CN106755388B - A kind of improved ARMS primer construction (Super-ARMS) and its application method - Google Patents
A kind of improved ARMS primer construction (Super-ARMS) and its application method Download PDFInfo
- Publication number
- CN106755388B CN106755388B CN201611159646.XA CN201611159646A CN106755388B CN 106755388 B CN106755388 B CN 106755388B CN 201611159646 A CN201611159646 A CN 201611159646A CN 106755388 B CN106755388 B CN 106755388B
- Authority
- CN
- China
- Prior art keywords
- primer
- arms
- sequence
- super
- improved
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000010276 construction Methods 0.000 title abstract 2
- 230000000295 complement effect Effects 0.000 claims abstract description 10
- 125000006850 spacer group Chemical group 0.000 claims abstract description 7
- 230000035772 mutation Effects 0.000 claims description 17
- 239000007850 fluorescent dye Substances 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 description 22
- 238000003753 real-time PCR Methods 0.000 description 17
- 239000000523 sample Substances 0.000 description 16
- 230000035945 sensitivity Effects 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 230000003321 amplification Effects 0.000 description 11
- 238000003199 nucleic acid amplification method Methods 0.000 description 11
- 238000001514 detection method Methods 0.000 description 9
- 238000011144 upstream manufacturing Methods 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 238000002844 melting Methods 0.000 description 6
- 230000008018 melting Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000012634 fragment Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000010355 oscillation Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- 102200124919 rs121913237 Human genes 0.000 description 3
- 102200124923 rs121913254 Human genes 0.000 description 3
- 101150039808 Egfr gene Proteins 0.000 description 2
- 101150105104 Kras gene Proteins 0.000 description 2
- 101150073096 NRAS gene Proteins 0.000 description 2
- 108700021358 erbB-1 Genes Proteins 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of improved ARMS primer construction (Super-ARMS) and its application methods, it include: positive special primer, it includes following three parts: 1) First ray, the length is 18-40 bases, wherein the 1st bit base of 3 ' end is mutated site, there are a base mismatch in 3 ' ends 2-4, remaining base and sequence to be measured are complementary；2) the second sequence, the length is 6-20 base, and 3 ' terminal sequence of First ray are complementary；3) spacer connects 5 ' ends of First ray and 3 ' ends of the second sequence；Reversed universal primer, the length is another chain complementations of 15-40 base and sequence to be measured；Wherein, the Tm value of the reverse primer is 3-8 DEG C lower than the Tm value of the First ray of forward primer.The present invention can be improved the specificity of ARMS.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an improved ARMS primer structure (in the following content of the invention, referred to as Super-ARMS for short) and a using method thereof.
Background
The ARMS technology is a new method developed on the basis of PCR for detecting DNA point mutation, and a mutation amplification system (ARMS) is also called Allele Specific Amplification (ASA) and is a method established by Newton and the like for detecting known mutation firstly. The basic principle is as follows: taq DNA polymerase lacks 3 ' → 5 ' exonuclease activity, so for a primer with a mismatched 3 ' end, the primer is extended at a speed lower than that of a normal end pairing primer, when the number of mismatched bases reaches a certain degree or the conditions reach a certain stringency degree, the 3 ' end base cannot be extended due to difficulty in phosphodiester bond formation, the reaction is terminated, and an amplified fragment with specific length cannot be obtained, thereby indicating that the template DNA does not have a mutation corresponding to the 3 ' end of the primer; if the PCR result can obtain an amplified fragment of a specific length, it is indicated that the template DNA has a mutation corresponding to the 3' -end of the primer.
However, the existing ARMS primers have limited ability to distinguish some mutations and limited detection sensitivity, and therefore, the detection sensitivity of the technology needs to be improved.
Disclosure of Invention
The present invention aims to provide an improved ARMS primer structure (Super-ARMS) to solve the above problems in the prior art.
The technical scheme provided by the invention is as follows:
an improved ARMS primer structure (Super-ARMS), comprising:
the forward specific primer comprises the following three parts:
1) the length of the first sequence is 15-40 bases, the 1 st base at the 3 'end is a mutation position, the 2 nd-4 th base at the 3' end is a mismatched base, and the rest bases are complementary with the sequence to be detected;
2) a second sequence, 6-20 bases in length, which is complementary to the first sequence;
3) a spacer linking the 5 'end of the first sequence and the 3' end of the second sequence;
the reverse universal primer has the length of 15-40 basic groups and is complementary with the other strand of the sequence to be detected;
wherein the Tm value of the reverse universal primer is 3-8 ℃ lower than the Tm value of the first sequence of the forward primer.
