WO2016137031A1 - 덤벨 구조 올리고뉴클레오티드, 이를 포함한 핵산 증폭용 프라이머 및 이를 이용한 핵산 증폭 방법 - Google Patents
덤벨 구조 올리고뉴클레오티드, 이를 포함한 핵산 증폭용 프라이머 및 이를 이용한 핵산 증폭 방법 Download PDFInfo
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Definitions
- the present invention relates to a dumbbell structure oligonucleotide (DSO), a nucleic acid amplification primer comprising the same, and a nucleic acid amplification method using the same. More specifically, in performing a polymerase chain reaction, a template is performed every first cycle. A multi-gene amplification and monobasic polymorphism analysis method using dumbbell structure oligonucleotides that can exclude nonspecific amplification products prior to binding.
- DSO dumbbell structure oligonucleotide
- a nucleic acid amplification primer comprising the same
- a nucleic acid amplification method using the same More specifically, in performing a polymerase chain reaction, a template is performed every first cycle.
- the polydeoxyinosine linker Since it is a universal base, at certain temperatures, the polydeoxyinosine linker forms a "bubble like structure" that blocks the nonspecific binding of the primer to the template, thereby inhibiting nonspecific amplification of PCR.
- this technique has become somewhat cheaper in its implementation than the prior art described above, in PCR practice the temperature of the binding step in the first cycle (PCR reaction fit temperature) and the second cycle There was an inconvenience that the temperature should be different. This is to allow for priming from the second PCR cycle up to the sequence further introduced into the primer.
- this "application of different temperatures” is not necessarily required, but the application of other temperatures is required for efficient PCR.
- the technique also has the constraint that the 5'-terminal pre-selection aviary nucleotide sequence must be added and that it must not be complementary to any position of the template. This leads to additional inconvenience and further makes it impossible to be certain of the success of the application of the technique when all the gene sequences of the template are unknown. Therefore, it may be necessary to develop a new method that is cheaper and easier to implement than the prior art.
- This non-specific amplification suppression technique is of course important for all PCR, especially in the PCR (diagnosis) that performs genetic tests, disease tests, etc. will be more important.
- Patent document 1 KR10-0649165 B
- an object of the present invention is to solve the problems of the prior art and the technical problem that has been requested from the past.
- the present inventors have diligently researched to develop a method for amplifying a plurality of genes by only one polymerase chain reaction.
- a dumbbell structure is formed.
- Dumbbell structure oligonucleotide (DSO) that adds a random base sequence capable of complementarily binding to the 3-end of 3 to 5 bp and a universal base pair of 3 to 5 bp connecting the two sites to the ⁇ -terminus
- DSO Dumbbell structure oligonucleotide
- the polymerase chain reaction was confirmed that it is possible to amplify a large number of different genes at the same time quickly and accurately, to complete the present invention.
- the main object of the present invention is a primer having a 3 to 5 bp universal base pair added to the 3 '-terminal complementary random sequence of 3 to 5 bp additionally inserted at the 5' -terminus and a template specific nucleotide sequence.
- dumbbell structure oligonucleotide represented by the following general formula:
- A represents a 5′-low T m specificity site comprising a nucleotide having a nucleotide sequence complementary to the 3′-terminal sequential sequence
- B is a fragment comprising a nucleotide having a universal base Site
- C represents a 3′-high Tm specific site comprising nucleotides having a nucleotide sequence complementary to a particular contiguous sequence of template nucleic acid, wherein p, q and r represent the number of nucleotides.
- p contains 3-5 nucleotides.
- q contains 3-5 nucleotides.
- r contains 18-30 nucleotides.
- the 5'-low T m specificity portion is of T m is preferably the 3 ' and lower than T m T m specificity portion.
- the T m of the cleavage site is preferably lower than the T m of the 5'-low T m specificity portion and the 3 ' of T m and T m specificity portion.
- the T m of the said 5'-low T m specific site is 10-30 degreeC.
- the T m of the divided regions is preferably 3 ⁇ 10 °C.
- the T m of the said 3'-high T m specific site is 50-65 degreeC.
- the universal base consists of deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine and combinations thereof It is preferably selected from the group.
- dumbbell structure oligonucleotide is preferably selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 35.
