with C, and then B is added into mixture；(II) A and B mixing resulting mixtures；B is removed from mixture, so C is added into mixture afterwards；(iii) A is added into B and C mixture." reactant mixture and template contacts " include Any or all of following situations：(i) template contacts generation mixture with first component of the reactant mixture；So Other components of reactant mixture are added into mixture in any order or in combination afterwards；(ii) reactant mixture is template Is thoroughly mixed before mixing

Terms used herein " mixture " refers to the combination without any specific order of composition.Mix ingredients is complicated, Different compositions can not be spatially separated into.Mixture examples include the different elements being dissolved in the identical aqueous solution, perhaps It is many spatially can not be respectively with different elements that are random or being attached to without any specific order in solid support.In other words, One mixture is unaddressable.Specifically, the oligonucleotide arrays that surface known in the art is combined, are not surfaces With reference to oligonucleotide mixture because surface combine oligonucleotides species be that spatially different and array is addressable 's.

A kind of diagnosis of cancer, cancer hair are referred to by " diagnosis " or " assessment " cancer (such as lung cancer or melanoma) The diagnosis in exhibition stage, the type of cancer or the diagnosis of classification, the diagnosis or detection of cancer return, the diagnosis or inspection of cancer progression Survey, the prognosis of cancer, or cancer is to operation or the assessment of the reaction of non-operative treatment.

Generally, the diagnosis of disease or illness is based on commenting to one or more factors and/or the symptom for indicating the disease Valency.That is, diagnosis is the presence based on the factor for representing disease or illness presence or absence, is not present or measures.It is each to be considered as indicating The factor or symptom of specified disease simultaneously need not be uniquely related to specified disease；Namely, it is possible to diagnose factor or symptom from one It is inferred to different diagnostic results.Similarly, it is understood that there may be such situation, indicate that the factor or symptom of specified disease are deposited It is and does not have in the individual of the disease.Diagnostic method can be used alone, or with it is known to certain disease in medical field Or other diagnostic methods and/or the method combined use by stages of illness, for example, lung cancer or melanoma.

Disclosed herein is some number ranges.It is appreciated that each median, unless expressly stated otherwise, to lower limit Unit 1/10th, the upper and lower bound of the scope is also specifically disclosed.In in the scope or in the scope Between each less scope between value and any other described value or median be also contained within the present invention.These smaller models The upper and lower bound enclosed can be included or excluded independently within the range, and be included in any boundary in smaller range, nothing Boundary, or each scope of two boundaries are also included in the present invention, the boundary of any exclusion submitted in the scope.When When the scope includes one or two boundary, the scope including either one or two boundary is not also included within invention.

Term " about " generally refers to up and down the 10% of number.For example, " about 20 " refer between 18 to 22；" about 1 " Refer between 0.9-1.1." about " the meaning may be apparent within a context, such as be rounded up, therefore, and " about 1 " can also Refer between 0.5 to 1.4.

The invention discloses preferred embodiment, but those are skilled artisan will recognize that except of the present invention In addition, other components and condition can be also used in method described herein.

Previous examples and preferred embodiment are construed as explaining that the present invention is wanted without being constrained to claims The present invention asked.It is readily appreciated that, the change and combination of features described above can be not being departed from required by claims of the present invention Scope in use.Such change should not be regarded as departing from the scope of the present invention, and all these changes are intended to be wrapped Include within the scope of following claims.

Embodiment 1

In this embodiment, three mutantional hotspot EGFR T790M, EGFR L858R, EGFR exon 19Del and BRAF V600E/V600K/V600D/V600G/V600A/V600R implements method disclosed above as example.To several types PCR chemistry and nucleic acid modification is tested.Following result proves that enrichment system disclosed by the invention is feasibility.

Table 1A is the blocker used, wherein, EGFR-19del-F-4 is represented for EGFR exon 19deletion's Blocker sequence.

Table 1A blocker sequences

*:Phosphorothioate bond；+:LNA

Table 1B is the primer used；Wherein EGFR_e19 represents the primer sequence for expanding EGFR exon 19deletion Row.