The improved ARMS primer structure (Super-ARMS) of the invention, under normal conditions or suitable conditions (e.g. room temperature), the first sequence and the second sequence of the forward primer are combined into a double strand; when the primer structure recognizes and hybridizes with the target sequence, the double-stranded binding portion is opened, the two are connected by Spacer, and the primer binds with the target sequence to obtain DNA extension capability.
In the present invention, the sequence to be tested may be a wild-type gene sequence or a mutant-type gene sequence. That is, the first sequence base may be fully complementary to the wild-type gene sequence, but not to the mutant; or may be complementary to the mutant type but not to the wild type.
Preferably, the mismatch type of the bases requires the introduction of a "weak" mismatch (C/A or G/T) if the 3 'end is a "strong" mismatch (A/G or C/T) or blocks the amplification of the 3' end without the introduction of a mismatch, and vice versa; similarly, when the 3' end is a "moderate" mismatch (A/A, C/C, G/G or T/T), it is desirable to introduce a "moderate" mismatch.
Preferably, the first sequence is 18 to 30 bases in length.
Preferably, the second sequence is 8-16 bases in length.
Preferably, the spacer may be one selected from C18, C3, C6, C12, and the like.
Use of unnatural nucleotides: any unnatural nucleotide, such as LNA, PNA, can be used at any position of the primer.
The invention provides an improved ARMS primer structure (Super-ARMS), which can be used for the amplification detection of specific target sequences in real-time PCR. The real-time PCR instrument suitable for the primers at present comprises: 7300, 7500, 7700, 7900, Stepone Plus, CFX96 from Bio-Rad, IQ Cycler, LightCycler2.0 from Roche, LightCycler480, Rotor-Gene Q from Qiagen, Mx3000P and Mx3005P from Stratagene, and the like, Applied Biosystems (ABI).
The invention also adopts the technical scheme that:
an improved ARMS-PCR system comprising the improved ARMS primer structure (Super-ARMS) described above.
Preferably, the improved ARMS-PCR system further comprises a fluorescent probe.
Preferably, the improved ARMS-PCR system further comprises a Block primer, and the length of the Block primer is 15-50 bp. Under a proper annealing temperature, the Block primer is preferentially combined with a wild template so as to Block the combination of the ARMS primer and the wild template, the ARMS primer can be combined with a mutation target sequence, and the PCR reaction is used for enriching and amplifying the DNA of the mutation template so as to improve the detection sensitivity.
Preferably, the Block primer is 25-45bp in length, is modified and blocked with-NH 2 at the 3 ' end of the Block primer, perfectly matches with the wild-type template, is as long as the 5 ' end of the ARMS primer, and has a mutation site at the 0 th-5 th base position of the 3 ' end of the Block primer. In addition to the-NH 2 modification, phosphorylation, dideoxy base modification, and the like can be employed.
The invention also adopts a technical scheme that:
an AMRS-PCR method, which adopts an improved ARMS primer structure (Super-ARMS) in the PCR process.
Preferably, the method further comprises the Block primer described above.
Preferably, the method further comprises the use of a fluorescent probe.
The type of the fluorescent probe of the present invention is not limited, and various existing structures may be adopted, for example: linear probes, stem-loop structure probes, and the like.
The fluorescent probes are also not limited in the type of fluorescer and quencher, and include: FAM, HEX, VIC, ROX, BHQ, TAMRA, etc
The invention has the following advantages: by adopting the Super-ARMS primer structure, the nonspecific amplification can be obviously reduced, and the specificity can be improved. The Super-ARMS primer structure improves the distinguishing capability between the mutant type and the wild type, so that the amplification efficiency of a reaction system can be adjusted by a plurality of factors, and the detection sensitivity can be as high as 1 copy/mu L.
Drawings
FIG. 1 is a schematic diagram of a primer structure according to the present invention;
FIG. 2 is a schematic diagram of the present invention;
FIG. 3 is a schematic diagram of the reaction in the presence of Block;
FIG. 4 is a melting curve of the Super-ARMS primer of example 1;
FIG. 5 is a comparison of ARMS primers with Super-ARMS primers of example 1 (upper panel-ARMS primers, lower panel)
-Super-ARMS primer)
FIG. 6 is a graph showing the results of sensitivity examination of the Super-ARMS primer of example 1;
FIG. 7 is the melting curve of the Super-ARMS primer of example 2
FIG. 8 is a comparison of ARMS primers with Super-ARMS primers in example 2 (upper panel-ARMS primers, lower panel-Super-ARMS primers)
FIG. 9 is a graph showing sensitivity examination of the Super-ARMS primer in example 2.
