CN117904263A - Primer pair and method for specifically amplifying target gene by OTARMS system - Google Patents
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- 230000000903 blocking effect Effects 0.000 claims abstract description 14
- 230000035772 mutation Effects 0.000 claims description 28
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Abstract
The invention belongs to the technical field of biology, and discloses a primer pair and a method for specifically amplifying a target gene by utilizing OTARMS systems. The primer pair comprises a first primer and an amplification blocking primer which is matched with the first primer, wherein a first part of the amplification blocking primer is complementarily combined with a target gene, and a second part of the amplification blocking primer is an open tail end. The amplification blocking primer anneals with the first amplification product as a whole primer to form a completely complementary fragment from the 2 nd amplification cycle, and the binding force between the amplification blocking primer and the first amplification product is greater than the binding force between the first part and the target gene. The amplification blocking primer can specifically bind with a target gene to complete extension amplification, and block mutant sequence or non-target sequence amplification, so that high-efficiency specific amplification of a highly similar gene is realized. The introduction of the second part of the amplification blocking primer greatly improves the binding force of the amplification primer and the amplified product, and further ensures the specificity of amplification.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to a gene amplification method, and particularly relates to a primer pair and a method for specifically amplifying a target gene by utilizing OTARMS systems.
Background
Genes are basic units of inheritance, and nucleic acid sequences carrying genetic information transfer genetic information to the next generation by replication, and control the expression of traits in biological individuals by directing the synthesis of proteins. The gene detection technique is a technique for detecting DNA or RNA by blood, tissue or other nucleic acid-carrying samples, and is a technique for amplifying nucleic acids in a sample to be detected and displaying nucleic acid information by a specific means or a specific device. In practical applications, it is often necessary to base-identify or differentially amplify a nucleic acid at a certain site (e.g., SNP site). If the amplified target sequences need to be identified or distinguished, however, when sequences similar to or even highly similar to the sequences to be detected exist in the sample to be detected, the actual detection is not challenged, and particularly when only few or even several base-inconsistent sequences exist, the differential amplification is more difficult.
Therefore, it is necessary to design a simple, easy-to-use, low-cost detection system that can accurately identify different sequences of a single site difference.
Disclosure of Invention
The present invention aims to overcome at least one of the disadvantages of the prior art and provides a primer pair and a method for specifically amplifying a target gene using OTARMS (Open Terminal Amplification Refractory Mutation System, open-end amplification block mutation system) system.
The technical scheme adopted by the invention is as follows:
In a first aspect of the invention, there is provided: a primer pair for specifically amplifying a target gene, comprising a first primer and an amplification blocker primer for use in pairing, the amplification blocker primer consisting of a first portion and a second portion, wherein: the first part is complementarily combined with the target gene, and can guide base synthesis to initiate template amplification; the second part is an open end and cannot form a complementary fragment with the target gene, the first part and the second part are used as an integral primer to anneal with the first amplified product to form a completely complementary fragment from the 2 nd amplification cycle, and the binding force between the amplification blocking primer and the first amplified product is greater than the binding force between the first part and the target gene.
In some examples of primer pairs, the first portion of the amplification blocker primer is 13 to 23 bases in length.
In some primer pair examples, the 3' end of the first portion of the amplification blocker primer is disposed at a position where the target gene is not base identical to the non-target gene.
In some primer pair examples, the 3 'end of the first portion of the amplification blocker primer introduces at least 1 base different from both the target gene and the non-target gene base to the 5' end in the direction of the 2 nd to 5 th base.
In some examples of primer pairs, the 3 'end of the first portion of the amplification blocker primer introduces 1 to 3, 1 or 2 bases different from both the target gene and the non-target gene bases to the 5' end in the direction of the 2 nd to 5 th base.
In some primer pair examples, the first portion of the amplification blocker primer is 13-23 bases in length and the 3' end of the first portion of the amplification blocker primer is disposed at a position where the target gene is not identical to the non-target gene base.
In some examples of primer pairs, the base that is different from both the target gene and the non-target gene base is C.
In some examples of primer pairs, the second portion of the amplification blocker primer is 6 to 15 bases in length.
In some examples of primer pairs, the amplification blocker primer second portion balances the GC content of the target fragment.
In some examples of primer pairs, the temperature at which the first portion of the amplification blocker primer binds to the target gene to initiate amplification of the template is between 56 and 66 ℃.
In some primer pair examples, the second portion of the amplification blocker primer is 6-15 bases in length and balances the GC content of the target fragment.
