CN107419018A - A kind of method and kit based on Blocker primers and ARMS primer detection gene mutations - Google Patents
A kind of method and kit based on Blocker primers and ARMS primer detection gene mutations Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6813—Hybridisation assays
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Abstract
The invention discloses a kind of method and kit based on Blocker primers and ARMS primer detection gene mutations.In the present invention, corresponding blocker primers are designed according to annealing temperature, under blocker annealing temperature, Blocker primers are combined with wild-type template, block the amplification of wild type, while the amplification of wild type background is reduced using ARMS technologies.For the present invention by the way that Blocker beneficiation technologies and ARMS fluorescent quantitative PCR techniques are incorporated into a reaction system, a step is that the enrichment and identification to mutator can be achieved, and reduces operating procedure, improves detection sensitivity and specificity.
Description
The application be submit within 2 16th, 2012, application number 201210035665.7 and entitled " one kind is based on
The divisional application of Blocker primers and the method and kit of ARMS primer detection gene mutations " application.
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of method and kit for detecting gene mutation.
Background technology
Gene mutation (gene mutation) is due to that increasing, lacking or change and draw for base-pair occurs in DNA molecular
The change of the gene structure risen.Gene mutation is divided into two kinds, property cell mutation and somatic mutation.Property cell mutation refers to send out
The raw mutation in property cell, it is heritable mutation type.Except the mutation that the extracellular body cell of property occurs will not cause offspring
Hereditary change, can but cause the genetic structure of the present age some cells to change.Most somatic mutations are without phenotype
Effect.Somatic mutation is a kind of rare mutation, and somatic mutation is present in substantial amounts of wild type background dna sequence, relatively
It is seldom in the content of wild type background sequence, somatic mutation content.As contained on a small quantity in tumor patient tissue and peripheral blood
DNA of tumor cell, the bacterium of appearance at initial stage and viral resistance situation etc..Somatic mutation is often related to the morbidity of disease, can be with
The outstanding feature of mark, Index for diagnosis and the mark of medication guide as disease incidence.Therefore, somatic mutation
Detect the diagnosis and treatment for disease and prognostic evaluation has great importance.
Lung cancer is the common malignant tumour of countries in the world today, and turns into the main original of overwhelming majority of countries cancer mortality
Cause.It is wherein most common with non-small cell lung cancer (NSCLC).Targeted therapy has become non-small cell lung cancer clinical treatment at present
Important means.Iressa (Iressa/ Gefitinibs/Gefitinib, AstraZeneca) and Erlotinib (Tarceva/ Erlotinibs/
Erlotinib, Roche Group) EGF-R ELISA (EGFR) tyrosine kinase inhibitor (TKI) is used as, it is U.S. FDA
Ratify the key agents for NSCLC targeted therapies.But clinical test shows Iressa and Erlotinib only to 10-30%'s
Patient NSCLC has significant curative effect.Further investigation revealed that EGFR genetic mutation L858R and 19 Exon deletion (E746_
A750) there is correlation with the effect of NSCLC targeted therapies, the overwhelming majority carries EGFR genetic mutation L858R and 19 extrons
The patient for lacking (E746_A750) is evident in efficacy.But nearly all patient be intended to sooner or later occur EGFR-TKI resistances, middle position without
Evolution time only has 6-12 months.And there is obvious molecular target, i.e. T790M in EGFR-TKI resistances.EGFR wild type genes
Sequence is shown in Genbank No.NM_005228.3, and T790M mutation are that the 2369th base C sports T in the sequence.With reference to EGFR
The detection information of gene mutation makes the cancer immunotherapies for being adapted to NSCLC individual patients the most, is carried for clinician's medication
For medication scientific basis, medical treatment risk and patient economy burden are reduced.
The detection method of somatic mutation at present mainly has the technical method such as DNA sequencing method and mutant enrichment PCR.DNA
PCR sequencing PCR is the reliable method for carrying out abrupt climatic change, and most commonly used method.PCR sequencing PCR compares materials and technical requirements
It is high, it is often more important that, due to the limitation of sequence measurement in itself, sensitivity is not high, and 20% mutator can only be more than to content
Detected.Mutant enrichment PCR is a kind of two-step method PCR that wild type gene is digested with restriction enzyme enzyme selectivity, with
PCR sequencing PCR is compared, and because mutant enrichment PCR has twice PCR amplification, detection mutation sensitivity is higher, and high specificity is stronger, energy
A mutator is detected from 104~105 wild type copies.The key constraints of this method are appropriate restrictions
The selection of restriction endonuclease, complex operation, time-consuming and twice PCR easily pollutes.
Probe amplification retardance abruptly-changing system (amplification refractory mutation system, ARMS)
ARMS also known as allele specific pcr (allele specific PCR, AS-PCR).Principle is：Utilize Taq archaeal dna polymerases
Lack 3 ' -5 ' 5 prime excision enzyme activities, the 3 ' of PCR primer holds the original that last bit bases must could be expanded effectively with its template DNA complementation
Reason, for different known mutations, designs appropriate primer to detect mutator.This method can be 103~104It is individual
A mutator, detection sensitivity height are detected in wild type copies.The key constraints of this method are, if the position of mutation
Point is weak base mismatch sequence, and the specificity of this method will be affected.