FIG. 10 is the melting curve of the Super-ARMS primer of example 3
FIG. 11 shows the ARMS primers and the Super-ARMS primers in example 2 (upper panel-ARMS primers, lower panel-Super-ARMS primers)
FIG. 12 is a graph showing sensitivity examination of the Super-ARMS primers in example 3.
Detailed Description
Example 1: Super-ARMS primer for detecting T790M mutation of EGFR gene
In order to examine the influence of the Super-ARMS primer on the specificity and sensitivity of a real-time PCR system, 1 pair of specific primers aiming at the T790M mutation of the EGFR gene is designed, and the length of an amplified fragment is 105 bp. The PCR template is cell line genome DNA.
The formula of the reaction solution is as follows:
| material(s) | Concentration of | Dosage (mu L) |
| H2O | Purified water | 42.345 |
| PCR buffer solution | 10× | 8 |
| MgCl2 | 25mM | 13.6 |
| dNTPs | 25mM | 0.8 |
| Specific upstream primer F | 100μM | 0.03 |
| Specific downstream primer R | 100μM | 0.03 |
| Specific probe | 100μM | 0.08 |
| Blcok primer | 100μM | 0.04 |
| Internal control primer F | 100μM | 0.025 |
| Internal control primer R | 100μM | 0.025 |
| Internal control probe | 100μM | 0.025 |
| Total volume | / | 65 |
The method comprises the following operation steps: taking out the reaction solution with the corresponding volume from the reaction solution tube according to the ratio of 60 mu L of the reaction solution to 0.4 mu L of the mixed enzyme in each tube of reagent, adding the mixed enzyme with the corresponding volume according to the ratio, uniformly mixing for 15 seconds by oscillation, quickly centrifuging for 15 seconds, subpackaging the mixture into PCR reaction tubes according to the amount of 65.4 mu L in each tube, respectively adding 15 mu L of samples to be detected, carefully covering a PCR tube cover, and quickly centrifuging for a plurality of seconds. The PCR reaction tube was placed in a real-time PCR instrument.
Real-time PCR reactions were performed in Stratagene Mx3000PTMThe real-time PCR is carried out on a real-time PCR instrument.
The first stage is as follows: 10 minutes at 95 ℃ for 1 cycle;
and a second stage: at 95 ℃ for 40 seconds, at 64 ℃ for 40 seconds, at 72 ℃ for 30 seconds, for 15 cycles;
and a third stage: 28 cycles of 93 ℃ for 40 seconds, 60 ℃ for 45 seconds, 72 ℃ for 30 seconds;
wherein,
the upstream primer is as follows:
the downstream primer is: 5'-AGTTGAGCAGGTACTGGGAG-3' the flow of the air in the air conditioner,
Block：5′-TCACCTCCACCGTGCARCTCATCAC-3′-NH2
spacer is Spacer C18
Y represents a mixed base C + T
R represents a mixed base A + G
The specific probe is FAM-5'-AGCTCATGCCCTTCGGCTGCC-3' -BHQ1
The internal control primer F is 5'-TCCAGGAGGTGGCTGGTTAT-3'
The internal control primer R is 5'-CATATTTCCTCTGATGATCTGCAGGTT-3'
The internal control probe is HEX-5'-CTCATTGCCCTCAACACAGTGGAGC-3' -BHQ1
The result judgment method comprises the following steps: the smaller the Ct value of the cell line and the plasmid is, the higher the amplification efficiency is; a larger delta Ct difference between the cell line or plasmid and the non-specific amplification indicates better specificity.
Comparative example
Common ARMS upstream primer: 5 '-TCACCTCCACCGTGCARCTCATCTT-3'
A downstream primer: 5'-TTGAGCAGGTACTGGGAGCCAAT-3' the flow of the air in the air conditioner,
other conditions and operations were the same as those in example 1
The results are as follows:
FIG. 4 shows the experimental results of melting curves of the dye-based secondary structure analysis of the Super-ARMS primer designed according to the method of the present invention, and the results show that the Tm value of the primer is around 68 ℃.