Table 1B primer sequences

EGFR T790M mutation are recurrent gain mutations in patient's body of TKI targeted therapies, in EGFR 790 Mutation on position result in methionine and instead of threonine.The residue of this mutation adds the affinity to ATP and exceeded Affinity of the inhibitor to ATP.Even if the cancer cell for having 5% obtains this mutation, patient also begins to drop to TKI sensitiveness It is low.It is therefore desirable to close supervision T790M appearance.Can assist a physician handle of making decision in the treatment to T790M detection Drug diversion third generation EGFR TKI or pause medication.

The DNA extracted from the cell line being mutated containing EGFR T790M is mixed according to different ratios with wild type DNA And it is enriched with (table 2).Gained library is sequenced with Illumina MiSeq and uses CLC genomes workbench to analyze sequencing data. Enrichment times increase with sample mutation concentration reduction.Research finds, the method disclosed in the present and system can from containing The example enrichment T790M of 0.015% to 8.5% mutation content, is enriched 583 times, and can effectively recognize mutation.

The EGFR T790M of table 2 are enriched with result

EGFR L858R are carcinogenic driving mutation, account for the 43% of all EGFR activation lung cancer.From containing EGFR L858R The DNA extracted in the cell line of mutation is mixed according to different ratios with wild type DNA and according to the method disclosed in the present (table 3) is enriched with system.It is similar with T790M results, 863 times of enrichment can with it is more than sufficient detect 0.01% mutation equipotential Gene.

The EGFR L858R of table 3 are enriched with result

Because blocker is that method and system disclosed above can be in any specific position based on wild-type sequence design The mutation of the more than one type of point enrichment.For example, on the site of BRAF valines 600, method as disclosed above and system have Pass through a pair of blocker enrichments and V600E, V600K, V600D, V600G, V600A and V600R mutation.Table 4 shows, except Outside V600E, BRAF V600 blocker Sync enrichment also from the mixture for three BRAF gene mutations for having wild type background All mutation.

The BRAF valines 600 of table 4 mutation mixture enrichment result

* % is all is mutated the % under wild type background, and single allelic concentration is different

In order to prove repeatability, the enrichment to the EGFR T790M of 8 0.01% carries out repeating (table 5) and enrichment scope is 4.1-7.9%, the difference less than twice.These results are significantly higher than in wild type because the people that nucleotides mistake is mixed and is enriched with Work mutation (up to 1.2%), therefore variation identification is unaffected.In order to detect<The mutation of 0.01% sample, as described below may be used It is determined that self-confident threshold value.Concentration before enrichment and after enrichment is very similar to the monotonic increase relation of logarithmic function.In view of enrichment knot Fruit is highly repeatable, can use logarithmic relationship to estimate the concentration range of the ctDNA before enrichment,

Table 5 is enriched with T790M 8 repetitions from 0.01% sample

The method disclosed in the present can carry out multiplex amplification, show that all three above-mentioned sites can be jointly rich here Collect (table 6).Therefore, the method disclosed in the present can be not only now set up listed by multiple chip (panel) detection table 8 Mutation, can also be covered in the future as the clinical more mutation confirmed.

The triple enrichment results of table 6

Embodiment 2

RNA can more effectively detect various kinase fusions.One ALK or ROS1 fusion transcript can come from multiple dyeing Body weight is arranged, and these, which are reset, occurs including subregion.The cell line for expressing SLC34A2-ROS1 fusions is detected. By up to 100 times of wild type cDNA of RNA reverse transcriptions and dilution, in all cases, fusion transcript can be identified.

In a word, the above results show, system and method disclosed in this invention can detect 0.01% mutation, with height Repeatability.And system can detect multiple mutational sites simultaneously.

The EGFR T790M accumulation rates of table 7 are improved

Embodiment 3

To those ratified therapy in the esoteric mutation design primer of >=1%NSCLC patient and blocker, can Detected with contrived experiment.

Similar with primer, the blocker in each site is confirmed also according to experience, but is usually needed to be optimized.This hair It is bright to have discovered that centralized way to adjust the composition of blocker, therefore optimize blocking efficiency.Table 7 shows each EGFR The effect of T790M blocker and how to adjust raising bioaccumulation efficiency.

