WO2010108325A1 - 一种用于核酸扩增的环形引物及其应用 - Google Patents
一种用于核酸扩增的环形引物及其应用 Download PDFInfo
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- the present invention relates to the design and application of nucleic acid amplification primers for use in polymerase chain reaction (PCR), and more particularly to the use in real-time PCR reactions, particularly in mutation detection and rare mutation detection.
- PCR polymerase chain reaction
- PCR Polymerase chain reaction
- probes such as Taqman probes used in 5'-exonuclease technology, molecular beacons, fluorescent energy transfer probes (adjacent probes), and tweezers Primers, light probes, etc.
- the dye method is simple, it does not recognize non-specific amplification, especially due to non-specific amplification caused by primer dimers, and thus is greatly limited in practical applications.
- the probe method has a second recognition step for the amplified product, thereby avoiding non-specific amplification and the result is more reliable.
- probes generally have the disadvantages of complicated design and high synthesis cost. The root cause of the shortcomings of these two methods is that they are not effective in preventing non-specific amplification.
- Rare mutations refer to a small number of mutant genes in the context of a large number of wild-type genes, particularly genes with single base mutations.
- the ratio of the general mutant gene to the wild type gene is less than 1/1000.
- the ability to accurately detect such rare mutations poses a challenge to existing detection techniques.
- the key to the PCR method for mutation detection is to design a pair of highly stringent primers complementary to the target gene DNA or RNA according to the target gene sequence.
- the Amplification Regulatory Mutation System (ARMS) technology was established in 1989, also known as Allele-specific PCR (AS-PCR), also known as PCR with Sequence Specific PCR.
- Primers, PCR-SSP is the most widely used in many mutation detection methods.
- the basic idea is to design two ARMS upstream primers and share one downstream primer to form a PCR reaction system.
- the allele-specific base is placed at the 3' end of the primer because the thermotolerant Taq DNA polymerase lacks the 3'-5' exo-correction activity, and the specific base at the 3' end of the primer is present during the PCR reaction.
- the present invention relates to the use of a novel primer for nucleic acid PCR amplification.
- the primer is fundamentally different in design from the current primers, and the specificity of the primer is greatly improved by clever design.
- the primer has high specificity, no primer dimer formation, simple design and easy synthesis, and is suitable for detection of gene expression, SP detection and rare mutation detection.
- the primers of the invention include two kinds, one is a single amplification circular primer, which can be used for ordinary amplification of genes; the other is a circular primer with double amplification function, which is particularly suitable for mutation detection systems, especially rare mutation detection. system.
- the single-amplified circular primer technique involved in the present invention is based on direct hybridization to a target nucleic acid, which has a ring structure complementary to the 3 end of the primer, and can form a ring structure, and the ring structure is easy to open, thus the target sequence Amplification efficiency is not affected.
- the bases for the mutated sequences may be at the end of the primer 3 or inside the primer, and the number of loop-forming sequences may be adjusted according to the sequence, thereby adjusting the specificity of the recognition of the mutant sequence.
- the double-amplified circular primer of the present invention introduces a universal universal tag sequence inside the primer, and the tag sequence and the circular primer are in a certain ratio (the tag sequence is generally 10 times or more of the amount of the circular primer), and the PCR amplification system is simultaneously added in the PCR cycle.
- the circular primer recognizes the mutant target sequence with high specificity and performs initial amplification.
- the large amount of the tag primer is amplified to rapidly amplify the initial accumulation of specific PCR products. Amplification, thereby maintaining extremely high specificity and extremely high sensitivity to rare mutant target sequences.
- the circular primer of the present invention can be used for detection of target nucleic acid in real-time PCR.
- the nucleotide used in the probe of the present invention may be DNA, RNA, LNA, or PNA, or any non-natural nucleoside composition.
- the invention also relates to the use of circular primer technology.
- the present invention relates to the use of a novel primer for PCR amplification of nucleic acids.
- Oligonucleotide Glycosyl acid, 5 to 20 complementary bases at the 5th and 3rd ends of the oligonucleotide, respectively, can be combined into a double strand under certain conditions, so that the primer forms a circular structure, and the recognition of the mutation can be placed in 3
- the first base of the terminal can also be placed inside the primer.