As can be seen from FIG. 5, the amplification efficiency and specificity of the Super-ARMS primer are better than those of the ordinary ARMS primer, the delta Ct value of the ordinary ARMS primer is about 3, and the delta Ct value of the Super-ARMS primer can reach 7. As shown in the results of FIG. 6, the detection sensitivity of the Super-ARMS primer pair T790M positive cell line (H1975) can be as low as 1 copy/. mu.L.
Example 2: application of Super-ARMS primer in detection of KRAS gene G12D mutation
In order to examine the influence of the Super-ARMS primer on the specificity and sensitivity of a real-time PCR system, 1 pair of specific primers aiming at the KRAS gene G12D mutation is designed, and the length of an amplified fragment is 145 bp. The PCR template is cell line genome DNA.
The formula of the reaction solution is as follows:
| material(s) | Concentration of | Dosage (mu L) |
| H2O | Purified water | 38.438 |
| PCR buffer solution | 10× | 8 |
| MgCl2 | 25mM | 17.1 |
| dNTPs | 25mM | 1 |
| Internal control primer F | 100μM | 0.04 |
| Internal control primer R | 100μM | 0.04 |
| Internal control probe | 100μM | 0.04 |
| Specific upstream primer F | 100μM | 0.1 |
| Specific downstream primer R | 100μM | 0.1 |
| Specific probe | 100μM | 0.092 |
| Block primer | 100μM | 0.05 |
| Total volume | 65 |
The method comprises the following operation steps: taking out the reaction solution with the corresponding volume from the reaction solution tube according to the ratio of 60 mu L of the reaction solution to 0.4 mu L of the mixed enzyme in each tube of reagent, adding the mixed enzyme with the corresponding volume according to the ratio, uniformly mixing for 15 seconds by oscillation, quickly centrifuging for 15 seconds, subpackaging the mixture into PCR reaction tubes according to the amount of 65.4 mu L in each tube, respectively adding 15 mu L of samples to be detected, carefully covering a PCR tube cover, and quickly centrifuging for a plurality of seconds. The PCR reaction tube was placed in a real-time PCR instrument.
Real-time PCR reactions were performed in Stratagene Mx3000PTMThe real-time PCR is carried out on a real-time PCR instrument.
The first stage is as follows: 10 minutes at 95 ℃ for 1 cycle;
and a second stage: at 95 ℃ for 40 seconds, at 64 ℃ for 40 seconds, at 72 ℃ for 30 seconds, for 15 cycles;
and a third stage: 30 cycles of 93 ℃ for 40 seconds, 60 ℃ for 45 seconds, 72 ℃ for 30 seconds;
upstream primer
The downstream primer is: 5'-TGCACCAGTAATATGCATATTAAAACAAGATT-3' the flow of the air in the air conditioner,
Block：5′-TGACTGAATATAAACTTGTGGTAGTTGGAGCTGG-3′-NH2
the specific probe is FAM-5'-AGGCAAGAGTGCCTTGACGATACAGCTAA-3' -BHQ1
The internal control primer F is 5'-TCTCGGCCTCCCAAGGTGTT-3'
The internal control primer R is 5'-TCCACACAGCAGCAAGTGATAGT-3'
The internal control probe is HEX-5'-TAGGTGCCTAGCCCATGGTGCC-3' -BHQ1
Comparative example
Common ARMS upstream primer: 5'-TGACTGAATATAAACTTGTGGTAGTTGGAGCTCA-3'
A downstream primer: 5'-TCCTGCACCAGTAATATGCATATTAAAACAAGATTTAC-3' the flow of the air in the air conditioner,
other conditions and operations were the same as in example 2
The results are as follows:
FIG. 7 shows the experimental results of melting curves of the dye-based secondary structure analysis of the Super-ARMS primer designed according to the method of the present invention, and the results show that the Tm value of the primer is about 67 ℃.
FIG. 8 shows that the amplification efficiency and specificity of the Super-ARMS primer are better than those of the common ARMS primer when the common ARMS primer and the Super-ARMS primer are used for detecting the same sample.
From the results in FIG. 9, the detection sensitivity of the Super-ARMS primer pair G12D positive cell line (LS174T) can be as low as 1 copy/. mu.L.
Example 3: Super-ARMS primer for detecting NRAS gene Q61K mutation
In order to examine the influence of the Super-ARMS primer on the specificity and sensitivity of a real-time PCR system, 1 pair of specific primers aiming at the NRAS gene Q61K mutation is designed, and the length of an amplified fragment is 98 bp. The PCR template is cell line genome DNA.