Table 8 lists the exercisable driving mutation with clear and definite clinical application, and wherein EGFR is c.2231_ C.2235_2246del12 etc. 2232ins18, EGFR are c.2237_2251del15, EGFR occurs in EGFR exon 19Del.Substance reaction assay is used in each mutation, and those cell line dnas for containing the single mutation in these mutation are titrated to 1%, 0.1% and 0.01% is used to be enriched with.Saltant type and wild type DNA are quantitative with Qubit, and the concentration of 1% sample uses qPCR Being sequenced with Illumina MiSeq confirms.Then the sample dilution from 1% obtains 0.1% and 0.01% sample.

To be enriched with the having of table 8 targets medicine mutation list

Embodiment 4

In this embodiment, enriched multiple and compatibility are detected.

Primer pond is tested and optimized uniformly to expand in uninterrupted thing first.It is permissible in Patients with Non-small-cell Lung body The one group of mutation coexisted is used for instructing Multiple detection.Cell line dna containing these compatibility mutation is mixed.Enriched multiple is anti- 1%, 0.1% and 0.01%DNA mixtures should be carried out respectively, the level of ctDNA in blood is represented respectively.All mutational sites All by Sync enrichment.

The ALK of table 9 and ROS1 fusion lists

Embodiment 5

In this embodiment, the target area enrichment with frequent fusion site is detected.

Primer is designed to be directed to ALK and ROS1 fusion transcripts (table 9).CDNA comprising these cell lines merged is entered Row test.Primer is used for testing amplification efficiency first, then cDNA and wild type cDNA different proportions dilute, and determine Compare believable required the smallest cell amount.

Embodiment 6

To wild type DNA initially enrichment test display occur in that up to 1.2%T790M mutation alleles, this be by The mistake for mixing and amplifying in nucleotides.However, in the elevated error rate observed by EGFR 790 in the Hes of EGFR 858 It is much less on BRAF 600.A large amount of tests have all been carried out to every group of blocker to determine its credible threshold value.In multiple repeating samples 50 wild-type cell systems in this are used as negative control, to determine the scope of the human error on each site.

Cell free DNA (cfDNA) standard items are used to test panel.In brief, above-mentioned mutant DNA titre passes through Ultrasound interrupt toSimilar cfDNA, and tested, to estimate required for each titre carries out reliable detection CfDNA amounts.Cell free RNA standard items are also produced and tested in a similar way.

The sample of NSCLC (non-small cell lung cancer) patient of numbering is removed in liquid biopsy to obtained from BioServe companies 20 Product have carried out preliminary identification.Sample is a pair of samples, and portion is the blood plasma before operation or treatment, and another is FFPE tumor groups Knit.The sample collection of BioServe companies also includes 5 drug resistances obtained from targeted therapy.

The blocker of listed driving mutation is individually tested to reach at least 100 times of enrichment in table 8.Then, they Carry out enriched multiple test.Primer is mixed and optimized so as to expand homogeneous.The uniformity of covering, is defined as the total base existed> 0.2 × average coverage rate, is 90% or higher.Possibility between blocker pond and then progress enriched multiple PCR detection amplified productions Oligomer interference.Problematic primer/blocker can be adjusted.The primer for being used for expanding fusion site in table 9 is all carried out Test to ensure optimum detection.The mutation recognition threshold of each of 56 mutation is individually detected.Sample is handled To determine the uniformity between the pairing tissue and blood sample.

According to preliminary data and to PCR and chemical understanding is blocked, all these mutational sites can be enriched with jointly. But, incompatible if there is blocker/primer, the reaction is divided into two.The mutation list that can be coexisted in NSCLC patient It can be edited (compiled), at least these mutation that can and deposit can be enriched with jointly.

Cross pollution and the generation of false positive should be rare, but be not without possibility.Because simple workflow And low cost, two libraries from same patient can be built parallel to reduce as far as possible, if not elimination, rare pollution With the situation of false positive.

The preferred embodiment of the application is the foregoing is only, the application is not limited to, for the skill of this area For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.