- the 3 ends of the primer form a double strand, and a loop structure is formed inside the primer.
- the primer recognizes and hybridizes with the target sequence, the double stranded structure is opened, the loop structure disappears, and the primer changes from its own double strand to the target sequence.
- the DNA extension ability is obtained by binding the double strands.
- the primer can form a loop structure upon annealing, forming a double strand at the 3' end, and blocking the DNA extension ability of the 3' end of the primer, so that non-specific amplification, especially primer dimerization, is not produced. body. See Figure la and Figure lb.
- the single-amplified circular primer invention system contains two primers, a single amplified circular upstream primer F and a single amplified circular downstream primer R, respectively.
- the single-amplified circular upstream primer F has three different functional regions 31, 32, and 33, wherein the 31 region is named as the target sequence recognition region, the 32 region is named as the mutation recognition region, and the mutation recognition region can be designed at the 3' end or The position farther from the 3' end can be determined according to the type of rare mutation, and the 33 region is named as a complementary sequence.
- the single-amplified circular downstream primer R has two different functional regions 34, 35, of which 34 regions are named target sequence recognition regions, and 35 regions are named complementary sequences.
- the primer sequence of the primers designed by the present invention is used to amplify the target sequence of the mutation, thereby effectively solving the problem of primer dimer caused by ordinary SPI detection, common PCR detection and multiplex PCR detection.
- the upstream and downstream primers are in a loop shape, so as to avoid the formation of primer dimers. Only when the temperature is denatured, the loops are opened, and the target sequence is combined during annealing, as shown in the single-amplified annular upstream primer.
- the mutated target sequence 41 binds, and the single amplified circular downstream primer binds to the target sequence 42 and extends to give the product 43, 44.
- the product sequence 43 becomes the target sequence of the single amplified circular downstream primer, and the single amplified circular downstream primer is extended with the product sequence 43 to obtain the target product 45.
- the present invention relates to the use of a novel primer for PCR detection and real-time PCR detection of gene mutations, particularly rare mutations.
- the primer is an oligonucleotide, and the 5th and 3rd ends of the oligonucleotide are respectively 3-20 bases, and under certain conditions, can be combined into a double strand, so that the primer forms a loop structure, and the primer is There is a universal tag sequence inside, and the recognition of the mutation can be placed at the 3rd end of the 1st base or inside the primer. Under certain conditions, the 3 ends of the primer form a double strand, and a loop structure is formed inside the primer.
- the primer When the primer recognizes and hybridizes with the target sequence, the double stranded structure is opened, the loop structure disappears, and the primer changes from its own double strand to the target sequence. DNA extension ability obtained by binding double strands; tag sequence and ring
- the primers are in a certain ratio (the tag sequence is generally more than 10 times the amount of the circular primer), and are present in the PCR amplification system.
- the circular primers In the first two cycles of the PCR cycle, the circular primers specifically recognize the mutant target sequence and perform initial amplification. Starting from the third cycle, due to the large number of tag primers, the large number of tag primers rapidly amplify and amplify the initial accumulation of specific PCR products, thereby maintaining extremely high specificity and high sensitivity to rare mutant target sequences.
- the primer When no target sequence is present, the primer can form a loop structure upon annealing, forming a double strand at the 3' end, and blocking the DNA extension ability of the 3' end of the primer, so that non-specific amplification, especially primer dimerization, is not produced.
- the recognition of the sequence of the mutation point can be based on the characteristics of the target sequence, and the recognition site can be placed at the 3' end or inside the primer.
- the strength of the primer specificity can be adjusted by the length of the 3'-end double-stranded region of the primer itself.
- the circular primer of the present invention comprises two primer designs, respectively, for different needs, one is a single amplification circular primer for common gene amplification, and the primer dimer can be eliminated; the other is a double amplification circular primer For high specific detection of rare mutations.
- the primer is an oligonucleotide, and the 5th and 3rd ends of the oligonucleotide respectively have 3-20 bases, and under certain conditions, can be combined into a double strand, so that the primer forms a cyclic structure under certain conditions.