In some primer pair examples, the second portion of the amplification blocker primer is 6-15 bases in length and the temperature at which the first portion of the amplification blocker primer binds to the target gene to trigger template amplification is 56-66 ℃.
In some primer pair examples, the second portion of the amplification blocker primer is 6-15 bases in length, the second portion of the amplification blocker primer balances the GC content of the target fragment, and the temperature at which the first portion of the amplification blocker primer binds to the target gene initiates template amplification is 56-66 ℃.
In some examples of primer pairs, the first primer also features the amplification blocker primer.
The above technical features may be combined arbitrarily without collision.
In a second aspect of the invention, there is provided: a method for specifically amplifying a target gene using an open-end amplification block mutation system, comprising the steps of:
s1) extracting to obtain a gene template to be detected;
s2) adding the primer pair of the first aspect of the invention into a template, and amplifying to obtain a target gene.
The beneficial effects of the invention are as follows:
The primer group of some examples of the invention can specifically bind with a target gene to complete extension amplification, and simultaneously has the capability of blocking amplification of a mutant sequence or a non-target sequence, thereby realizing efficient and specific amplification of highly similar genes such as SNP.
According to the primer group provided by the embodiment of the invention, the second part of the amplification blocking primer (OTARMS primer) is introduced to greatly improve the binding force of the amplification primer and an amplified product, the amplification primer and a mutant gene or a non-target gene have differences of more than or equal to 1 and 2 bases, the amplified primer and the target product can be annealed normally and complemented and extended at a higher temperature, the possibility of combining the amplification primer and the mutant gene or the non-target gene is greatly reduced, and the purpose of further blocking the amplification of the mutant gene or the non-target gene is achieved.
The primer group of some examples of the invention, the open sequence introduced by the amplification blocking primer gradually becomes the main amplification template in the subsequent amplification, and OTARMS primer and the template containing the open sequence can be completely matched, so that the amplification efficiency of the primer of the completely matched template is increased, namely the system ensures that the amplification of mutant genes or non-target gene sequences can be blocked by the earlier cycle, and after the absolute advantage of the specific sequence is ensured, another set of sequence primers for triggering the amplification is started, so that the target template amplification can be completed with higher efficiency in the subsequent amplification.
The primer group of some examples of the invention has both open sequence and open design, so that the primer group can be conveniently applied to different technical platforms, including but not limited to the following platforms and detection methods:
1) The high-throughput sequencing platform is characterized in that the sequence is set to be complementary with a sequencing-used adapter or a general amplification primer through the design of an open end, multiple steps such as end repair and addition of 'A', addition of 'adapter' and the like are not needed, and a complete library structure can be constructed by introducing the sequencing-related adapter or the general amplification primer through PCR amplification.
2) The QPCR detection platform is used for qualitative detection: and adding fluorescent fuel or fluorescent probe into the amplification system to perform real-time fluorescent detection.
3) The QPCR detection platform is used for quantitatively detecting: and an internal standard gene amplification system is added into the amplification system, so that whether the detection target is single copy or double copy can be determined by the ratio of the detection target to the internal standard gene. The results may be reported by fluorescent fuel or fluorescent probe.
4) In the PCR detection method, after amplification of the amplification system is finished, a result report can be carried out through agarose gel electrophoresis, when a target gene is contained in a to-be-detected object, the agarose gel electrophoresis has a band with a corresponding size, and when the target gene is not contained in the to-be-detected object, the agarose gel electrophoresis does not have a band with a corresponding size; the results can also be reported by Sanger sequencing.
Drawings
FIG. 1 is an electrophoresis chart of the detection result of example 1.
FIG. 2 is an electrophoretogram of the detection result of example 2.
FIG. 3 is an electrophoretogram of the detection result of example 3.
Detailed Description
An open-ended amplification block mutation system (Open Terminal Amplification Refractory Mutation System, OTARMS) for gene amplification and detection of interest, wherein the OTARMS system consists essentially of OTARMS primers with specifically designed and capable of blocking mutant sequences or non-target sequences, allowing for 3 combinations of the system: ① Forward OTARMS primer + normal reverse primer; ② Forward OTARMS primer + reverse OTARMS primer; ③ Forward normal primer + reverse OTARMS primer.
The OTARMS primer structure consists of 2 parts of an amplification part (first part) and an open end (second part).