- the present invention provides a primer for nucleic acid amplification comprising a dumbbell structure oligonucleotide represented by the following general formula:
- A represents a 5′-low T m specificity site comprising a nucleotide having a nucleotide sequence complementary to the 3′-terminal sequential sequence
- B is a fragment comprising a nucleotide having a universal base Site
- C represents a 3′-high Tm specific site comprising nucleotides having a nucleotide sequence complementary to a particular contiguous sequence of template nucleic acid, wherein p, q and r represent the number of nucleotides.
- p contains 3-5 nucleotides.
- q contains 3-5 nucleotides.
- r contains 18-30 nucleotides.
- the 5'-low T m specificity portion is of T m is preferably the 3 ' and lower than T m T m specificity portion.
- the T m of the cleavage site is preferably lower than the T m of the 5'-low T m specificity portion and the 3 ' of T m and T m specificity portion.
- the T m of the said 5'-low T m specific site is 10-30 degreeC.
- the T m of the divided regions is preferably 3 ⁇ 10 °C.
- the T m of the said 3'-high T m specific site is 50-65 degreeC.
- the universal base consists of deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine and combinations thereof It is preferably selected from the group.
- dumbbell structure oligonucleotide is preferably selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 35.
- the present invention is a method for amplifying a nucleic acid by performing a polymerase chain reaction from a mixture comprising a template, a primer and a polymerase, a nucleic acid amplification primer comprising a dumbbell structure oligonucleotide represented by the following general formula
- nucleic acid amplification methods characterized by:
- A represents a 5′-low T m specificity site comprising a nucleotide having a nucleotide sequence complementary to the 3′-terminal sequential sequence
- B is a fragment comprising a nucleotide having a universal base Site
- C represents a 3′-high Tm specific site comprising nucleotides having a nucleotide sequence complementary to a particular contiguous sequence of template nucleic acid, wherein p, q and r represent the number of nucleotides.
- p contains 3-5 nucleotides.
- q contains 3-5 nucleotides.
- r contains 18-30 nucleotides.
- the 5'-low T m specificity portion is of T m is preferably the 3 ' and lower than T m T m specificity portion.
- the T m of the cleavage site is preferably lower than the T m of the 5'-low T m specificity portion and the 3 ' of T m and T m specificity portion.
- the T m of the said 5'-low T m specific site is 10-30 degreeC.
- the T m of the divided regions is preferably 3 ⁇ 10 °C.
- the T m of the said 3'-high T m specific site is 50-65 degreeC.
- the universal base consists of deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine and combinations thereof It is preferably selected from the group.
- dumbbell structure oligonucleotide it is preferable to use a nucleic acid amplification primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 35.
- the nucleic acid amplification method is preferably a multiplex polymerase chain reaction using two or more types of templates.
- the present invention relates to an arbitrary sequence designed to complementarily bind a 5'-terminal oligonucleotide and a 3'-terminal oligonucleotide before binding to a template in every first cycle during a polymerase chain reaction (pcr).
- Dumbbell structure oligonucleotides created by adding 3 ⁇ -terminal template-dependent specific sequences and universal base pairs linking the two sequences inhibit unintended amplification products at room temperature.
- a plurality of genes can be amplified by only one polymerase chain reaction, and single nucleotide polymorphism analysis can be more easily detected, thereby contributing to the advancement of R & D related to genes. will be.
- 1 is a diagram showing the structural characteristics of the primer used in the multiple gene simultaneous amplification method.
- FIG. 2 is a diagram schematically showing that amplification using a PCR product as a template is superior to amplification based on an initial template from a third cycle during PCR.
- Figure 3 illustrates the principle of dumbbell oligonucleotides of the present invention in target dependent extension reactions. (a) shows that the environment cannot be amplified due to the high hybridization specificity of dumbbell-structured oligonucleotides under high stringency conditions, and (b) shows the successful extension of dumbbell-structured oligonucleotides.
- Figure 4 is an electrophoresis picture of the causative bacteria causing amplification using amplification method gene.
- FIG. 5 is a diagram showing the SNP reading of the MTHFR gene C677T by dumbbell-structure oligonucleotide and allele specific polymerase chain reaction.