- the 3 ends of the primer form a double strand, and a loop structure is formed inside the primer.
- the primer recognizes and hybridizes with the target sequence, the double strand structure is opened, the loop structure disappears, and the primer changes from the double strand to the target sequence. Chain to obtain DNA extension ability.
- the primer can form a loop structure upon annealing, forming a double strand at the 3' end, and blocking the DNA extension ability of the 3' end of the primer, so that non-specific amplification, especially primer dimerization, is not produced. body. See Figure la and Figure lb.
- Double amplified circular primers The primer is an oligonucleotide, and the 5th and 3rd ends of the oligonucleotide are respectively 3-20 bases, and under certain conditions, can be combined into a double strand, so that the primer forms a loop structure, and the primer is The inside has a universal tag sequence. Under certain conditions, the 3 ends of the primer form a double strand, and the inside of the primer forms a ring structure. When the primer recognizes and hybridizes with the target sequence, the double strand structure is opened, the ring structure disappears, and the primer is removed.
- the self-double strand becomes a double strand that binds to the target sequence to obtain DNA extension ability;
- the tag sequence and the circular primer are in a certain ratio (the tag sequence is generally more than 10 times the amount of the circular primer), and are present in the PCR amplification system, in the PCR
- the primer When no target sequence is present, the primer can form a loop structure upon annealing, forming a double strand at the 3' end, and blocking the DNA extension ability of the 3' end of the primer, so that non-specific amplification, especially primer dimerization, is not produced.
- the recognition of the sequence of the mutation point can be based on the characteristics of the target sequence, and the recognition site can be placed at the 3' end or inside the primer.
- the strength of the primer specificity can be adjusted by the length of the 3'-end double-stranded region of the primer itself.
- the present invention creatively proposes a method for designing primers for rare mutations.
- the present invention contains three primers, a double amplified circular upstream primer F, a double amplified circular downstream primer R, and a universal primer.
- the double-amplified circular upstream primer F has four different functional regions 11, 12, 13, and 14, wherein 11 regions are named target sequence recognition regions, 12 regions are named mutation recognition regions, and mutation recognition regions can be designed at 3
- the end of the 'end or the 3' end can be determined according to the type of the rare mutation, the 13 region is named as the tag sequence, and the 14 region is named as the complementary sequence.
- the double-amplified circular downstream primer R has three different functional regions 13, 15, and 16, wherein the 13 region is named as a tag sequence, which is identical to the 13 region sequence in the double-amplified circular upstream primer, and the 15 region is named as a target.
- the sequence recognition region, the 16 region is named as a complementary sequence.
- the universal primer T is completely matched with the 13-region tag sequence in the double-amplified circular upstream and downstream primers F, R.
- Figure 2b shows the target sequence for double amplification of rare mutations using primers designed in accordance with the present invention.
- the amplification consists of two stages. In the first stage of amplification, the target sequence with rare mutations is in the presence of double-amplified circular upstream and downstream primers F and R. At a higher annealing temperature, the optimal annealing temperature at this stage is 60 66 degrees. At this temperature, the universal primer T cannot be matched due to the lower TM value, and the double-amplified circular upstream primers F, R can be matched with the target sequences 21, 22 with high specificity.
- sequences are extended to obtain sequences 23, 24, and sequence 23 is matched by the double-amplified circular downstream primer R at a higher annealing temperature, and extended to obtain sequence 25, thereby realizing accumulation of rare mutant sequences. Since the binding efficiency of the primer to the target sequence is lowered at a higher annealing temperature, the accumulation of this stage does not achieve efficient exponential accumulation.
- the present invention uses the universal primer T in the second stage of amplification, and the rare mutation accumulated in the first stage Sequence 3 has a sequence at the 3' end that matches the universal primer T.
- the optimum annealing temperature for this stage is 54 to 58 degrees.
- the universal primer ⁇ can efficiently bind to the rare mutation sequence 25, and the sequence 26 with a rare mutation can be extended, and the sequences 25 and 26 can be matched by the universal primer ,, thereby achieving exponential amplification and finally obtaining A large number of sequences 27 with mutations are used to effect amplification of rare mutant sequences.