Corresponding document report has also been had based on Blocker technologies at present to detect mutational site.Blocker primers are
One section of nucleotide sequence for matching complementation completely with wild type.The application method of Blocker primers mainly has following two at present,
One kind is that Blocker primers and amplimer be not overlapping, and Blocker primers are located at mutational site region, and mutant primer closing is wild
Raw type DNA, without closing saltant type template, therefore saltant type template amplification；Another kind is that Blocker primers and amplimer have
It is overlapping, wild type DNA is closed using Blocker primers, reduces wild-type template DNA amplification.This method can be effectively
The amplification of wild-type template is reduced, improves the Detection capability of mutant DNA template in wild type background.But this method is also deposited
In a problem, during Blocker primers are closed, Blocker can not effectively distinguish saltant type template and wild type
Template, in spite of the difference (being directed to mutational site) of a base, but (the melting under the annealing temperature of primer
Temperature, Tm) Blocker primers are still able to engage with the template of saltant type, or primer also can be with wild pattern
Plate engages, so as to reduce the detection efficiency of the efficiency of wild type closing and saltant type.Therefore, one is provided for mutational site
The detection method that kind is easy, quick, sensitive, specificity is high turns at present clinically urgent need.
Therefore, in order to further improve the closing efficiency of Blocker primers in ARMS+Blocker technologies, what we designed
The annealing temperature of Blocker primers is higher than the annealing temperature of ARMS primers.So, under the annealing temperature of Blocker primers,
Blocker primers are preferentially combined with wild-type template, because Blocker primers differ a base with saltant type template, annealing
Temperature is also variant, so under this condition, Blocker primers are preferentially combined with wild type, without being combined with saltant type, together
When amplimer also do not combined with wild type.Under the annealing temperature condition of amplimer, amplimer will be with saltant type template
With reference to.The amplification of wild type background can be effectively reduced by the above method, improves the detection efficiency of saltant type.
The content of the invention
It is an object of the present invention to provide a kind of side based on Blocker primers and ARMS primer detection gene mutations
Method, this method incorporate Blocker technologies and ARMS fluorescent quantitative PCR techniques.The mutation type that the present invention can detect is main
Including：Point mutation, deletion mutation and insertion mutation etc..
It is a further object to provide a kind of detection kit of gene mutation.The mutation that the present invention can detect
Type mainly includes：Point mutation, deletion mutation and insertion mutation etc..
Therefore, according to the first aspect of the invention, there is provided a kind of Blocker primers for detection in Gene Mutation,
The Blocker primers by with detection sudden change region wild-type sequence complete complementary and 3 ' ends modified to prevent it
The oligonucleotides of extension, wherein, the annealing temperature (Tm values) of the Blocker primers is higher than the annealing temperature of abrupt climatic change primer
(Tm values).Therefore, after DNA profiling denaturation double-strand is opened, under the annealing temperature (Tm) of Blocker primers, Blocker draws
Thing is preferentially combined so as to block abrupt climatic change primer to be combined with wild-type template with wild-type template, then in abrupt climatic change primer
Annealing temperature (Tm) under, abrupt climatic change primer is combined with saltant type template, by PCR react Enrichment Amplification sample DNA in treat
The fragment of cls gene mutation.
In the present invention, it is preferable that the length of the Blocker primers is 15~35bp.
In the present invention, it is preferable that the relevant parameter of the Blocker primers can be：65.0 DEG C -85.0 DEG C of Tm values, GC values
40.0%-65.0%, 25 ± 10bp of primer size.
In the present invention, it is preferable that carry out double deoxidation base modification in 3 ' ends of the Blocker primers, the end is repaiied
Base and the base complementrity of the wild-type sequence of institute's detection sequence are adornd, or is modified such as using other chemical groups：
C3Spacer or phosphorylation modification.
In the present invention, it is preferable that the Blocker primers are introduced directly into chemical group in building-up process in its sequence
Its Tm value is improved, for example, MGB is modified.
In the present invention, it is preferable that dephosphorylation modification is carried out in 5 ' ends of the Blocker primers, to prevent its quilt
Taq polymerase digestion is degraded.
In the present invention, it is preferable that make the annealing temperature of the Blocker primers higher than the annealing temperature of abrupt climatic change primer
5~25 DEG C.It is highly preferred that the Tm values of the Blocker primers are higher than the Tm values of the abrupt climatic change primer 20~25 DEG C.
In one embodiment, the invention provides a kind of Blocker for being used to detect EGFR gene point mutation T790M
Primer, the sequence of the Blocker primers is 5 '-TGCAGCTCATCACGCAGCTCATG-3 ' ddC (SEQ ID NO：1).
In another embodiment, it is used to detect the Exon deletion (E746_ of EGFR gene 19 the invention provides one kind
A750 deletion mutations) Blocker primers, the sequences of the Blocker primers for 5 '-
CAAGGAATTAAGAGAAGCAACATC-3’ddC(SEQ ID NO：9).