- Figure 6 shows the results of reading the SNPs of the BRAF gene V600E by allele-specific polymerase chain reaction with dumbbell structure oligonucleotides.
- FIG. 7 is a diagram showing the results of reading the SNPs of the APC gene by the dumbbell structure oligonucleotide and allele specific polymerase chain reaction.
- each of the genes used as templates and primers that can bind to each other are complementary Note that if specific binding is possible, multiple genes can be amplified by one polymerase chain reaction, and the specific selectivity of the primers for the template genes will be improved.
- dumbbell structure oligonucleotide for preparing nucleic acid molecules by a template-dependent extension reaction.
- the present invention relates to dumbbell structure oligonucleotides and various methods of using the same.
- Dumbbell-structured oligonucleotides of the present invention allow primers or probes to be annealed to target nucleic acids with improved specificity, thereby greatly improving the specificity of nucleic acid amplification (particularly PCR).
- dumbbell structure oligonucleotides represented by the following general formula:
- A represents a 5′-low T m specificity site comprising a nucleotide having a nucleotide sequence complementary to the 3′-terminal sequential sequence
- B is a fragment comprising a nucleotide having a universal base Site
- C represents a 3′-high Tm specific site comprising nucleotides having a nucleotide sequence complementary to a particular contiguous sequence of template nucleic acid, wherein p, q and r represent the number of nucleotides.
- p contains 3-5 nucleotides.
- q contains 3-5 nucleotides.
- r contains 18-30 nucleotides.
- the 5'-low T m specificity portion is of T m is preferably the 3 ' and lower than T m T m specificity portion.
- the T m of the cleavage site is preferably lower than the T m of the 5'-low T m specificity portion and the 3 ' of T m and T m specificity portion.
- the T m of the said 5'-low T m specific site is 10-30 degreeC.
- the T m of the divided regions is preferably 3 ⁇ 10 °C.
- the T m of the said 3'-high T m specific site is 50-65 degreeC.
- the universal base consists of deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine and combinations thereof It is preferably selected from the group.
- dumbbell structure oligonucleotide is preferably selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 35.
- the present invention provides a primer for nucleic acid amplification comprising a dumbbell structure oligonucleotide represented by the following general formula:
- A represents a 5′-low T m specificity site comprising a nucleotide having a nucleotide sequence complementary to the 3′-terminal sequential sequence
- B is a fragment comprising a nucleotide having a universal base Site
- C represents a 3′-high Tm specific site comprising nucleotides having a nucleotide sequence complementary to a particular contiguous sequence of template nucleic acid, wherein p, q and r represent the number of nucleotides.
- p contains 3-5 nucleotides.
- q contains 3-5 nucleotides.
- r contains 18-30 nucleotides.
- the 5'-low T m specificity portion is of T m is preferably the 3 ' and lower than T m T m specificity portion.
- the T m of the cleavage site is preferably lower than the T m of the 5'-low T m specificity portion and the 3 ' of T m and T m specificity portion.
- the T m of the said 5'-low T m specific site is 10-30 degreeC.
- the T m of the divided regions is preferably 3 ⁇ 10 °C.
- the T m of the said 3'-high T m specific site is 50-65 degreeC.
- the universal base consists of deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine and combinations thereof It is preferably selected from the group.
- dumbbell structure oligonucleotide is preferably selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 35.
- the present invention is a method for amplifying a nucleic acid by performing a polymerase chain reaction from a mixture comprising a template, a primer and a polymerase, a nucleic acid amplification primer comprising a dumbbell structure oligonucleotide represented by the following general formula
- nucleic acid amplification methods characterized by:
- A represents a 5′-low T m specificity site comprising a nucleotide having a nucleotide sequence complementary to the 3′-terminal sequential sequence
- B is a fragment comprising a nucleotide having a universal base Site
- C represents a 3′-high Tm specific site comprising nucleotides having a nucleotide sequence complementary to a particular contiguous sequence of template nucleic acid, wherein p, q and r represent the number of nucleotides.
- p contains 3-5 nucleotides.
- q contains 3-5 nucleotides.
- r contains 18-30 nucleotides.
- the 5'-low T m specificity portion is of T m is preferably the 3 ' and lower than T m T m specificity portion.