The formula of the reaction solution is as follows:
| material(s) | Concentration of | Dosage (mu L) |
| H2O | Purified water | 23.225 |
| PCR buffer solution | 10× | 5 |
| MgCl2 | 25mM | 11 |
| dNTPs | 25mM | 0.5 |
| Internal control primer F | 100μM | 0.03 |
| Internal control primer R | 100μM | 0.03 |
| Internal control probe | 100μM | 0.03 |
| Specific upstream primer F | 100μM | 0.05 |
| Specific downstream primer R | 100μM | 0.05 |
| Specific probe | 100μM | 0.045 |
| Block primer | 100μM | 0.04 |
| Total volume | 40 |
The method comprises the following operation steps: taking out the reaction solution with the corresponding volume from the reaction solution tube according to the ratio of 40 mu L of reaction solution to 0.6 mu L of mixed enzyme in each tube of reagent, adding the mixed enzyme with the corresponding volume according to the ratio, uniformly mixing for 15 seconds by oscillation, quickly centrifuging for 15 seconds, subpackaging the mixture into PCR reaction tubes according to the amount of 40.6 mu L in each tube, respectively adding 10 mu L of samples to be detected, carefully covering a PCR tube cover, and quickly centrifuging for a plurality of seconds. The PCR reaction tube was placed in a real-time PCR instrument.
Real-time PCR reactions were performed in Stratagene Mx3000PTMThe real-time PCR is carried out on a real-time PCR instrument.
The first stage is as follows: 10 minutes at 95 ℃ for 1 cycle;
and a second stage: at 95 ℃ for 40 seconds, at 64 ℃ for 40 seconds, at 72 ℃ for 30 seconds, for 15 cycles;
and a third stage: 30 cycles of 93 ℃ for 40 seconds, 60 ℃ for 45 seconds, 72 ℃ for 30 seconds;
the upstream primer is as follows: 5'-AGGATTCTTACAGAAAACAAGTGGT-3' the flow of the air in the air conditioner,
downstream primer
Block：5′-GTCTCTCATGGCACTGTACTCTTCTTGTC-3′-NH2
The specific probe is FAM-5'-CCAGCTGTATCCAGTATGTCCAACAAACAGG-3' -BHQ1
The internal control primer F is 5'-TCATTGCACCAGTAACTCCAGC-3'
The internal control primer R is 5'-CTGAGTTTTCCAGTCATGGTAAAGG-3'
The internal control probe is HEX-5'-CTGACCTCTTCTTTCCTTGCAGGGCTA-3' -BHQ1
Comparative example
An upstream primer: 5'-CCAGGATTCTTACAGAAAACAAGTGGT-3' the flow of the air in the air conditioner,
common ARMS downstream primers: 5'-GTCTCTCATGGCACTGTACTCTTCTGT-3'
Other conditions and operations were the same as those in example 3
The results are as follows:
FIG. 10 shows the results of melting curve experiments of the dye-based secondary structure analysis of the Super-ARMS primer designed according to the method of the present invention, and the results show that the Tm value of the primer is around 66 ℃.
FIG. 11 shows that the amplification efficiency and specificity of the Super-ARMS primer are better than those of the common ARMS primer when the common ARMS primer and the Super-ARMS primer are used for detecting the same sample.
From the results in FIG. 12, the detection sensitivity of the Super-ARMS primer pair Q61K positive cell line (NCI-H1299) can be as low as 1 copy/. mu.L.