- Binding region to target sequence Tm value the region in the primer that specifically binds to the target sequence.
- the Tm value can be maintained by the Tm value of the common primer, and can be determined according to the GC content of the target sequence.
- the general Tm value is about 55-65 °C.
- the annealing temperature during amplification may be the same as the primer Tm value or lower than the primer TM value of 3-5 V.
- the complementary base of the primer is 3-20 bp, and its Tm value is equivalent to or higher than the annealing primer Tm value of 2-5 °C.
- the Tm value of the circular primer complementary region of real-time PCR for single-base mutation discrimination detection can be higher than the annealing temperature of 3-5 °C.
- the Tm value of the double-stranded junction can be higher than the annealing temperature of 5-12 °C.
- the position of the base of the mutation recognition for a target sequence such as a common point mutation, the mutation recognition site may be located at the first base of the 3' end of the primer; for the deletion and insertion mutation, it may be a few bases of the continuity of the 3' end For the close proximity of the target gene mutation point, there may be many other similar mutations.
- the mutation recognition base is usually placed inside the primer instead of the first base at the 3' end of the primer.
- Primer length In general, the total length of the primer is 20-80 bp, the target sequence binding region is 16-40 bp, and the internal complementary region of the primer, that is, the double-stranded binding region is 3-20 bp at the 5 and 3 ends, respectively, and the length of the circular region is about 5-60 bp.
- the universal tag sequence is approximately 16-30 bp.
- non-natural nucleotides Any non-natural nucleotides, such as LNA, PNA, can be used anywhere in the primer.
- Circular primers can be used for amplification detection of specific target sequences in real-time PCR.
- Current real-time PCR instruments for circular primers include: Applied Biosystems (ABI) 7300, 7500, 7700, Bio-Rad's IQ Cycler, Roche's LightCycler 2.0, LightCycler 480, Corbett Research's Rotor-Gene 3000, 6000 , Strategene's MX3000P and MX3005P and more. Positive effects of circular primers
- the primer 3 end sequence is part of the amplified target sequence, and the 5 end has several bases complementary to the primer 3 end sequence.
- the tag sequence can be added or not added to the primer, and the mutation is recognized.
- the base may be at the 3' end of the first base or within the primer as long as the Tm value of the double stranded binding region of the primer is consistent with or higher than the annealing temperature used in the corresponding PCR reaction system.
- Primer-free dimer production Before the PCR pre-denaturation temperature is raised to the denaturation temperature, the primer's own double-stranded binding blocks the primer DNA extension; in the subsequent PCR cycle, maintaining a certain annealing temperature, A primer that fails to bind to a target sequence preferentially forms a double strand by itself due to the tendency of its own double-stranded binding, and does not form a double strand with another primer, and thus does not amplify a primer dimer.
- the primer Since the primer has its own reverse complementary double-stranded structure, the primer can hybridize to the target sequence only when the binding of the target sequence to the binding region of the primer target sequence is greater than the binding energy of the reverse complementary double strand.
- the circular structure can be opened, and the primer can obtain DNA extension ability. If there is a mismatch between the target sequence and the primer, such as a single base mismatch, the mismatched position is located at the 3' end or inside the primer, the primer preferentially maintains the stability of its own circular structure, and the primer DNA extension ability will be Blocked.
- the length of the complementary region can be adjusted during design to adjust the specificity of the primer. For rare mutations, such as somatic mutations in the face of a large number of wild-type DNA backgrounds, the primer further ensures its specificity, except that the sequence specificity from the 3' end of the primer is simultaneously competing by the complementary sequence.
- Double-amplified circular primers can have two amplifications for the target sequence due to the universal tag sequence.
- the content of the rare mutant DNA is low, and the specific primer has a large amount of wild background, so the amplification efficiency is not high, and as long as one copy is amplified, the high concentration of the label primer in the system can efficiently This target sequence is rapidly amplified and amplified.
- Suitable for multiplex PCR systems Since the primer itself can guarantee extremely high specificity, no primer-dimer is produced and has a tag sequence. After two rounds of initial amplification, all amplification products are all amplified by the same tag sequence. There is no problem that the amplification efficiency of each primer is different and the primers will affect each other, so it is very suitable for multiplex PCR system, and is suitable for some quantitative detection systems with multiple gene expression.