- the T m of the cleavage site is preferably lower than the T m of the 5'-low T m specificity portion and the 3 ' of T m and T m specificity portion.
- the T m of the said 5'-low T m specific site is 10-30 degreeC.
- the T m of the divided regions is preferably 3 ⁇ 10 °C.
- the T m of the said 3'-high T m specific site is 50-65 degreeC.
- the universal base consists of deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine and combinations thereof It is preferably selected from the group.
- dumbbell structure oligonucleotide it is preferable to use a nucleic acid amplification primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 35.
- the nucleic acid amplification method is preferably a multiplex polymerase chain reaction using two or more types of templates.
- the present invention provides a dumbbell structure oligonucleotide for synthesizing a nucleic acid molecule by a template-dependent extension reaction represented by the following general formula.
- A is a nucleotide sequence capable of substantially complementary binding to a partial sequence consecutive from the 3′-end of the general formula
- B represents a cleavage site including a universal base pair
- C is one of the template nucleic acids hybridized
- p, q, and r are the number of nucleotides
- A, B, and C are deoxyribonucleotides or ribonucleotides
- the cleavage site is one of three sites: A, B, and C Has the lowest T m ;
- the cleavage site forms a non-base pair hairpin structure under the condition that A and B bind to the template nucleic acid, so that A is separated from B in terms of binding specificity for the template nucleic acid, and the binding specificity of the oligonucleotide Determined by both A and B, it can be seen that the specific selectivity of the primer for the template gene can be improved.
- Dumbbell-structured oligonucleotides of the present invention are found in a variety of fields, including Miller, HI method (WO 89/06700) and Davey, C. et al. (EP329, 822), ligase chain reaction (LCR, Wu, DY et al., Genomics 4 : 560 (1989)), polymerase ligase chain reaction (Barany, PCR Methods and Applic., 1: 5-16 (1991)), gap-LCR (WO 90/01069), repair chain reaction (EP 439,182) Primer-related nucleic acid amplification methods such as 3SR (Kwoh et al., PNAS, USA, 86: 1173 (1989)) and NASBA (US Pat.
- 3SR Zaoh et al., PNAS, USA, 86: 1173 (1989)
- NASBA US Pat.
- FIG. 1 is a diagram showing the structural characteristics of the primer used in the multiple gene simultaneous amplification method.
- Figure 1 when constructing the primer of the gene to be amplified by adding a random base sequence of 3bp to 5bp complementary to the 3 ⁇ -end to the 5 ⁇ -end does not bind complementarily with the template base sequence every first cycle Therefore, it inhibited nonspecific binding, and when hybridized with the template gene during the polymerase chain reaction, it was replaced with a universal base pair of 3bp to 5bp to form a bulge in the center, and 3 to 5bp base sequence of the 5'-terminal part.
- nucleotide sequence of the 3′-terminal portion is a dumbbell structure oligonucleotide that can complementarily bind to the gene to be amplified
- a primer consisting of was constructed.
- template genes are normally amplified even when the annealing temperature is changed.
- PCR amplification using conventional primers under the condition that the annealing temperature is changed the amplification rate increases with annealing temperature. It was confirmed that this was lowered and the template gene was not normally amplified (see FIG. 2).
- the multi-gene simultaneous amplification method of the present invention comprises the steps of: (i) selecting each region to be amplified from 2 to 30 target genes; (ii) determining a random 5'-terminal sequence capable of complementarily binding to the 3'-terminal sequence of each selected site, and adding a 3 to 5 bp universal base pair located at the center of the determined base sequence Constructing the prepared sense primer; (iii) determining a nucleotide sequence capable of complementarily binding to the 3'-terminal sequence of each of the selected sites, and constructing an antisense primer added with a universal string of ⁇ 5 bp located at the center of the determined base sequence step; (iv) mixing the 2 to 30 target genes with the 2 to 30 sense primers and antisense primers corresponding to the target genes, respectively, and performing one polymerase chain reaction using the mixture. step; And (v) identifying the amplified product obtained by the polymerase chain reaction. At this time, the temperature and time conditions in the polyme
- a plurality of genes can be amplified by only one polymerase chain reaction, and a single base polymorphic analysis can be easily implemented, thus contributing to the advancement of R & D related to genes. .
- DNA was extracted from samples obtained from 20 suspected patients with susceptible disease and 10X polymerase chain reaction buffer solution (750mM Tris-HCl (pH 9.0), 20mM MgCl 2 , 500mM KCl, 200mM (NH) 4) 2 SO 4) 2 ⁇ l, 2.5mM dNTP mix (2.5mM dATP, 2.5mM dGTP, 2.5mM dTTP, 2.5mM dCTP) 2 ⁇ l, Taq polymerase (Biotools, Spain) 2.0unit and SEQ ID NO: 1 to 26, 36 and 1 ⁇ l of DS primer (0.5 ⁇ M) having a nucleotide sequence of 37 was mixed, titrated to 20 ⁇ l by adding tertiary distilled water, and then subjected to a polymerase chain reaction (10 minutes at 94 ° C., 30 seconds at 94 ° C., 60 at 65 ° C.).
- 10X polymerase chain reaction buffer solution 750mM Tris-HCl (pH 9.0
- FIG. 4 is an electrophoresis photograph of 12 kinds of causative bacteria causing causative wave disease amplified through polymerase chain reaction.
- a first amplification image of a CA-infected clinical sample is shown, and a second second is a clinical sample infected with UP or GV.
- the amplified image is shown, 3 shows an amplified image of GV-infected clinical sample, 4 shows an amplified image of CT-infected clinical sample, 5 shows an amplified image of GV-infected clinical specimen, and 6 Amplified images of clinical samples infected with UP and CA are shown, 7 is amplified images of clinical samples infected with MH, UP, and GV, 8 is amplified images of negative samples, and 9 is infected with GV and CT.
- An amplified image of the clinical sample is shown, 10 shows an image of the amplified clinical sample infected with UP, HSV2, CA, 11 shows an amplified image of the clinical sample infected with CA, 12 and 13 Represents an amplified image of a negative sample, 14 represents an amplified image of a clinical sample infected with GV, CT, 15 represents an amplified image of a clinical sample infected with CT, TV, 16, 17 and 18 and 19 shows an amplified image of negative sample, 20 shows an amplified image of UP and GV infected clinical sample, 21 shows an amplified image of negative sample, 22 shows an amplified image of UP infected sample 23 shows an image of amplified clinical sample infected with UP, GV, CA, 24 shows an image of amplified negative sample, 25 shows an amplified image of MH, GV infected clinical sample, and 26 shows UP.
- number 27 represents an amplified image of MH
- number 28 represents an amplified image of UP
- 29 shows amplified images of clinical samples infected with MH, UU, and GV
- 30 shows amplified images of clinical samples infected with CA
- 31 shows amplified images of negative samples
- 32 shows amplified negative controls. Represents an image.
- Example 2 Monobasic polymorphism analysis of MTHFR gene, BRAF gene and APC gene using DSO primer
- the conditions of the polymerase chain reaction was carried out 35 cycles under the conditions of 10 minutes at 94 °C, 30 seconds at 94 °C, 60 seconds at annealing, 60 seconds at 72 °C, annealing temperature is 60 °C, 55 °C and 62 °C, respectively
- the amplified sections were electrophoresed on a 2% (w / v) agarose gel (see FIGS. 5, 6, 7).
- M is to confirm the presence or absence of mutations in the MTHFR gene that is closely related to cardiovascular disease, targeting 16 clinical specimens whose type was confirmed by the polymerase chain reaction-restriction fragment fragment polymorphism method, The result of allele specific pcr analysis is shown.
- M as image is a size marker identifying the amplification product size.
- Clinical specimens 1, 3, 5, 7, 8, 9, 11, 12, and 13 were clinical samples with high homocysteine concentrations and were found to have both wild and mutant mutations in this analysis. 2, 4, 10, 14 and 15 were patients with cardiovascular disease and only mutants were identified in this analysis.
- Clinical specimens 6 and 16 were normal and only wild (wild type) in this analysis.
- Figure 6 is to confirm the presence or absence of mutations in the BRAF gene that is closely related to thyroid cancer, alleles of 16 clinical specimens whose type was identified by the polymerase chain reaction-restriction fragment fragment polymorphism analysis method The result of the gene specific PCR analysis is shown.
- M as image is a size marker identifying the amplification product size.
- Clinical specimens 1, 5, 6, 7, 10, 12, and 16 were normal and were found to have only wild (wild type) in this analysis, and clinical specimens 3, 8, 11, 13, and 15 showed abnormal thyroid function.
- the clinical specimens of this study were found to have both wild (mutant) and mutant mutations, and clinical specimens 2, 4, 9, and 14 patients with thyroid cancer were identified as having only mutants. .
- Figure 7 is to determine the presence or absence of mutations in the APC gene that is closely related to familial polyposis (colorectal cancer), 16 clinical trials of the type was confirmed by the polymerase chain reaction-restriction fragment fragment polymorphism analysis method Allele-specific PCR analysis is performed on the sample. M as image is a size marker identifying the amplification product size. Clinical specimens 1, 2, 3, 4, 8, 9, 10, 11, 12, 14 and 16 were normal and found to have only wild (wild type) in this analysis. As a result of colon polyscopic findings, it was confirmed that this analysis had both wild (wild type) and mutant (mutant), and clinical sample 5 was a stage 0 epithelial cancer patient with only mutant (mutant) in this analysis. Confirmed.
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- 다음의 일반식으로 나타내어지는 덤벨 구조 올리고뉴클레오티드:일반식 : 5'-Ap-Bq-Cr-3'여기에서, 상기 A는 상기 3'-말단의 연속된 염기서열과 상보적인 염기서열을 갖는 뉴클레오티드를 포함하는 5'-저 Tm 특이성 부위를 나타내고, 상기 B는 유니버설 염기를 갖는 뉴클레오티드를 포함하는 분할 부위를 나타내고, 상기 C는 주형 핵산의 특정 연속된 염기서열과 상보적인 염기서열을 갖는 뉴클레오티드를 포함하는 3'-고 Tm 특이성 부위를 나타내고, 상기 p, q 및 r은 뉴클레오티드의 수를 나타낸다.
- 제 1항에 있어서,상기 p는 3~5개의 뉴클레오티드를 포함하는 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 q는 3~5개의 뉴클레오티드를 포함하는 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 r은 18~30개의 뉴클레오티드를 포함하는 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 5'-저 Tm 특이성 부위의 Tm은 상기 3'-고 Tm 특이성 부위의 Tm보다 낮은 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 분할 부위의 Tm은 상기 5'-저 Tm 특이성 부위의 Tm 및 상기 3'-고 Tm 특이성 부위의 Tm보다 낮은 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 5'-저 Tm 특이성 부위의 Tm은 10~30℃인 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 분할 부위의 Tm은 3~10℃인 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 3'-고 Tm 특이성 부위의 Tm은 50~65℃인 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 유니버설 염기는 디옥시이노신, 이노신, 7-디아자-2'-디옥시이노신, 2-아자-2'-디옥시이노신, 2'-OMe 이노신, 2'-F 이노신 및 이들의 조합으로 이루어지는 군으로부터 선택되는 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 제 1항에 있어서,상기 덤벨 구조 올리고뉴클레오티드는 서열번호 1 내지 서열 번호 35로 이루어지는 군으로부터 선택되는 것을 특징으로 하는 덤벨 구조 올리고뉴클레오티드.
- 다음의 일반식으로 나타내어지는 덤벨 구조 올리고뉴클레오티드를 포함하는 핵산 증폭용 프라이머:일반식 : 5'-Ap-Bq-Cr-3'여기에서, 상기 A는 상기 3'-말단의 연속된 염기서열과 상보적인 염기서열을 갖는 뉴클레오티드를 포함하는 5'-저 Tm 특이성 부위를 나타내고, 상기 B는 유니버설 염기를 갖는 뉴클레오티드를 포함하는 분할 부위를 나타내고, 상기 C는 주형 핵산의 특정 연속된 염기서열과 상보적인 염기서열을 갖는 뉴클레오티드를 포함하는 3'-고 Tm 특이성 부위를 나타내고, 상기 p, q 및 r은 뉴클레오티드의 수를 나타낸다.