SEQUENCE LISTING

<110>Suzhou Ida health medical science and technology Co., Ltd

<120>Enrichment system and its application based on blocker

<130>

<160> 25

<170> PATENTIN VERSION 3.51

<210> 1

<211> 17

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (16)…(16)

<223>The G of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (17)…(17)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base guanine

<400> 1

cgtgcaactc atcacgg 17

<210> 2

<211> 13

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (12)…(12)

<223>The T of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (13)…(13)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base guanine

<400> 2

catgagctgc gtg 13

<210> 3

<211> 15

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (14)…(14)

<223>The T of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (15)…(15)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base guanine

<400> 3

ggcatgagct gcgtg 15

<210> 4

<211> 15

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (14)…(14)

<223>The T of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (14)…(14)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base thymidine

<400> 4

ggcatgagct gcgtg 15

<210> 5

<211> 18

<212> DNA/RNA

<213>Artificial sequence

<400> 5

tcacagattt tgggctgg 18

<210> 6

<211> 15

<212> DNA/RNA

<213>Artificial sequence

<400> 6

aaactgctgg ttgac 15

<210> 7

<211> 18

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (17)…(17)

<223>The G of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (17)…(17)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base guanine

<400> 7

tcacagattt tgggctgg 18

<210> 8

<211> 14

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (13)…(13)

<223>The G of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (13)…(13)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base guanine

<400> 8

gcagtttggc cagc 14

<210> 9

<211> 27

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (26)…(26)

<223>The C of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (26)…(26)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base cytimidine

<400> 9

gtcgctatca aggaattaag agaagca 27

<210> 10

<211> 15

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (14)…(14)

<223>The A of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (14)…(14)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base adenine

<400> 10

tgcctacgcc accag 15

<210> 11

<211> 16

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (15)…(15)

<223>The T of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (15)…(15)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base thymidine

<400> 11

gtagttggag ctggtg 16

<210> 12

<211> 19

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (19)…(19)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base adenine

<400> 12

tttggtctag ctacagtga 19

<210> 13

<211> 19

<212> DNA/RNA

<213>Artificial sequence

<400> 13

actccatcga gatttcact 19

<210> 14

<211> 21

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (20)…(20)

<223>The guanine of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (20)…(20)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base guanine

<400> 14

attttggtct agctacagtg a 21

<210> 15

<211> 19

<212> DNA/RNA

<213>Artificial sequence

<220>

<221>The base of modification

<222> (18)…(18)

<223>The cytimidine of phosphorothioate bond modification

<220>

<221>The base of modification

<222> (18)…(18)

<223>Lock nucleic acid,

2'-O, 4'-C- methylene Β-D-RIBOSE base cytimidine

<400> 15

actccatcga gatttcact 19

<210> 16

<211> 55

<212> DNA/RNA

<213>Primer

<400> 16

acactctttc cctacacgac gctcttccga tctgtcatag ggactctgga tccca 55

<210> 17

<211> 54

<212> DNA/RNA

<213>Primer

<400> 17

gactggagtt cagacgtgtg ctcttccgat ctccacacag caaagcagaa actc 54

<210> 18

<211> 53

<212> DNA/RNA

<213>Primer

<400> 18

acactctttc cctacacgac gctcttccga tctctccagg aagcctacgt gat 53

<210> 19

<211> 52

<212> DNA/RNA

<213>Primer

<400> 19

gactggagtt cagacgtgtg ctcttccgat ctgtctttgt gttcccggac at 52

<210> 20

<211> 55

<212> DNA/RNA

<213>Primer

<400> 20

acactctttc cctacacgac gctcttccga tctggaacgt actggtgaaa acacc 55

<210> 21

<211> 54

<212> DNA/RNA

<213>Primer

<400> 21

gactggagtt cagacgtgtg ctcttccgat cttgcctcct tctgcatggt attc 54

<210> 22

<211> 57

<212> DNA/RNA

<213>Primer

<400> 22

acactctttc cctacacgac gctcttccga tctttcatga agacctcaca gtaaaaa 57

<210> 23

<211> 52

<212> DNA/RNA

<213>Primer

<400> 23

gactggagtt cagacgtgtg ctcttccgat ctccacaaaa tggatccaga ca 52

<210> 24

<211> 53

<212> DNA/RNA

<213>Primer

<400> 24

acactctttc cctacacgac gctcttccga tctaggcctg ctgaaaatga ctg 53

<210> 25

<211> 54

<212> DNA/RNA

<213>Primer

<400> 25

gactggagtt cagacgtgtg ctcttccgat ctttgttgga tcatattcgt ccac 54

Claims (10)

1. a kind of composition for being used to be enriched with the allelic mutation of target nucleic acid sequence, it is characterised in that the composition includes：

A pair of primer pairs including forward primer and reverse primer, for specific amplification target nucleic acid sequence；

First blocker, includes the first paragraph sequence with following functions：(i) with the wild-type allele of target nucleic acid sequence Match somebody with somebody or complementary；(ii) it can be extended by archaeal dna polymerase.