- Simple synthesis no special instrument is required, just a normal DNA synthesizer can be used for synthesis. Wide range of applications: It has a good applicability to difficult templates in AT-rich and GC-rich regions, as well as point mutations, mutations, and insertion mutations.
- Figure lb Schematic diagram of the reaction principle of single-amplified circular primers in PCR cycle detection
- FIG. 2a schematic diagram of double amplified circular primer
- Figure 2b Schematic diagram of the reaction principle of double-amplified circular primers in PCR cycle detection
- FIG. 4 Circular primer-binding probes for real-time PCR detection.
- the template is continuously diluted 10 times (Example 2)
- Example 1 Circular primers for real-time PCR detection of Arabidopsis thaliana SUC2 gene
- PCR template is DNA extracted from Arabidopsis leaf tissue.
- the total volume of the reaction system is 50 ⁇ l, including 5 ⁇ 10 X buffer (160 mM [ H 4 ] 2 S0 4 , 670 mM Tris-HCl pH 8.8, and 0.1 % w/v Tween 20), 1.5 ⁇ 25 mM MgCl 2 , per dNTP 400 ⁇ , upstream and downstream primers 0.4 ⁇ , Eve Green 0.5 ⁇ 1, 1.0 U Taq DNA polymerase, 5 ⁇ l template DNA.
- Real-time PCR reactions were performed on a RotorGene 3000 real-time PCR machine.
- the reaction conditions were: pre-denaturation at 96 °C for 3 min, 94 °C for 3 min , 94 °C for 15 s, 55 °C for 20 s (detecting FAM fluorescence signal), 72 °C for 15 s, 35 cycles in total; then melting curve analysis.
- H 2 0 was used as a negative control.
- the circular primer includes a nucleic acid sequence that is complementary to the amplification product.
- the upstream primers are: 5'- AACCAGCTCATCGTCGCTGGAGCTGGTT-3 ', and the downstream primer is: 5 ' -ATCAGCTTGCGGCGGTTTGTCAAGCTGAT-3 ', primers 5 and 3 are underlined
- the indicated bases are bases involved in the formation of a double-stranded binding region within the probe.
- a is a NTC (blank control) result of fluorescent PCR amplification of primers designed according to the method of the present invention and dyes designed according to conventional methods.
- Line 51 is a primer-amplified NTC result designed according to the conventional method, and it is apparent that more primer dimers are formed.
- Line 52 is a primer-amplified NTC result designed in accordance with the methods of the present invention, and it is apparent that primers designed in accordance with the methods of the present invention are effective in reducing the formation of primer dimers and avoiding unnecessary amplification.
- Figure 3b shows the results of the melting curve analysis of the primers designed according to the method of the present invention after fluorescent PCR amplification of the dyes.
- the primers designed by the traditional method will form a primer dimer, and two peaks will be formed in the melting curve analysis, which are the primer dimer peak and the product peak, respectively. However, only one product peak is formed when the primers designed in accordance with the present invention are expanded.
- Example 2 Circular primer binding probe for real-time PCR quantification of hepatitis B virus detection
- PCR templates were a series of diluted standard DNA samples.
- the total volume of the reaction system was 50 ⁇ l, including 5 ⁇ 10 X buffer (160 mM [ H 4 ] 2 S0 4 670 mM Tris-HCl pH 8.8, and 0.1 % w/v Tween 20), 1.5 ⁇ 25 mM MgCl 2 , each dNTP 400 ⁇ , upstream and downstream primers 0.4 ⁇ , probe 0.1 ⁇ , 1.0 U Taq DNA polymerase, 5 ⁇ l template DNA.
- Real-time PCR reactions were performed on a RotorGene 3000 real-time PCR machine.
- the reaction conditions were: pre-denaturation at 94 °C for 5 min, followed by denaturation at 94 °C for 15 s, 58 °C for 25 s, 72 °C for 15 s, 10 cycles; 94 V 15 s, 58 °C for 20 s (detection of FAM, fluorescence) Signal), 72V 15 s, a total of 35 cycles.