The amplification part of OTARMS primer is the complementary binding part with target gene and has length of 13-23 bases, and the part can guide base synthesis at the priming temperature to prime template amplification. Furthermore, the 3' end of the amplification part is arranged at a position where the base of the target gene is inconsistent with that of the mutant gene or the non-target gene, so that the specificity of amplification is improved, and non-specific amplification is reduced; further, at least 1 base different from the base of the target gene and the base of the mutant gene or the base of the non-target gene is introduced into the position of the 2 nd to 5 th bases in the direction from the 3 'end to the 5' end of the amplification part (for example, the base of the target gene and the mutant gene which are point mutation A to T and the base of the target gene and the base of the mutant gene or the base of the non-target gene which are different can be C or G), and preferably the base is C. By introducing at least 1, preferably 1 to 3, more preferably 1 to 2 bases different from both the bases of the target gene and the non-target gene, a greater steric resistance is introduced, achieving a greater retarding effect. The C base is more sterically hindered than the other A, C, T bases and is a better choice.
The open end of OTARMS primer has length of 6-15 bases, and the fragment can not form complementary fragment with target gene, and the sequence has no special requirement.
The first and second portions of the OTARMS primer anneal to the round 1 product as a single primer on cycle 2 to form a fully complementary fragment, the first portion of the OTARMS primer and round 1 amplification product having a binding force to each other > OTARMS primer binding to the target genome, so that starting with cycle 3 of the amplification reaction, primer OTARMS as a single primer priming amplification is the dominant amplification and the second portion as part of the amplified fragment, the product located most exotic to the product will become the dominant product. The difference between the product and the original mutant sequence or non-target sequence is more than or equal to 1 and 2 bases, and the primer is completely matched with the product, so that the amplification of the mutant sequence or non-target sequence is more likely to be blocked at a higher annealing temperature. Meanwhile, the sequence of the second part can balance the GC content of the target fragment, and the advantages are more obvious in fragments with too high or too low GC content; the method is beneficial to reducing the nonspecific amplification of complex target fragments, especially repetitive region fragments and improving the amplification efficiency thereof; in amplification, the second part, which is part of the amplified fragment, is located most exogenously in the product, avoiding the effect of the ultralong genome on the amplification process.
The first and second portions of OTARMS primers may be directly ligated or may be ligated using a Spacer commonly used in the art. The usual spacers are straight carbon chains or ethylene glycol (C3 or C6).
① Number combination: forward OTARMS primer + normal reverse primer forward OTARMS primer contains 2 parts: a forward amplification portion and an open end, the amplification portion being complementary to the annealing of the target fragment, initiating amplification; common reverse primers are primers that anneal to complementary fragments of the target fragment to complement the primer that initiates synthesis.
② Number combination: forward OTARMS primer + reverse OTARMS primer, forward OTARMS primer contains 2 parts, forward amplified part and open ends: annealing complementation of the amplification part and the target fragment, and initiation of amplification; the reverse OTARMS primer contained 2 parts, the reverse amplified part and the open end: the reverse amplified portion and the complementary fragment of the target fragment are capable of annealing to complement-prime synthesis.
③ Number combination: forward common primer + reverse OTARMS primer, the forward common primer is the primer which is annealed and complemented with the target fragment to trigger synthesis; the reverse OTARMS primer contained 2 parts: reverse amplified portion and open end, reverse amplified portion and complementary fragment of target fragment are capable of annealing complementary priming synthesis.
The technical scheme of the invention is further described below by combining examples.
Example 1: detection of KRAS Gene G12A mutation
The mutation site G12A detection is carried out by designing OTARMS primer amplification system aiming at KRAS genes.
1. Sample DNA extraction and concentration determination:
And extracting genomic DNA of the KRAS G12A positive sample and the negative sample from the FFPE sample to be detected by using a genome extraction kit, measuring the concentration and the purity by using an ultraviolet spectrophotometer, and determining that the quality of the extracted DNA is qualified.
2. Gene detection site selection and OTARMS primer design:
Obtaining KRAS genes, selecting 500bp sequences at the upstream and downstream of a G12A mutation site, designing amplification primers aiming at the mutation site, selecting OTARMS primers for designing the upstream primers, completely matching the tail end with the genome of the mutation site, simultaneously adding 1 mismatched base at the 4 th position inwards at the 3' end, designing 6bp in the second part, and connecting the first part and the second part through 1 spacer. The specific primer sequences are as follows:
G12A-OTARMS forward primer: GGACTC-Spacer-GCACTCTTGCCTACGCCAC (SEQ ID NO.: 1)
G12A-OTARMS reverse primer: GTACAGTTCATTACGATACACGTCT (SEQ ID NO.: 2).