2. the composition described in claims 1, it is characterised in that

The composition also includes the second blocker, including second segment sequence, its complementary genes with wild-type allele Match somebody with somebody or complementary.

3. the composition described in claims 1 or 2, it is characterised in that

Wherein the first blocker or the second blocker do not have overlapping with forward primer or reverse primer.

4. the composition described in claims 1 or 2, it is characterised in that

First blocker or the second blocker include one or more modification of nucleic acids or key；It is preferred that, the first blocker Or second blocker ' end has one or more modification of nucleic acids or key 3；It is preferred that, wherein modification of nucleic acids or key Including-O- methyl the nucleic acid of PNA, LNA, 2 ', 2 '-O- alkyl nucleic acid, 2 '-fluorescence labeling nucleic acid, phosphorothioate bond and they Any combination.

5. the composition described in claims 1 or 2, it is characterised in that

Wherein target nucleic acid sequence covering coding EGFR T790M, EGFR L858R, EGFR exon 19del, BRAFV600E, That is listed in BRAF V600K, BRAF V600D, BRAF V600G, BRAF V600A, BRAF V600R or table 8 and 9 is any The region of one；Wherein table 8 is：

Table 9 is：

6. the composition described in claims 1 or 2, it is characterised in that

The forward primer length is 10-50 nucleotides, and reverse primer length is 10-50 nucleotides；First or second Blocker length is 5-100 nucleotides.

7. a kind of kit, including：

Composition in claims 1-4 described in any one；

With the reagent needed for one or more amplified reactions；It is preferred that, the reagent includes buffer solution, archaeal dna polymerase, RNase It is one or more kinds of in inhibitor, extension nucleotides and probe.

8. a kind of method of the allelic mutation of enrichment target area, it is characterised in that

Methods described is included using the nucleic acid molecules composition described in claims any one of 1-4, the first blocker and/ Or second blocker exist under conditions of use forward primer and reverse primer amplifying target nucleic acid sequence the step of；It is preferred that, it is described Method target nucleic acid sequential covering coding EGFR T790M, EGFR L858R, EGFR exon 19del, BRAF V600E, BRAF Any one listed in V600K, BRAF V600D, BRAF V600G, BRAF V600A, BRAF V600R or table 8 and 9 Region.

9. a kind of method that allelic mutation detected in target nucleic acid sequence whether there is, it is characterised in that methods described bag Include：

(1) by described in claim 6 be enriched with target area allelic mutation method be enriched with allelic variants come Prepare amplified production；

(2) it whether there is allelic mutation in detection amplified production.

It is preferred that, wherein detecting step is verified by gene sequencing；Methods described target nucleic acid sequential covering is encoded EGFR T790M、EGFR L858R、EGFR exon 19del、BRAF V600E、BRAF V600K、BRAF V600D、BRAF The region of any one listed in V600G, BRAF V600A, BRAF V600R or table 8 and 9.

10. a kind of method for assessing subject's cancer condition, it is characterised in that methods described includes：

(1) biological sample of subject is obtained；With

(2) method in claims 14 carries out detection to biological sample and determines whether there is one or more equipotential Gene mutation；It is preferred that, cancer is adenocarcinoma of lung；It is furthermore preferred that adenocarcinoma of lung is non-cell lung cancer；It is preferred that, biological sample refers to Serum, blood plasma, whole blood, saliva, or phlegm；It is preferred that, methods described also includes being determined according to assessment result or recommends to treat The step of scheme；It is preferred that, the therapeutic scheme includes determining dosage regimen and according to appraisal procedure to controlling according to assessment result Treatment process is monitored.