- the circular primer includes a nucleic acid sequence that is complementary to the amplification product.
- the upstream and downstream primers amplify the S region gene of the hepatitis B virus (NC-003977) and amplify 174 bp.
- the upstream primer is: 5 -GGTCCTACAACATCAGGATTCCTAGGACC-3 '
- the downstream primer is: 5 -CAACCTCGGTGAGTGATTGGAGGTTG-3 '
- the target sequence of the probe is close to the upstream primer, and the sequence is: FAM-5 '-CAGAGTCTAGACTCGTGGTGGACTTC-3 '-BHQ.
- the bases underlined at the 5th and 3rd ends of the primer are the bases involved in the formation of the double-stranded binding region within the primer.
- Example 3 Fluorescence real-time detection was measured for 35 cycles during PCR amplification, and the results are shown in FIG.
- the initial target concentration is line 81 at the time when the dilution is the strongest, and lines 82, 83, and 84 represent the fluorescence curves after the template is diluted by 10 times for 3 consecutive times.
- Example 3 Detection of SNP by circular primers in real-time PCR
- HPA-4 gene of human platelet alloantigen (HPA) was selected, and a G>A mutation was present in the gene, resulting in HPA- 4a and HPA-4b two platelet antigens, HPA-4a is wild type, and HPA-4b is mutant type. Two-loop probes were designed for the gene sequences of the two antigens.
- the two probes differed by only one base, and three typical samples of known genotypes were selected for testing.
- a no (negative) sample ie, a 3 ⁇ 40 control, was made.
- the wild-type HPA-4a primer sequence is: 5 ' -
- the HPA-4b primer sequence is: 5 ' - ICGCATCT ACCAAGCTGGCCACCCAGATGCA - 3, , the underlined base is a mutation-generating base, and the outer-framed base is a loop-forming binding base.
- the general downstream primers are: 5 ' - TGCCCCGAAGCC AATCC -3 '.
- the reaction consisted of two PCRs, one containing wild-type primers and one containing mutant primers.
- the total reaction system is 25 ⁇ , including 2.5 ⁇ 10 X buffer (160 mM [ H 4 ] 2 S0 4 , 670 mM Tris-HCl pH 8.8, and 0.1 % w/v Tween 20), 1.5 ⁇ 25 mM MgCl 2 , per dNTP 400 ⁇ , various primers 0.4 ⁇ , Eve Green 0.5 ⁇ 1, 1.0 U Tag DNA polymerase, 20 ng of template DNA.
- Real-time PCR reactions were performed on a RotorGene 3000 real-time PCR machine.
- reaction conditions were: pre-denaturation at 96 °C for 2 min, followed by denaturation at 96 °C for 15 s, 68 °C-59 °C (temperature drop of 1 °C per cycle) for 15 s, extension at 72 °C for 15 s, 10 cycles; 94 ° C 3 min; 94V 15 s, 58 ° C 20 s (detect FAM fluorescence signal), 72V 15 s, a total of 35 cycles.
- Figure 5 shows the results of real-time fluorescent PCR for S P analysis cycle data, consisting of two PCRs, one for wild type and one for mutant. Primer amplification results relative to wild-type targets are shown in solid squares, and primer amplification results relative to mutant heterologous targets are indicated by solid circles.
- the K-ras gene plays an important role in regulating cell growth and differentiation. About 70% of tumors are involved in mutations at the 12th and 13th codons to varying degrees, and other codon mutations are at positions 59 and 61.
- the 3 and 13 codons may be mutated in three bases. Most of the codons are mutated at bases 1 and 2, and can be mutated to one of any three bases other than itself, or the first one. Simultaneous mutation with 2 bases.
- the first base mutation of the 12 codon is A, that is, GT>IGT (Cosmic ID: 480).
- This mutation is a hotspot mutation, which is related to the drug resistance of the colorectal cancer treatment drug Erbitux; and the 12 codon Simultaneous mutations of 1 and 2 bases, such as GGT>TGC (Cosmic ID: 513), GGT>TT (Cosmic ID: 512), GGT>TAT (Cosmic ID: 25081), these three mutations are common mutations, not yet Determine whether it is related to the drug resistance of colorectal cancer treatment drug Erbitux.