According to the second aspect of the invention, there is provided one kind is dashed forward based on Blocker primers and ARMS primer detection genes
The method of change, same anti-including sample DNA, Blocker primers, ARMS primers, TaqMan probe, polymerase and buffer solution
Answer in system, carry out blocking enrichment PCR reactions and the reaction of ARMS quantitative fluorescent PCRs successively, this method comprises the following steps：
1) first under the annealing temperature (Tm) of Blocker primers, Blocker primers and wild-type template preferentially combine from
And block ARMS primers to be combined with wild-type template, and then under the annealing temperature (Tm) of ARMS primers, ARMS primers and mutation
The hardened conjunction of pattern, the fragment for including testing gene sudden change region in Enrichment Amplification sample DNA is reacted by PCR, wherein, it is described
Blocker primers by with detection sudden change region wild-type sequence complete complementary and 3 ' ends modified to prevent its extension
Oligonucleotides, the end of ARMS primers 3 ' is located at sudden change region, the amplifiable fragment for including testing gene sudden change region
And it is consistent with mutant sequences, wherein, the annealing temperature of the Blocker primers is higher than the annealing temperature of ARMS primers.Its
In, mutant sequences refer to point mutation, missing or the nucleotide sequence for inserting base.
2) on the basis of reaction 1), the Taqman probes of ARMS primers and 5 ' end FAM marks are anti-by quantitative fluorescent PCR
Fluoroscopic examination should be carried out to the mutator.
Blocker primers
Process of the present invention it is preferred ground, the Blocker primers (include sudden change region) near sudden change region and set
Meter, and Blocker primers and the wild-type sequence complete complementary of sudden change region and 3 ' ends are modified to prevent its extension.
Process of the present invention it is preferred ground, the length of the Blocker primers is 15~35bp.
Process of the present invention it is preferred ground, the relevant parameter of the Blocker primers can be：65.0 DEG C -85.0 of Tm values
DEG C, GC value 40.0%-65.0%, 25 ± 10bp of primer size.
Process of the present invention it is preferred ground, double deoxidation base modification is carried out in 3 ' ends of the Blocker primers, or
Person is modified such as using other chemical groups：C3Spacer or phosphorylation modification.
In the present invention, it is preferable that the Blocker primers are introduced directly into chemical group in building-up process in its sequence
Its Tm value is improved, for example, MGB is modified.
In the present invention, it is preferable that dephosphorylation modification is carried out in 5 ' ends of the Blocker primers, to prevent its quilt
Taq polymerase digestion is degraded.
ARMS primers
Process of the present invention it is preferred ground, the product of the amplifiable one section of 60-150bp fragment of ARMS primers is described
The relevant parameter of ARMS primers can be：55.0 DEG C -60.0 DEG C of Tm values, GC value 40.0%-60.0%, 20 ± 5bp of primer size.
Process of the present invention it is preferred ground, the terminal bases of ARMS primers 3 ' and the mutating alkali yl of the mutator
It is completely the same.
Process of the present invention it is preferred ground, 3 ' ends of the ARMS primers can be located at sudden change region, and and saltant type
The mutating alkali yl sequence of gene is consistent.To improve specificity, it is preferable that can be in 3 ' ends of the ARMS primers the 2nd, the 3rd
A base mismatch is re-introduced at individual base or the 4th base.Specifically, when it is described sport point mutation when, ARMS primers
3 ' terminal bases can be mutational site at base；When it is described sport deletion mutation when, 3 ' terminal bases of ARMS primers can
For the next base adjacent with missing base；When it is described sport insertion mutation when, 3 ' terminal bases of ARMS primers can be
Insert base.
Process of the present invention it is preferred ground, the annealing temperature of the annealing temperatures of the Blocker primers than the ARMS primers
Spend high 5~25 DEG C.It is highly preferred that the annealing temperature of the Blocker primers it is higher by 20 than the annealing temperature of the ARMS primers~
25℃。
Probe
Probe is designed in ARMS primer amplified fragments, the relevant parameter of the probe is：68.0 DEG C -70.0 DEG C of Tm values,
GC value 40.0%-70.0%, carry out 5 ' end FAM marks to probe, 3 ' ends mark corresponding quenching fluorescence group TAMAR or
BHQ。
In one embodiment, the invention provides an a kind of EGFR gene point mutation T790M step detection method, its
It is characterised by, the sequence of the Blocker primers is 5 '-TGCAGCTCATCACGCAGCTCATG-3 ' ddC (SEQ ID NO：
1), the sequence of the upstream ARMS primers is：5’-CACCGTGCAGCTCATTAT-3’(SEQ ID NO：2), anti-sense primer
Sequence is：5’-CACACACCAGTTGAGCAGGTACT-3’(SEQ ID NO：3)；The sequence of the Taqman probes is：5’-
CCTTCGGCTGCCTCCTGGACTATGT-3’(SEQ ID NO：4).Preferably, reaction condition is：1) 95 DEG C 5 minutes；95℃
15 seconds, 80 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 20 seconds, totally 5 circulation；2) (collect glimmering within 45 seconds within 20 seconds, 60 DEG C within 15 seconds, 80 DEG C for 95 DEG C
Light), totally 40 circulations.
In another embodiment, the invention provides a kind of Exon deletion of EGFR gene 19, (E746_A750 is lacked
Mutation) a step detection method, it is characterised in that the sequence of the Blocker primers is：5’-
CAAGGAATTAAGAGAAGCAACATC-3’ddC(SEQ ID NO：9), the sequence of upstream ARMS primers is：5’-
AATTCCCGTCGCTATCAAAAC-3’(SEQ ID NO：10), the sequence of anti-sense primer is：5’-
ACCCCCACACAGCAAAGC-3’(SEQ ID NO：11), the sequence of the Taqman probes is：5’-
CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3’(SEQ ID NO：12).Preferably, reaction condition is：1) 95 DEG C 5 points
Clock；95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 20 seconds, totally 5 circulation；2) 95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 45 seconds
(collection fluorescence), totally 40 circulations.