- 제 12항에 있어서,상기 p는 3~5개의 뉴클레오티드를 포함하는 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 q는 3~5개의 뉴클레오티드를 포함하는 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 r은 18~30개의 뉴클레오티드를 포함하는 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 5'-저 Tm 특이성 부위의 Tm은 상기 3'-고 Tm 특이성 부위의 Tm보다 낮은 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 분할 부위의 Tm은 상기 5'-저 Tm 특이성 부위의 Tm 및 상기 3'-고 Tm 특이성 부위의 Tm보다 낮은 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 5'-저 Tm 특이성 부위의 Tm은 10~30℃인 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 분할 부위의 Tm은 3~10℃인 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 3'-고 Tm 특이성 부위의 Tm은 50~65℃인 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 유니버설 염기는 디옥시이노신, 이노신, 7-디아자-2'-디옥시이노신, 2-아자-2'-디옥시이노신, 2'-OMe 이노신, 2'-F 이노신 및 이들의 조합으로 이루어지는 군으로부터 선택되는 것을 특징으로 하는 핵산 증폭용 프라이머.
- 제 12항에 있어서,상기 덤벨 구조 올리고뉴클레오티드는 서열번호 1 내지 서열 번호 35로 이루어지는 군으로부터 선택되는 것을 특징으로 하는 핵산 증폭용 프라이머.
- 주형, 프라이머 및 중합효소를 포함하는 혼합물로부터 중합효소 연쇄반응을 수행하여 핵산을 증폭시키는 방법에 있어서, 다음의 일반식으로 나타내어지는 덤벨 구조 올리고뉴클레오티드를 포함하는 핵산 증폭용 프라이머를 이용하는 것을 특징으로 하는 핵산 증폭 방법:일반식 : 5'-Ap-Bq-Cr-3'여기에서, 상기 A는 상기 3'-말단의 연속된 염기서열과 상보적인 염기서열을 갖는 뉴클레오티드를 포함하는 5'-저 Tm 특이성 부위를 나타내고, 상기 B는 유니버설 염기를 갖는 뉴클레오티드를 포함하는 분할 부위를 나타내고, 상기 C는 주형 핵산의 특정 연속된 염기서열과 상보적인 염기서열을 갖는 뉴클레오티드를 포함하는 3'-고 Tm 특이성 부위를 나타내고, 상기 p, q 및 r은 뉴클레오티드의 수를 나타낸다.
- 제 23항에 있어서,상기 p는 3~5개의 뉴클레오티드를 포함하는 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 q는 3~5개의 뉴클레오티드를 포함하는 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 r은 18~30개의 뉴클레오티드를 포함하는 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 5'-저 Tm 특이성 부위의 Tm은 상기 3'-고 Tm 특이성 부위의 Tm보다 낮은 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 분할 부위의 Tm은 상기 5'-저 Tm 특이성 부위의 Tm 및 상기 3'-고 Tm 특이성 부위의 Tm보다 낮은 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 5'-저 Tm 특이성 부위의 Tm은 10~30℃인 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 분할 부위의 Tm은 3~10℃인 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 3'-고 Tm 특이성 부위의 Tm은 50~65℃인 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 유니버설 염기는 디옥시이노신, 이노신, 7-디아자-2'-디옥시이노신, 2-아자-2'-디옥시이노신, 2'-OMe 이노신, 2'-F 이노신 및 이들의 조합으로 이루어지는 군으로부터 선택되는 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 덤벨 구조 올리고뉴클레오티드는 서열번호 1 내지 서열 번호 35로 이루어지는 군으로부터 선택되는 핵산 증폭용 프라이머를 사용하는 것을 특징으로 하는 핵산 증폭 방법.
- 제 23항에 있어서,상기 핵산 증폭 방법은 2종 이상의 주형을 이용한 다중 중합효소 연쇄반응인 것을 특징으로 하는 핵산 증폭 방법.
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US15/509,179 US20180073082A1 (en) | 2015-02-25 | 2015-02-25 | Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same |
CN201580046667.8A CN107075576B (zh) | 2015-02-25 | 2015-02-25 | 哑铃结构寡核苷酸、包含其的核酸扩增用引物及利用该引物的核酸扩增方法 |
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