- Commonly used ARMS primers can easily detect a mutation in a class as GT>IGT.
- a pair of primers and probes were designed according to the wild type gene sequence of the K-ras gene 12 codon published by Cosmic data and the GT>IGT mutant gene sequence (Cosmic ID: 480). It is K-ras-Ml-F, K-ras-Ml-R, K-ras-P. According to the wild type gene sequence analysis of HGH gene published by Cosmic data, a pair of primers and probes were designed: HGH-F, HGH-R, HGH-P.
- the method for detecting a clinically collected paraffin tissue sample K-ras gene GT>IGT mutation using the above fluorescent PCR system comprises the following steps:
- Sample processing and template extraction receiving samples of paraffin tissue from clinical samples, the samples are cut into 5-10 Mm, de-waxed by adding 1 ml of xylene, and the precipitate is collected by centrifugation, and 1 ml of absolute ethanol is added to the precipitate, room temperature or 37°.
- the underlined base of K-ras-Ml-F is a tag sequence, the base with a border at the 5-end is a sequence complementary to the 3-terminal base, and the bold base is a mutation recognition base;
- K-ras- Ml-R GCAAGGGGTCAGTAAAGCGTCGTCCACAAAATGATTCTG, wherein the underlined base is a tag sequence;
- the tag sequence T is GCAAGGGGTCAGTAAAGCG.
- K-ras-P FAM-5'TGCCTTGACGATACAGCT3'-BHQ.
- HGH-F 5 '-GCAAGGGGTCAGTAAAGCGGCAGTGCCTTCCCAACCATT-3 '
- HGH-R Underlined base is the tag sequence
- HGH-P HEX-5'TTGACAACGCTATGCTCCGC3 ' - BHQ.
- Real-time PCR reaction conditions were: pre-denaturation at 96 °C for 3 minutes, 15 cycles at 95 °C for 15 seconds, annealing at 64 °C for 25 seconds, 72 °C for 10 seconds, 35 cycles at 95 °C for 15 seconds, 58 ° C was annealed for 25 seconds, extended at 72 °C for 10 seconds, 35 cycles, and the last 35 cycles detected FAM and HEX fluorescence signals during annealing.
- Quantitative analysis was carried out using a synthetic plasmid DNA containing the K-ras gene TG>IGT mutation, and the DNA of the sample having a concentration of 1000 copies was quantified, and 10-fold dilution was performed for 3 dilutions, followed by 4 dilutions. 5 ⁇ 1 of the degree, 8 parallel groups were subjected to a fluorescent PCR reaction.
- the fluorescent PCR method of the present invention has high sensitivity and can be detected by 1-10 copys/ ⁇ .
- Fluorescent PCR was carried out in the background of 1 ng, 5 ng, 10 ng, 50 ng, 100 ng, 200 ng of plasmid DNA containing the K-ras gene TG>IGT mutation. See Figure 6.
- Figure 6 shows the detection results of rare mutant double-amplified circular primers designed according to the present invention for amplification of rare mutant templates in different concentrations.
- the 5 copies of the mutant template were detected in lng, 5ng, 10ng, 50ng, 100ng, 200ng background, line 71 was 5 copies in lng background, line 72 was 5 copies in 5ng background, line 73 was 5 copies in 10ng background, line 74 for 5 copies on 50ng background, line 75 for 5 copies on lOOng background, line 76 for 5 copies on 200ng background.
- the double-amplified circular bow designed in accordance with the present invention is highly selective.
- the invention relates to a circular primer for nucleic acid amplification and the application thereof, which has high primer specificity, no primer dimer formation, simple design, suitable for detecting gene expression, SNP detection and rare mutation detection, and has good industrialization. Practicality.
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Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/142,874 US9644233B2 (en) | 2009-03-26 | 2009-03-26 | Loop-shaped primer used in nucleic acid amplification and the use thereof |
DK09842062.3T DK2412718T3 (en) | 2009-03-26 | 2009-03-26 | CIRCUIT PRIMES USED BY NUCLEIC ACID AMPLIFICATION AND USE THEREOF |
PCT/CN2009/071026 WO2010108325A1 (zh) | 2009-03-26 | 2009-03-26 | 一种用于核酸扩增的环形引物及其应用 |
JP2012501111A JP5818018B2 (ja) | 2009-03-26 | 2009-03-26 | 核酸増幅を用いる環状プライマー及びその応用 |
PT98420623T PT2412718T (pt) | 2009-03-26 | 2009-03-26 | Iniciador em forma de anel utilizado na amplificação de ácido nucleico e a sua utilização |
ES09842062.3T ES2610616T3 (es) | 2009-03-26 | 2009-03-26 | Cebador con conformación de bucle empleado en la amplificación de ácidos nucleicos y el uso del mismo |
PL09842062T PL2412718T3 (pl) | 2009-03-26 | 2009-03-26 | Primer w kształcie pętli stosowany w amplifikacji kwasów nukleinowych i jego zastosowanie |
EP09842062.3A EP2412718B1 (en) | 2009-03-26 | 2009-03-26 | Loop-shaped primer employed in nucleic acid amplification and the use thereof |
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KR102351434B1 (ko) | 2013-02-07 | 2022-01-17 | 러트거즈,더스테이트유니버시티오브뉴저지 | 고도로 선택적인 핵산 증폭 프라이머 |
KR102124058B1 (ko) * | 2013-09-16 | 2020-06-17 | 삼성전자주식회사 | 폴리뉴클레오티드 및 그의 용도 |
KR101525495B1 (ko) * | 2013-11-15 | 2015-06-03 | (주)다이오진 | 덤벨 구조 올리고뉴클레오티드, 이를 포함한 핵산 증폭용 프라이머 및 이를 이용한 핵산 증폭 방법 |
KR101723207B1 (ko) * | 2015-01-29 | 2017-04-05 | (주)다이오진 | 덤벨 구조의 올리고뉴클레오티드 및 이를 이용한 유전자 변이 검출 방법 |
KR101742681B1 (ko) * | 2015-01-30 | 2017-06-01 | 에스디 바이오센서 주식회사 | 상보적 염기서열 내지는 미스-매치된 염기를 포함하는 상보적인 염기서열과 연결된 pcr 프라이머 및 이를 이용한 핵산 증폭 방법 |
US20180073082A1 (en) * | 2015-02-25 | 2018-03-15 | Diogene Co., Ltd | Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same |
KR101785687B1 (ko) * | 2016-07-20 | 2017-10-17 | (주)다이오진 | 다중 증폭 이중 시그널 증폭에 의한 타겟 핵산 서열의 검출 방법 |
CN111235261B (zh) * | 2019-12-20 | 2022-11-04 | 江苏伟禾生物科技有限公司 | 用于检测人类血小板特异性抗原hpa 1~29基因分型的试剂盒 |
CN114134219A (zh) * | 2021-12-14 | 2022-03-04 | 广州市金圻睿生物科技有限责任公司 | 一种多重核酸检测系统及其制备方法与应用 |
CN114032337A (zh) * | 2021-12-14 | 2022-02-11 | 广州市金圻睿生物科技有限责任公司 | 一种呼吸道病原体检测试剂盒及其制备方法与应用 |
CN116121358A (zh) * | 2022-11-11 | 2023-05-16 | 重庆浦济生命科技有限公司 | 用于精神药物基因检测的组合物、试剂盒及使用方法 |
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US20110269192A1 (en) | 2011-11-03 |
EP2412718B1 (en) | 2016-10-12 |
US9644233B2 (en) | 2017-05-09 |
PT2412718T (pt) | 2016-12-30 |
JP2013518559A (ja) | 2013-05-23 |
EP2412718A1 (en) | 2012-02-01 |
EP2412718A4 (en) | 2013-02-06 |
ES2610616T3 (es) | 2017-04-28 |
PL2412718T3 (pl) | 2017-06-30 |
DK2412718T3 (en) | 2017-01-30 |
JP5818018B2 (ja) | 2015-11-18 |
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