According to the third aspect of the present invention, there is provided a kind of detection kit of gene mutation, including：Blocker draws
The Taqman probes of thing, ARMS primers and 5 ' end FAM marks, wherein, the Blocker primers is with being detected sudden change region
Wild-type sequence complete complementary and 3 ' ends modified with prevent its extend oligonucleotides, the end of ARMS primers 3 '
At sudden change region, the amplifiable fragment comprising testing gene sudden change region and consistent with mutant sequences, wherein, it is described
The annealing temperature of Blocker primers is higher than the annealing temperature of ARMS primers.Wherein, mutant sequences refer to point mutation, missing or
Person inserts the nucleotide sequence of base.
Blocker primers
In the kit of the present invention, it is preferable that the Blocker primers (include sudden change region) near sudden change region and set
Meter, and blocker primers and the wild-type sequence complete complementary of sudden change region and 3 ' ends are modified to prevent its extension.
In the kit of the present invention, it is preferable that the length of the Blocker primers is 15~35bp.
In the kit of the present invention, it is preferable that the relevant parameter of the Blocker primers can be：65.0 DEG C -85.0 of Tm values
DEG C, GC value 40.0%-65.0%, 25 ± 10bp of primer size.
In the kit of the present invention, it is preferable that carry out double deoxidation base modification in 3 ' ends of the Blocker primers.
In kit of the present invention, it is preferable that the Blocker primers being introduced directly into its sequence in building-up process
Learn group and improve its Tm value, for example, MGB is modified.
In the kit of the present invention, it is preferable that dephosphorylation modification is carried out in 5 ' ends of the Blocker primers, with
It is prevented to be degraded by Taq polymerase digestion.
ARMS primers
In the kit of the present invention, it is preferable that the product of the amplifiable one section of 60-150bp fragment of ARMS primers, it is described
The relevant parameter of ARMS primers can be：55.0 DEG C -60.0 DEG C of Tm values, GC value 40.0%-60.0%, 20 ± 5bp of primer size.
In the kit of the present invention, it is preferable that 3 ' ends of the ARMS primers are located at sudden change region, and and saltant type
Gene order is consistent., can be in 3 ' ends the 2nd of the ARMS primers or the 3rd base or the 4th base to improve specificity
Place is re-introduced into a base mismatch.Specifically, when it is described sport point mutation when, 3 ' terminal bases of ARMS primers can be prominent
Become base at site；When it is described sport deletion mutation when, 3 ' terminal bases of ARMS primers can be adjacent with missing base
Next base；When it is described sport insertion mutation when, 3 ' terminal bases of ARMS primers can be insertion base.
In the present invention, it is preferable that the annealing temperature of the Blocker primers is higher by 5 than the annealing temperature of the ARMS primers
~25 DEG C.It is highly preferred that the annealing temperature of the Blocker primers is higher than the annealing temperature of the ARMS primers 20~25 DEG C.
Probe
Probe is designed in ARMS primer amplified fragments, the relevant parameter of the probe is：68.0 DEG C -70.0 DEG C of Tm values,
GC value 40.0%-70.0%, carry out 5 ' end FAM marks to probe, 3 ' ends mark corresponding quenching fluorescence group TAMAR or
BHQ。
In one embodiment, the invention provides an a kind of EGFR gene point mutation T790M step detection kit,
Characterized in that, the sequence of the Blocker primers is 5 '-TGCAGCTCATCACGCAGCTCATG-3 ' ddC (SEQ ID NO：
1), the sequence of the upstream ARMS primers is：5’-CACCGTGCAGCTCATTAT-3’(SEQ ID NO：2), anti-sense primer
Sequence is：5’-CACACACCAGTTGAGCAGGTACT-3’(SEQ ID NO：3)；The sequence of the Taqman probes is：5’-
CCTTCGGCTGCCTCCTGGACTATGT-3’(SEQ ID NO：4).Preferably, reaction condition is：1) 95 DEG C 5 minutes；95℃
15 seconds, 80 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 20 seconds, totally 5 circulation；2) (collect glimmering within 45 seconds within 20 seconds, 60 DEG C within 15 seconds, 80 DEG C for 95 DEG C
Light), totally 40 circulations.
In another embodiment, the invention provides a kind of Exon deletion of EGFR gene 19, (E746A750 is lacked
Mutation) a step detection kit, it is characterised in that the sequence of the Blocker primers is：5’-
CAAGGAATTAAGAGAAGCAACATC-3’ddC(SEQ ID NO：9), the sequence of upstream ARMS primers is：5’-
AATTCCCGTCGCTATCAAAAC-3’(SEQ ID NO：10), the sequence of anti-sense primer is：5’-
ACCCCCACACAGCAAAGC-3’(SEQ ID NO：11), the sequence of the Taqman probes is：5’-
CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3’(SEQ ID NO：12).Preferably, reaction condition is：1) 95 DEG C 5 points
Clock；95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 20 seconds, totally 5 circulation；2) 95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 45 seconds
(collection fluorescence), totally 40 circulations.
Terms used herein " mutant sequences " refers to point mutation, missing or the nucleotide sequence for inserting base.
Terms used herein " sudden change region " refers to mutational site, sequence deletion or Sequence Insertion Site.