<110> Xiamen Ed biomedical science and technology Co., Ltd
<120> an improved ARMS primer structure (SUper-ARMS) and methods of use thereof
<160>27
<210>1
<211>39
<212>DNA
<213> Artificial sequence
<400>1
AAGATGAGYT GCACTCACCT CCACCGTGCA RCTCATCTT 39
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<400>2
AGTTGAGCAG GTACTGGGAG 20
<210>3
<211>25
<212>DNA
<213> Artificial sequence
<400>3
TCACCTCCAC CGTGCARCTC ATCAC 25
<210>4
<211>21
<212>DNA
<213> Artificial sequence
<400>4
AGCTCATGCC CTTCGGCTGC C 21
<210>5
<211>20
<212>DNA
<213> Artificial sequence
<400>5
TCCAGGAGGT GGCTGGTTAT 20
<210>6
<211>27
<212>DNA
<213> Artificial sequence
<400>6
CATATTTCCT CTGATGATCT GCAGGTT 27
<210>7
<211>25
<212>DNA
<213> Artificial sequence
<400>7
CTCATTGCCC TCAACACAGT GGAGC 25
<210>8
<211>25
<212>DNA
<213> Artificial sequence
<400>8
TCACCTCCAC CGTGCARCTC ATCTT 25
<210>9
<211>23
<212>DNA
<213> Artificial sequence
<400>9
TTGAGCAGGT ACTGGGAGCC AAT 23
<210>10
<211>48
<212>DNA
<213> Artificial sequence
<400>10
TGAGCTCCAA CTACTGACTG AATATAAACT TGTGGTAGTT GGAGCTCA 48
<210>11
<211>32
<212>DNA
<213> Artificial sequence
<400>11
TGCACCAGTA ATATGCATAT TAAAACAAGA TT 32
<210>12
<211>34
<212>DNA
<213> Artificial sequence
<400>12
TGACTGAATA TAAACTTGTG GTAGTTGGAG CTGG 34
<210>13
<211>29
<212>DNA
<213> Artificial sequence
<400>13
AGGCAAGAGT GCCTTGACGA TACAGCTAA 29
<210>14
<211>20
<212>DNA
<213> Artificial sequence
<400>14
TCTCGGCCTC CCAAGGTGTT 20
<210>15
<211>23
<212>DNA
<213> Artificial sequence
<400>15
TCCACACAGC AGCAAGTGAT AGT 23
<210>16
<211>22
<212>DNA
<213> Artificial sequence
<400>16
TAGGTGCCTA GCCCATGGTG CC 22
<210>17
<211>34
<212>DNA
<213> Artificial sequence
<400>17
TGACTGAATA TAAACTTGTG GTAGTTGGAG CTCA 34
<210>18
<211>38
<212>DNA
<213> Artificial sequence
<400>18
TCCTGCACCA GTAATATGCA TATTAAAACA AGATTTAC 38
<210>19
<211>25
<212>DNA
<213> Artificial sequence
<400>19
AGGATTCTTA CAGAAAACAA GTGGT 25
<210>20
<211>43
<212>DNA
<213> Artificial sequence
<400>20
ACAGAAGAGT ACAGTGGTCT CTCATGGCAC TGTACTCTTC TGT 43
<210>21
<211>29
<212>DNA
<213> Artificial sequence
<400>21
GTCTCTCATG GCACTGTACT CTTCTTGTC 29
<210>22
<211>31
<212>DNA
<213> Artificial sequence
<400>22
CCAGCTGTAT CCAGTATGTC CAACAAACAG G 31
<210>23
<211>22
<212>DNA
<213> Artificial sequence
<400>23
TCATTGCACC AGTAACTCCA GC 22
<210>24
<211>25
<212>DNA
<213> Artificial sequence
<400>24
CTGAGTTTTC CAGTCATGGT AAAGG 25
<210>25
<211>27
<212>DNA
<213> Artificial sequence
<400>25
CTGACCTCTT CTTTCCTTGC AGGGCTA 27
<210>26
<211>27
<212>DNA
<213> Artificial sequence
<400>26
CCAGGATTCT TACAGAAAAC AAGTGGT 27
<210>27
<211>27
<212>DNA
<213> Artificial sequence
<400>27
GTCTCTCATG GCACTGTACT CTTCTGT 27
Claims (10)
1. An improved ARMS primer structure, comprising:
the forward specific primer comprises the following three parts:
1) the length of the first sequence is 18-40 bases, wherein the 1 st base at the 3 'end is a mutation position, the 2 nd-4 th base at the 3' end is a mismatched base, and the rest bases are complementary with the sequence to be detected;
2) a second sequence, 6-20 bases in length, which is complementary to the first sequence;
3) a spacer linking the 5 'end of the first sequence and the 3' end of the second sequence;
the reverse universal primer has the length of 15-40 basic groups and is complementary with the other strand of the sequence to be detected;
wherein the Tm value of the reverse primer is 3-8 ℃ lower than the Tm value of the first sequence of the forward primer.