3. Amplification reaction system:
The system formulation was carried out according to the following table
| Reagent name | Concentration of | 1 Human consumption/. Mu.L |
| PCR Buffer | 10× | 2.5 |
| dNTP MIX | 10 mM | 0.5 |
| Upstream primer | 10 μM | 1 |
| Downstream primer | 10 μM | 1 |
| dd H2O | / | 15 |
| DNA template | 1 copy/μL | 5 |
| Total | / | 25 |
4. Amplification reaction conditions:
Denaturation at 95℃for 5min was followed by 40 cycles of 15s at 95℃and 30s at 60 ℃.
5. And (3) judging results:
Amplification product determination by agarose gel electrophoresis
When the sample to be detected has the strip consistent with the positive sample, judging that the sample to be detected carries the G12A mutation, and when the sample to be detected does not have the strip consistent with the positive sample, judging that the sample to be detected does not carry the G12A mutation. In the experimental process, the negative sample cannot be provided with a strip consistent with the positive sample, each of the yin-yang templates is provided with 2 compound holes, 1-2 holes are KRAS G12A negative samples, and 3-4 holes are KRAS G12A negative samples. The result of electrophoresis is shown in FIG. 1.
Example 2: detection of KRAS Gene G13D mutation
And designing OTARMS primer amplification system aiming at KRAS genes to detect mutation site G13D.
1. Sample DNA extraction and concentration determination:
And extracting genomic DNA of the KRAS G13D positive sample and the negative sample from the FFPE sample to be detected by using a genome extraction kit, measuring the concentration and the purity by using an ultraviolet spectrophotometer, and determining that the quality of the extracted DNA is qualified.
2. Gene detection site selection and OTARMS primer design:
Obtaining KRAS genes, selecting 500bp sequences at the upstream and downstream of a G13D mutation site, designing amplification primers aiming at the mutation site, selecting OTARMS primers for designing the upstream primers, completely matching the tail end with the genome of the mutation site, simultaneously adding 1 mismatched base at the 4 th position inwards at the 3' end, designing 6bp in the second part, and connecting the first part and the second part through 1 spacer. The specific primer sequences are as follows:
G13D-OTARMS forward primer: CCTAGA-Spacer-TTGTTGGATCATATTCCTC (SEQ ID NO.: 3)
G13D-OTARMS reverse primer: TAGCTC-Spacer-ATACACGTCTGCAGTCAACTG (SEQ ID NO.: 4).
3. Amplification reaction system:
The system formulation was carried out according to the following table
| Reagent name | Concentration of | 1 Human consumption/. Mu.L |
| PCR Buffer | 10× | 2.5 |
| dNTP MIX | 10 mM | 0.5 |
| Upstream primer | 10 μM | 1 |
| Downstream primer | 10 μM | 1 |
| dd H2O | / | 15 |
| DNA template | 1 copy/μL | 5 |
| Total | / | 25 |
4. Amplification reaction conditions:
Denaturation at 95℃for 5min was followed by 40 cycles of 15s at 95℃and 30s at 60 ℃.
5. And (3) judging results:
Amplification product determination by agarose gel electrophoresis
When the sample to be detected has the strip consistent with the positive sample, judging that the sample to be detected carries the G13D mutation, and when the sample to be detected does not have the strip consistent with the positive sample, judging that the sample to be detected does not carry the G13D mutation. In the experimental process, the negative sample cannot be provided with a strip consistent with the positive sample, each of the positive and negative templates is provided with 2 complex holes 1 and 3 holes which are KRAS G13D positive samples, the 2 holes and 4 holes are KRAS G13D negative samples, and the electrophoresis result is shown in figure 2.
Example 3: detection of EGFR gene L858R mutation
Mutation site L858R detection was performed against EGFR gene design OTARMS primer amplification system.
1. Sample DNA extraction and concentration determination:
and extracting genome DNA of the EGFR L858R positive sample and the negative sample from the FFPE sample to be detected by using a genome extraction kit, measuring the concentration and the purity by using an ultraviolet spectrophotometer, and determining that the quality of the extracted DNA is qualified.
2. Gene detection site selection and OTARMS primer design:
The EGFR gene is obtained, a 500bp sequence at the upstream and downstream of the L858R mutation site is selected, an amplification primer design is carried out aiming at the mutation site, a OTARMS primer is selected, the tail end of the primer is completely matched with the genome of the mutation site, meanwhile, 1 mismatched base is added at the 4 th position inwards at the 3' end, 6bp is designed in the second part, and the first part and the second part are connected through 1 spacer. The specific primer sequences are as follows:
L858R-OTARMS Forward primer: CCCTGAATTCGGATGCAGAGC (SEQ ID NO.: 5)
L858R-OTARMS reverse primer: GTTACT-Spacer-TCCGCACCCAGCAGTTTCGCCA (SEQ ID NO.: 6).
3. Amplification reaction system:
The system formulation was carried out according to the following table
| Reagent name | Concentration of | 1 Human consumption/. Mu.L |
| PCR Buffer | 10× | 2.5 |
| dNTP MIX | 10 mM | 0.5 |
| Upstream primer | 10 μM | 1 |
| Downstream primer | 10 μM | 1 |
| dd H2O | / | 15 |
| DNA template | 1 copy/μL | 5 |
| Total | / | 25 |
4. Amplification reaction conditions:
Denaturation at 95℃for 5min was followed by 40 cycles of 15s at 95℃and 30s at 60 ℃.
5. And (3) judging results:
Amplification product determination can be performed by agarose gel electrophoresis. When the sample to be detected has the strip consistent with the positive sample, the sample to be detected is judged to carry the L858R mutation, and when the sample to be detected does not have the strip consistent with the positive sample, the sample to be detected is judged not to carry the L858R mutation. In the experimental process, the negative sample cannot be provided with a strip consistent with the positive sample, each of the positive and negative templates is provided with 2 compound holes 1,3 holes which are EGFR L858R positive samples, 2 and 4 holes which are EGFR L858R negative samples, and the electrophoresis result is shown in figure 3.
The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.
Claims (10)
1. A primer pair for specifically amplifying a target gene, comprising a first primer and an amplification blocker primer for use in pairing, the amplification blocker primer consisting of a first portion and a second portion, wherein: the first part is complementarily combined with the target gene, and can guide base synthesis to initiate template amplification; the second part is an open end and cannot form a complementary fragment with the target gene, the first part and the second part are used as an integral primer to anneal with the first amplified product to form a completely complementary fragment from the 2 nd amplification cycle, and the binding force between the amplification blocking primer and the first amplified product is greater than the binding force between the first part and the target gene.
2. The primer pair of claim 1, wherein the first portion of the amplification block primer is 13-23 bases in length.
3. The primer pair of claim 1, wherein the 3' end of the first portion of the amplification block primer is disposed at a position where the target gene is not base identical to the non-target gene.
4. The primer set according to any one of claims 1 to 3, wherein the 3 '-end of the first portion of the amplification block primer introduces at least 1 base different from the bases of the target gene and the non-target gene to the position of the 2 nd to 5 th bases in the 5' -end direction.
5. The primer pair of claim 4, wherein the base different from both the base of the target gene and the base of the non-target gene is C.
6. The primer pair of claim 1, wherein the second portion of the amplification blocker primer is 6-15 bases in length.
7. The primer pair of claim 1, wherein the amplification blocker primer second portion balances GC content of the target fragment.
8. The primer pair of claim 1,6 or 7, wherein the temperature at which the first portion of the amplification blocker primer binds to the target gene to trigger template amplification is between 56 and 66 ℃.
9. The primer pair of claim 1, wherein the first primer also features the amplification block primer.
10. A method for specifically amplifying a target gene using an open-end amplification block mutation system, comprising the steps of:
s1) extracting to obtain a gene template to be detected;
s2) adding the primer pair as set forth in any one of claims 1 to 9 into a template, and amplifying to obtain a target gene.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000047766A1 (en) * | 1999-02-11 | 2000-08-17 | Astrazeneca Ab | Method for detecting variant nucleotides using arms multiplex amplification |
| CN106755388A (en) * | 2016-11-24 | 2017-05-31 | 厦门艾德生物医药科技股份有限公司 | A kind of improved ARMS primer constructions (Super ARMS) and its application method |
| CN111118119A (en) * | 2019-12-18 | 2020-05-08 | 杭州瑞普基因科技有限公司 | Method for detecting target mutation by carrying out retardation substitution amplification enrichment based on blocker introducing extra base mismatch |
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- 2024-03-01 CN CN202410236490.9A patent/CN117904263A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000047766A1 (en) * | 1999-02-11 | 2000-08-17 | Astrazeneca Ab | Method for detecting variant nucleotides using arms multiplex amplification |
| CN106755388A (en) * | 2016-11-24 | 2017-05-31 | 厦门艾德生物医药科技股份有限公司 | A kind of improved ARMS primer constructions (Super ARMS) and its application method |
| CN111118119A (en) * | 2019-12-18 | 2020-05-08 | 杭州瑞普基因科技有限公司 | Method for detecting target mutation by carrying out retardation substitution amplification enrichment based on blocker introducing extra base mismatch |
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