Terms used herein " consistent " refers to that the nucleotide sequence of the sudden change region with detecting is identical or complementary, herein
It is the nucleotide sequence identical situation with the sudden change region of detection listed by embodiment.
In the present invention, corresponding blocker primers are designed according to annealing temperature, under blocker annealing temperature,
Blocker primers are combined with wild-type template, block the amplification of wild type, while reduce wild type background using ARMS technologies
Amplification.
The present invention by Blocker beneficiation technologies and ARMS fluorescent quantitative PCR techniques by being incorporated into a reaction system
In, a step is that enrichment and identification to mutator can be achieved, and reduces operating procedure, improves detection sensitivity and special
Property.
Brief description of the drawings
Fig. 1 shows T790 site wild plasmids in the case where adding Blocker primers and not adding Blocker primers
Amplification curve.A：Blocker primers, B are not added under 107 copies of T790 sites wild plasmid/μ l backgrounds：T790 sites
Blocker primers are added under 107 copies of wild plasmid/μ l backgrounds.
Fig. 2 shows amplification curve of the T790 site mutation type plasmids in the case where not adding Blocker primers.A：T790
Site mutation type plasmid 104Individual copy/μ l, B：T790 site mutation types plasmid 103Individual copy/μ l, C：T790 site mutation type matter
Grain 102 copies/μ l, D：10 copies of T790 site mutation types plasmid/μ l.
Fig. 3 shows amplification curve of the T790 site mutation type plasmids in the case where adding Blocker primers.A：T790 positions
104 copies of point mutation type plasmid/μ l, B：103 copies of T790 site mutation types plasmid/μ l, C：T790 site mutation type matter
Grain 102 copies/μ l, D：10 copies of T790 site mutation types plasmid/μ l.
Fig. 4 shows that T790 site mutation type plasmids are blended in 107 copies/μ l wild types background plasmids and added
Blocker primer condition amplification curves.A：T790 site mutation types plasmid 104Individual copy/μ l, B：T790 site mutation type plasmids
103Individual copy/μ l, C：102 copies of T790 site mutation types plasmid/μ l, D：10 copy/μ of T790 site mutation types plasmid
l。
Fig. 5 shows that T790 site mutation type plasmids are blended in 107Individual copy/μ l wild types background plasmids are not adding
Blocker condition amplification curves.A：104 copies of T790 site mutation types plasmid/μ l, B：T790 site mutation types plasmid 103
Individual copy/μ l, C：T790 site mutation types plasmid 102Individual copy/μ l, D：10 copies of T790 site mutation types plasmid/μ l.
Fig. 6 shows that 19 deletion segment wild plasmids are adding Blocker primers and do not adding the situation of Blocker primers
Under amplification curve.A：19 deletion segment wild plasmids 107Blocker primers, B are not added under individual copy/μ l backgrounds：19 lack
Unsceptered wild plasmid 107Blocker primers are added under individual copy/μ l backgrounds.
Fig. 7 shows amplification curve of the 19 deficient mutant plasmids in the case where not adding Blocker primers.A：19 missings
Mutant plasmids 104Individual copy/μ l, B：19 deficient mutant plasmids 103Individual copy/μ l, C：19 deficient mutant plasmids 102It is individual
Copy/μ l, D：19 copies of deficient mutant plasmid 10/μ l.
Fig. 8 shows amplification curve of the 19 missing modification plasmids in the case where adding Blocker primers.A：19 deletion mutations
Type plasmid 104Individual copy/μ l, B：19 deficient mutant plasmids 103Individual copy/μ l, C：19 deficient mutant plasmids 102Individual copy/
μ l, D：19 copies of deficient mutant plasmid 10/μ l.
Fig. 9 shows that 19 deficient mutant plasmids are blended in 107 copies/μ l wild types background plasmids and are adding Blocker
Primer condition amplification curve.A：T790 site mutation types plasmid 104Individual copy/μ l, B：T790 site mutation types plasmid 103 is copied
Shellfish/μ l, C：T790 site mutation types plasmid 102Individual copy/μ l, D：10 copies of T790 site mutation types plasmid/μ l.
Figure 10 shows that 19 deficient mutant plasmids are blended in 107 copies/μ l wild types background plasmids and do not added
Blocker condition amplification curves.A：T790 site mutation types plasmid 104Individual copy/μ l, B：T790 site mutation types plasmid 103It is individual
Copy/μ l, C：T790 site mutation types plasmid 102Individual copy/μ l, D：10 copies of T790 site mutation types plasmid/μ l.
Embodiment
With reference to specific embodiment, the invention will be further described, but the present invention is not limited to following examples.Under
State in embodiment, be conventional method unless otherwise specified.
Embodiment
Embodiment 1EGFR point mutations T790M mutation blocks the step detection method of enrichment ARMS quantitative fluorescent PCRs one
1. primer and probe designs
1) Blocker design of primers
(including mutational site) designs primer near mutational site, and 3 ' ends carry out double deoxidation base modification, to prevent
It extends and improves its annealing temperature.Relevant parameter is：63.0 DEG C -80.0 DEG C of Tm values, GC value 40.0%-65.0%, primer are big
Small 25 ± 10bp.