2. The improved ARMS primer structure of claim 1, wherein: the first sequence is 18-30bp in length.
3. The improved ARMS primer structure of claim 1, wherein: the second sequence is 8-16bp in length.
4. An improved ARMS-PCR system comprising the improved ARMS primer structure of claim 1.
5. An improved ARMS-PCR system as claimed in claim 4, further comprising a blocker primer.
6. An improved ARMS-PCR system as claimed in claim 5, wherein the blocker primer is 15-50bp in length.
7. An improved ARMS-PCR system according to claim 4, 5 or 6, further comprising a fluorescent probe.
8. An AMRS-PCR method, characterized in that: it uses an improved ARMS primer structure as described in claim 1.
9. The AMRS-PCR method of claim 8, wherein: a blocker primer was also used.
10. The AMRS-PCR method of claim 8, wherein: also included is the use of fluorescent probes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/112816 WO2018095401A1 (en) | 2016-11-24 | 2017-11-24 | Improved arms primer structure (super-arms) and usage method therefor |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611042783 | 2016-11-24 | ||
| CN2016110427835 | 2016-11-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106755388A CN106755388A (en) | 2017-05-31 |
| CN106755388B true CN106755388B (en) | 2019-08-23 |
Family
ID=58887597
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611159646.XA Active CN106755388B (en) | 2016-11-24 | 2016-12-15 | A kind of improved ARMS primer construction (Super-ARMS) and its application method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN106755388B (en) |
| WO (1) | WO2018095401A1 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755388B (en) * | 2016-11-24 | 2019-08-23 | 厦门艾德生物医药科技股份有限公司 | A kind of improved ARMS primer construction (Super-ARMS) and its application method |
| CN108048531B (en) * | 2017-11-16 | 2021-06-18 | 苏州吉玛基因股份有限公司 | Ultra-blocking fluorescent quantitative PCR method for detecting rare mutation with high sensitivity |
| CN108753935A (en) * | 2018-06-15 | 2018-11-06 | 上海优甲医疗科技有限公司 | A kind of method that primer dimer expands in reduction multiplex polymerase chain re-action |
| CN108823285A (en) * | 2018-07-09 | 2018-11-16 | 博奥生物集团有限公司 | A kind of the Loop formula enrichment detection primer and its application of low abundance mutant DNA |
| CN109136345A (en) * | 2018-09-21 | 2019-01-04 | 北京知光基因科技有限公司 | A kind of PCR method and its application expanded and detect low-content gene mutation |
| CN109628589A (en) * | 2018-11-20 | 2019-04-16 | 北京昱晟达医疗科技有限公司 | Detect primer, kit and the method for gene mutation |
| CN111534599A (en) * | 2020-06-18 | 2020-08-14 | 重庆浦洛通基因医学研究院有限公司 | Thyroid cancer auxiliary molecular diagnosis kit and using method thereof |
| CN111647650B (en) * | 2020-07-06 | 2023-06-27 | 河南赛诺特生物技术有限公司 | Primer, primer probe composition and kit for detecting V600E mutation of human B-raf gene |
| CN112094842B (en) * | 2020-11-06 | 2021-02-05 | 迈杰转化医学研究(苏州)有限公司 | Annular blocking probe, amplification blocking mutation system containing annular blocking probe and application of amplification blocking mutation system |
| CN112375808A (en) * | 2020-11-18 | 2021-02-19 | 合肥欧创基因生物科技有限公司 | Blocker design and screening method of ARMS-TaqMan Blocker system |
| CN112575072B (en) * | 2020-12-29 | 2021-12-31 | 苏州科贝生物技术有限公司 | Primer pair and kit for detecting BRAF gene V600E mutation in plasma free DNA |
| WO2022222937A1 (en) * | 2021-04-20 | 2022-10-27 | 南京金斯瑞生物科技有限公司 | Primer group and method for detecting single-base mutations |
| CN118374587A (en) * | 2024-03-01 | 2024-07-23 | 深圳精科生物技术有限公司 | Primer group and method for SMN gene amplification and copy number detection |
| CN117904263A (en) * | 2024-03-01 | 2024-04-19 | 深圳精科生物技术有限公司 | Primer pair and method for specifically amplifying target gene by OTARMS system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255201A (en) * | 2012-02-16 | 2013-08-21 | 北京宏微特斯生物科技有限公司 | Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0004232D0 (en) * | 2000-02-24 | 2000-04-12 | Zeneca Ltd | Diagnostic method |
| EP1325152A2 (en) * | 2000-07-14 | 2003-07-09 | F. Hoffmann-La Roche Ag | Method for detecting pre-disposition to hepatotoxicity |