Designed Blocker primer sequences are as follows：
Blocker primers：5’-TGCAGCTCATCACGCAGCTCATG-3’ddC(SEQ ID NO：1)
2) ARMS design of primers
The primer of amplifiable one section of 60-150bp fragment is designed, relevant parameter is：55.0 DEG C -60.0 DEG C of Tm values, GC values
40.0%-60.0%, 20 ± 3bp of primer size.3 ' ends of upstream ARMS primers are located at mutational site, and with saltant type base
Because consistent, to improve specificity, a base mismatch is re-introduced at the base of its 3 ' end the 3rd, designed ARMS draws
Thing sequence is as follows：
Upstream ARMS primers：5’-CACCGTGCAGCTCATTAT-3’(SEQ ID NO：2)
Anti-sense primer：5’-CACACACCAGTTGAGCAGGTACT-3’(SEQ ID NO：3)
3) probe designs
Probe is designed in ARMS primer amplified fragments, relevant parameter is：68.0 DEG C -70.0 DEG C of Tm values, GC values 40.0%-
70.0%, 5 ' end FAM marks are carried out to probe, its sequence is as follows：
Probe：5’-CCTTCGGCTGCCTCCTGGACTATGT-3’(SEQ ID NO：4)
Blocker primers in embodiment 1, ARMS primers, primer and probe is by Shanghai bioengineering Co., Ltd system
It is standby.
2. prepared by detected sample：
Wild plasmid is built：Use respectively containing T790 sites upstream primer sequence 1 (5 '-
TTCACAGCCCTGCGTAAAC-3’(SEQ ID NO：) and (the 5 '-TTTCCACATGCAGATGGGAC- of downstream primer sequence 1 5)
3’(SEQ ID NO：6)) using human genome as template PCR amplifications (PCR reaction conditions：1) 95 DEG C 5 minutes；95 DEG C 15 seconds, 55 DEG C
30 seconds, 72 DEG C 20 seconds, totally 35 circulation；72 DEG C 10 minutes) after, by product cloning to Takara companies pMD19 carriers, through sequencing
Will be standby after sample extraction after checking is correct.Reaction system is as shown in table 1.
Table 1
Mutant plasmids are built：Use respectively containing T790 sites upstream primer sequence 1 (5 '-
TTCACAGCCCTGCGTAAAC-3’(SEQ ID NO：5)) and downstream primer sequence 2 (5 '-
CATGAGCTGCaTGATGAGCTGCA-3’(SEQ ID NO：8)) using wild plasmid as template PCR amplifications after recovery product 1.
Then (5 '-TGCAGCTCATCAtGCAGCTCATG-3 ' (the SEQ ID NO of upstream primer sequence 2 are used：7)) with wild plasmid
For recovery product after template PCR amplifications 2.It is template with product 1 and product 2, uses the upstream primer sequence 1 containing T790 sites
(5’-TTCACAGCCCTGCGTAAAC-3’(SEQ ID NO：5)) and downstream primer sequence 1 (5 '-
TTTCCACATGCAGATGGGAC-3’(SEQ ID NO：6)) PCR is expanded.By product cloning to Takara companies pMD19 carriers,
After sequence verification is correct by sample extraction after standby (lowercase in upstream primer sequence 2 and downstream primer sequence 1 represents
Saltant type site 790).The PCR reaction conditions and reaction system and wild plasmid structure of mutant plasmids structure are identical.
3. detection method：The mutation of the present invention blocks one step detection method of enrichment that Blocker technologies are applied into mutation position
Point PCR blocks enrichment, ARMS quantitative fluorescent PCRs, enters performing PCR successively in a reaction system and blocks enrichment and ARMS fluorescence to determine
PCR identifications are measured, detailed process is that the reaction system that 20 μ l are included to component in following table on fluorescent PCR instrument (contains sample to be checked
DNA profiling) carry out first it is high-temperature denatured make genome double-strand open；Under 80 DEG C of annealing temperatures, Blocker primers and wild type
Gene template, which combines, blocks ARMS primers in connection；The combination of ARMS primers and wild-type template is obstructed during 60 DEG C of annealing,
ARMS primers can not expand wild-type template；And the combination of saltant type template and ARMS primers, 72 DEG C of extensions cause comprising mutation
Base wants detection part to be enriched with by selectivity amplification.Then the detection reaction in mutational site is run, also due to Blocker draws
Thing reduces the amplification of wild type gene, reduces its interference to mutated genes to the sealing process of wild type；Prolong at 60 DEG C
Stretch 45 seconds, collect the fluorescence signal of mutation amplification.Reaction system is as shown in table 2.
Table 2
Reaction condition is as follows：1) 95 DEG C 5 minutes；95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 20 seconds, totally 5 are followed
Ring；2) 95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 45 seconds (collection fluorescence), totally 40 circulation.Quantitative real time PCR Instrument used is ABI
STEPONE (Appliedbiosystem companies).
Experimentation sets wild plasmid template to be added without Blocker primers and adds Blocker primers respectively
Experimental group, while the mutant plasmids to being separately added into various concentrations under wild type background (107 copy/μ l) simulate mutation
Type sample is detected, and sets the experimental group for being added without Blocker primers and adding Blocker primers respectively；Set simultaneously
Mutant plasmids template use corresponding to putting is added without Blocker primer systems and detected.
Interpretation of result：Experimental result is as shown in figure 1, the wild type sample for not adding the group of Blocker primers has amplification
Curve, after adding Blocker primers, the amplification curve of wild plasmid is closed, without amplification curve.Fig. 2, Fig. 3, Fig. 4 and
Fig. 5 shows that wild plasmid background signal can be effectively blocked after adding Blocker primers, being capable of effective detection aggregate sample
Mutant plasmids in this；Blocker primers are not added, the wild plasmid and saltant type matter that reaction system can not be distinguished effectively
Grain sample；Meanwhile reaction system does not change for the detectability of wild plasmid after adding Blocker primers.
In summary, the reaction system of addition Blocker primers can be under the background concn of wild plasmid effectively
Detection saltant type.
The mutation of the Exon deletion E746_A750 deletion mutations of embodiment 2EGFR genes 19 blocks enrichment ARMS fluorescence to determine
Measure the step detection methods of PCR mono-
1. primer and probe designs
1) Blocker design of primers
Primer is designed in deletion mutation areas adjacent (including deletion sequence), 3 ' ends carry out double deoxidation base modification, with
Prevent it from extending and improve its annealing temperature.Relevant parameter is：65.0 DEG C -80.0 DEG C of Tm values, GC value 40.0%-65.0%, draw
25 ± 10bp of thing size.
Designed Blocker primer sequences are as follows：
Blocker primers：5’-CAAGGAATTAAGAGAAGCAACATC-3’ddC(SEQ ID NO：9)
2) ARMS design of primers
The primer of amplifiable one section of 60-150bp fragment is designed, relevant parameter is：55.0 DEG C -60.0 DEG C of Tm values, GC values
40.0%-60.0%, 20 ± 3bp of primer size.3 ' ends of upstream ARMS primers are located at mutational site, and with saltant type base
Because consistent, to improve specificity, a base mismatch, designed ARMS are re-introduced at the base of its 3 ' end the 3rd
Primer sequence is as follows：
Upstream ARMS primers：5’-AATTCCCGTCGCTATCAAAAC-3’(SEQ ID NO：10)
Anti-sense primer：5’-ACCCCCACACAGCAAAGC-3’(SEQ ID NO：11)
3) probe designs
Probe is designed in ARMS primer amplified fragments, relevant parameter is：68.0 DEG C -70.0 DEG C of Tm values, GC values 40.0%-
70.0%, 5 ' end FAM marks are carried out to probe, its sequence is as follows：
Probe：5’-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3’(SEQ ID NO：12)
Above-mentioned Blocker primers, ARMS primers, primer and probe is by Shanghai bioengineering Co., Ltd.
2. sample preparation：
Wild plasmid is built：Use respectively containing E746_A750 deletion segments upstream primer sequence 1 (5 '-
GCCTAGACGCAGCATCATTA-3’(SEQ ID NO：13)) and downstream primer sequence 1 (5 '-
ATGCCTCCATTTCTTCATCC-3’(SEQ ID NO：14)) using human genome as template PCR amplifications (PCR reaction conditions：1)
95 DEG C 5 minutes；95 DEG C 15 seconds, 55 DEG C 30 seconds, 72 DEG C 20 seconds, totally 35 circulation；72 DEG C 10 minutes) after, by product cloning extremely
Takara companies pMD19 carriers, after sequence verification is correct by sample extraction after it is standby.Reaction system is as shown in table 3.
Table 3
Mutant plasmids are built：Use respectively containing E746_A750 deletion segments upstream primer sequence 1 (5 '-
GCCTAGACGCAGCATCATTA-3’(SEQ ID NO：13)) and downstream primer sequence 2 (5 '-
CGGAGATGTttTGATAGCGACGGGAATT-3’(SEQ ID NO：16)) using wild plasmid as template PCR amplifications after reclaim
Product 1.Then (5 '-TCGCTATCAaaACATCTCCGAAAGCCAAC-3 ' (the SEQ ID NO of upstream primer sequence 2 are used：
) and (5 '-ATGCCTCCATTTCTTCATCC-3 ' (the SEQ ID NO of downstream primer sequence 1 15)：14)) using wild plasmid as mould
Recovery product 2 after plate PCR amplifications.
Be template with product 1 and product 2, using containing E746_A750 deletion segments upstream primer sequence 1 (5 '-
GCCTAGACGCAGCATCATTA-3’(SEQ ID NO：13)) and downstream primer sequence 1 (5 '-
ATGCCTCCATTTCTTCATCC-3’(SEQID NO：14)) PCR is expanded.By product cloning to Takara companies pMD19 carriers,
After sequence verification is correct by sample extraction after standby (lowercase in upstream primer sequence 2 and downstream primer sequence 1 represents
E746_A750 deletion segments).
The PCR reaction conditions and reaction system and wild plasmid structure of mutant plasmids structure are identical.
3. detection method：The mutation of the present invention blocks one step detection method of enrichment that Blocker technologies are applied into mutation position
Point PCR blocks enrichment, ARMS quantitative fluorescent PCRs, enters performing PCR successively in a reaction system and blocks enrichment and ARMS fluorescence to determine
PCR identifications are measured, detailed process is that the reaction system that 20 μ l are included to component in following table on fluorescent PCR instrument (contains sample to be checked
DNA profiling) carry out first it is high-temperature denatured make genome double-strand open；Under 80 DEG C of annealing temperatures, Blocker primers and wild type
Gene template, which combines, blocks ARMS primers in connection；The combination of ARMS primers and wild-type template is obstructed during 60 DEG C of annealing,
ARMS primers can not expand wild-type template；And the combination of saltant type template and ARMS primers, 72 DEG C of extensions cause comprising mutation
Base wants detection part to be enriched with by selectivity amplification.Then the detection reaction in mutational site is run, also due to Blocker draws
Thing reduces the amplification of wild type gene, reduces its interference to mutated genes to the sealing process of wild type；Prolong at 60 DEG C
Stretch 45 seconds, collect the fluorescence signal of mutation amplification.Reaction system is as shown in table 4.
Table 4
Reaction condition is as follows：1) 95 DEG C 5 minutes；95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 20 seconds, totally 5 are followed
Ring；2) 95 DEG C 15 seconds, 80 DEG C 20 seconds, 60 DEG C 45 seconds (collection fluorescence), totally 40 circulation.Quantitative real time PCR Instrument used is ABI
STEPONE (Appliedbiosystem companies).
Experimentation sets wild plasmid template to be added without Blocker primers and adds Blocker primers respectively
Experimental group, while the mutant plasmids to being separately added into various concentrations under wild type background (107 copy/μ l) simulate mutation
Type sample is detected, and sets the experimental group for being added without Blocker primers and adding Blocker primers respectively；Set simultaneously
Mutant plasmids template use corresponding to putting is added without Blocker primer systems and detected.
Interpretation of result：Experimental result is as shown in fig. 6, the wild type sample for not adding the group of Blocker primers has amplification
Curve, after adding Blocker primers, the amplification curve of wild plasmid is closed, without amplification curve.Fig. 7, Fig. 8, Fig. 9 and
Figure 10 shows that wild plasmid background signal can be effectively blocked after adding Blocker primers, being capable of effective detection aggregate sample
Mutant plasmids in this；Blocker primers are not added, the wild plasmid and saltant type matter that reaction system can not be distinguished effectively
Grain sample；Meanwhile reaction system does not change for the detectability of wild plasmid after adding Blocker primers.
In summary, the reaction system of addition Blocker primers can be under the background concn of wild plasmid effectively
Detection saltant type.
Sequence table
<110>Jiangsu Hong Weitesi Pharmaceutical Technology Co., Ltd
<120>A kind of method and kit based on Blocker primers and ARMS primer detection gene mutations
<130> 201707
<160> 16
<170> PatentIn version 3.3
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Claims (3)
1. a kind of method based on Blocker primers and ARMS primer detection gene mutations, draws including sample DNA, Blocker
In thing, ARMS primers, TaqMan probe, the same reaction system of polymerase and buffer solution, carry out blocking enrichment PCR reactions successively
Reacted with ARMS quantitative fluorescent PCRs, this method comprises the following steps：
1) first under the annealing temperature of Blocker primers, Blocker primers are preferentially combined so as to block with wild-type template
ARMS primers are combined with wild-type template, and then under the annealing temperature of ARMS primers, ARMS primers are combined with saltant type template,
The fragment for including testing gene sudden change region in Enrichment Amplification sample DNA is reacted by PCR, wherein, the Blocker primers are
Modified with the wild-type sequence complete complementary of detected sudden change region and 3 ' ends to prevent the oligonucleotides that it extends, institute
3 ' the ends for stating ARMS primers are located at sudden change region, amplifiable fragment and and saltant type comprising testing gene sudden change region
Sequence is consistent, wherein, it is re-introduced into a mistake at the base of 3 ' ends of the ARMS primers the 2nd, the 3rd or the 4th base
With base, wherein, the annealing temperature of the Blocker primers is higher than the annealing temperature of the ARMS primers 20~25 DEG C；
2) on the basis of reaction 1), the Taqman probes of ARMS primers and 5 ' end FAM marks pass through quantitative fluorescent PCR reaction pair
The mutator carries out fluoroscopic examination；
Wherein described gene mutation is EGFR gene point mutation T790M, wherein, the sequences of the Blocker primers for 5 '-
TGCAGCTCATCACGCAGCTCATG-3’ddC(SEQ ID NO:1), the sequence of the upstream ARMS primers is：5’-
CACCGTGCAGCTCATTAT-3’(SEQ ID NO:2), the sequence of anti-sense primer is：5’-
CACACACCAGTTGAGCAGGTACT-3’(SEQ ID NO:3)；The sequence of the Taqman probes is：5’-
CCTTCGGCTGCCTCCTGGACTATGT-3’(SEQ ID NO:4)。
2. the method for claim 1, wherein carry out double deoxidation base modification in 3 ' ends of the Blocker primers；
And/or dephosphorylation modification is carried out in 5 ' ends of the Blocker primers, to prevent it from being degraded by Taq polymerase digestion.
3. the method for claim 1, wherein the gene mutation is the Exon deletion E746_A750 of EGFR gene 19
Deletion mutation, wherein, the sequence of the Blocker primers is：5’-CAAGGAATTAAGAGAAGCAACATC-3’ddC(SEQ
ID NO：9), the sequence of upstream ARMS primers is：5’-AATTCCCGTCGCTATCAAAAC-3’(SEQ ID NO：10), downstream
The sequence of primer is：5’-ACCCCCACACAGCAAAGC-3’(SEQ ID NO：11), the sequence of the Taqman probes is：
5’-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3’(SEQ ID NO：12).
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| CN107419018B (en) | 2020-09-04 |
| CN103255201B (en) | 2017-07-14 |
| CN103255201A (en) | 2013-08-21 |
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