| CN105274220A (en) * | 2015-10-14 | 2016-01-27 | 北京晋祺生物科技有限公司 | CYP4F2*3 detection primer and detection system thereof |
| CN106755388B (en) * | 2016-11-24 | 2019-08-23 | 厦门艾德生物医药科技股份有限公司 | A kind of improved ARMS primer construction (Super-ARMS) and its application method |
-
2016
- 2016-12-15 CN CN201611159646.XA patent/CN106755388B/en active Active
-
2017
- 2017-11-24 WO PCT/CN2017/112816 patent/WO2018095401A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255201A (en) * | 2012-02-16 | 2013-08-21 | 北京宏微特斯生物科技有限公司 | Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit |
Non-Patent Citations (6)
| Title |
|---|
| ALEXANDRE HARLÉ等.Analysis of PIK3CA exon 9 and 20 mutations in breast cancers using PCR-HRM and PCR-ARMS: Correlation with clinicopathological criteria.《ONCOLOGY REPORTS》.2013,1043-1052. |
| ARMS技术联合Taqman探针检测100例非小细胞肺癌EGFR基因突变;赵静等;《中国肺癌杂志》;20130120;第16卷(第1期);25-32 |
| Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation by Thermophilic DNA Polymerases;Christopher J. Lacenere等;《Nucleosides, Nucleotides and Nucleic》;20060820;9-16 |
| Gefitinib Treatment in EGFR Mutated Caucasian NSCLC Circulating-Free Tumor DNA as a Surrogate for Determination of EGFR Status;Jean-Yves Douillard等;《Journal of Thoracic Oncology》;20140930;第9卷(第9期);1345-1353 |
| Modified Tetra-Primer ARMS PCR as a Single-Nucleotide Polymorphism Genotyping Tool;Hamzeh Mesrian Tanha等;《GENETIC TESTING AND MOLECULAR BIOMARKERS》;20150331;第19卷(第3期);1-6 |
| PCＲ 直接测序法与AＲMS 检测非小细胞肺癌EGFＲ 基因突变;宿杰阿克苏等;《临床与实验病理学杂志》;20140325;328-331 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755388A (en) | 2017-05-31 |
| WO2018095401A1 (en) | 2018-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106755388B (en) | A kind of improved ARMS primer construction (Super-ARMS) and its application method | |
| US10760118B2 (en) | Method for DNA amplification with a blocking oligonucleotide | |
| CN107034277B (en) | Method for detecting low-abundance gene mutation | |
| US20120225421A1 (en) | Kit and method for sequencing a target dna in a mixed population | |
| WO1999040219A1 (en) | Quantification by inhibition of amplification | |
| US7618773B2 (en) | Headloop DNA amplification | |
| JP2018516594A5 (en) | ||
| CN110791577A (en) | Kit and method for detecting mycobacterium tuberculosis isoniazid drug-resistant mutant gene | |
| US20240084363A1 (en) | Method for quantifying and/or detecting human male dna | |
| TWI780315B (en) | Method of quantifying mutant allele burden of target gene | |
| Lee et al. | Rapid ABO genotyping using whole blood without DNA purification | |
| JP2020182457A (en) | Method for specifically inhibiting nucleic acid amplification | |
| CN116103411A (en) | Primer probe composition and kit for detecting helicobacter pylori drug-resistant gene mutation in sample | |
| EP3682027A1 (en) | Hybridization-extension-ligation strategy for generating circular single-stranded dna libraries | |
| JPWO2009119331A1 (en) | Reagent containing primer for detection of cytokeratin 7 mRNA | |
| KR102126031B1 (en) | Method for detecting nucelic acids containing a genetic variation | |
| US20180073082A1 (en) | Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same | |
| EP1759017B1 (en) | Dna sequences for the detection of and differentiation amongst pathogenic e.coli strains | |
| CN112980932B (en) | Nucleotide for detecting gene mutation, composition and method thereof | |
| EP4253564A1 (en) | Target nucleic acid amplification method with high specificity and target nucleic acid amplifying composition using same | |
| CN112239791A (en) | Human immunodeficiency virus detection kit | |
| CN115678989A (en) | Use of markers for predicting the risk of recurrence and/or metastasis of colorectal cancer | |
| CN117448441A (en) | Single nucleotide polymorphism isothermal typing method based on RNase HII enzyme dependency-locked nucleic acid-blocking probe-loop-mediated isothermal amplification | |
| JP2011030514A (en) | Method for detecting single nucleotide polymorphism | |
| Oliver | Polymerase Chain Reaction and Reverse Transcription&## x2014; Polymerase Chain Reaction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |