WO2005005664A1 - Procede de detection et procede d'identification d'un acide nucleique - Google Patents

Procede de detection et procede d'identification d'un acide nucleique Download PDF

Info

Publication number
WO2005005664A1
WO2005005664A1 PCT/JP2004/010170 JP2004010170W WO2005005664A1 WO 2005005664 A1 WO2005005664 A1 WO 2005005664A1 JP 2004010170 W JP2004010170 W JP 2004010170W WO 2005005664 A1 WO2005005664 A1 WO 2005005664A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
specificity
stranded nucleic
base sequence
stranded
Prior art date
Application number
PCT/JP2004/010170
Other languages
English (en)
Japanese (ja)
Inventor
Ken Nemoto
Joji Oshima
Original Assignee
G & G Science Co., Ltd.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by G & G Science Co., Ltd. filed Critical G & G Science Co., Ltd.
Priority to JP2005511598A priority Critical patent/JPWO2005005664A1/ja
Publication of WO2005005664A1 publication Critical patent/WO2005005664A1/fr

Links

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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention provides a method for synthesizing a single-stranded nucleic acid having relatively low specificity under low-temperature annealing conditions to detect and identify a nucleic acid having an arbitrary base sequence with high sensitivity, and under high-temperature annealing conditions. Synthesize single-stranded nucleic acids with relatively high specificity, increase the physical quantity of single-stranded interfering substances by mixing the former and the latter, or by adding synthetic single-stranded nucleic acids to increase specificity and The present invention relates to a nucleic acid detection and identification method that achieves high sensitivity and implements them with one type of primer. Background art
  • nucleic acids have been amplified by using complementary specific primers that bind one end of one strand of a specific site of double-stranded DNA to the other end of the other strand using an enzyme DNA polymerase as a catalyst.
  • the polymerase chain reaction (PCR) method for amplifying a target nucleic acid is widely used.
  • the principle of the PCR method is to amplify a DNA fragment between primers by forming a double-stranded DNA into a ⁇ shape and repeating extension of the two primer strands sandwiching a specific region by DNA polymerase.
  • Various DNA and RNA analysis techniques have been devised based on the method (see, for example, Japanese Patent Publication No.
  • the above-mentioned method for synthesizing single-stranded nucleic acids using one type of primer and recognizing the dissociation curve of the mutual interference product as a dissociation curve waveform pattern to detect and identify nucleic acids is easy and diverse. Although useful as a method for analysis, there is room for improvement in the low efficiency of single-stranded nucleic acid synthesis and the resulting low detection sensitivity. In other words, in order to improve the low detection sensitivity due to low synthesis efficiency, a method of increasing the nucleic acid synthesis efficiency by adapting the low annealing temperature can be considered, but this method has a specificity for the detection and identification of the target base sequence.
  • An object of the present invention is to provide a method for improving the detection sensitivity and at the same time maintaining the specificity of a nucleic acid detection and identification method utilizing single-stranded nucleic acid synthesis, which is more effective.
  • a method for adding a synthetic nucleic acid or a chemical substance having a function was used as a means for solving the problem.
  • the present invention uses a single type of primer having specific complementarity to any specific nucleotide sequence region on a nucleic acid, and a single strand having a high relative specificity to the specific nucleotide sequence region under high temperature annealing conditions.
  • the present invention has realized a method for synthesizing a nucleic acid, detecting and identifying the nucleic acid, and improving the reduction in sensitivity due to the low efficiency of single-stranded nucleic acid synthesis. Specifically, (i) at the beginning of the synthesis cycle, set low-temperature annealing conditions and
  • a method of adding a separately synthesized single-stranded nucleic acid was devised. All of these methods are applicable to single-stranded synthetic nucleic acids that are synthesized under high-temperature annealing conditions and have relatively high specificity, but are insufficient in quantity and do not provide sufficient sensitivity in the detection step.
  • the physical quantity of hydrogen bonds is increased, and the effect of improving the sensitivity in the detection step is obtained.
  • This embodiment uses a single type of primer that amplifies only a single strand in response to a specific base sequence of a target double-stranded nucleic acid, and improves the sensitivity while improving the specificity.
  • anneal means that a nucleotide chain forms a double-stranded structure by base pair bonding based on the Watson-Crick law.
  • the annealing temperature is changed between a low temperature and a high temperature; Less likely 1 Synthesize single-stranded nucleic acids together with target single-stranded nucleic acids with high specificity.In high-temperature annealing, synthesize only single-stranded target nucleic acids with high specificity, and form a single-stranded nucleic acid mutual interference product in the presence of both.
  • FIG. Figure 1 shows the nucleic acid synthesis status during low-temperature annealing, where a single type of primer is used to increase the detection sensitivity for a large amount of low-specificity single-stranded nucleic acid, and the target base sequence X Both a synthetic nucleic acid of interest, a small amount of a target single-stranded nucleic acid with high specificity, and a synthetic nucleic acid of the target base sequence X are synthesized.
  • FIG. 1 (B) shows the synthesis status of nucleic acids in high-temperature annealing, in which only a single-stranded target nucleic acid having high specificity and a synthetic nucleic acid derived from the target base sequence X are synthesized with one kind of primer.
  • low-temperature annealing annealing occurs even if there is no perfect complementarity between the primer and type I nucleic acid, and nucleic acid synthesis proceeds
  • high-temperature annealing annealing occurs when there is no perfect complementarity between the primer and type II nucleic acid. This indicates that only a specific target nucleic acid is synthesized because no reaction is performed.
  • FIG. 2 schematically shows the state of incomplete complementary annealing between the primer and the type I nucleic acid during low-temperature annealing, and the mutual interaction in the case where various single-stranded synthetic nucleic acids synthesized by the above-mentioned temperature change annealing are mixed.
  • Figure 3 shows a schematic diagram of the interference products.
  • a single-stranded nucleic acid with low specificity to increase detection sensitivity coexists with the target single-stranded nucleic acid with high specificity, causing a reaction between the two and increasing the physical quantity of hydrogen bond.
  • the amount of intercalator increases, resulting in high sensitivity.
  • the hydrogen-bonding site is present when the target single-stranded nucleic acid with high specificity is present at the time of self-loop or hairpin structure formation by itself, or only at the time of dimer formation.
  • the physical quantity is also small, resulting in lower sensitivity.
  • the concept of the method for adding a synthetic single-stranded nucleic acid is the same as that in Fig. 3, and by separately synthesizing and adding a synthetic nucleic acid derived from outside the target base sequence X (synthetic nucleic acid for increasing the fluorescence intensity) in the same figure. An effect similar to the above-described method of performing low-temperature annealing and high-temperature annealing continuously or discontinuously is obtained.
  • the method of performing low-temperature annealing and high-temperature annealing continuously or discontinuously, and the method of adding a single-stranded nucleic acid use primers in the presence of the enzyme DNA polymerase.
  • a surfactant such as a nonionic type or a sulfuric acid compound such as sodium sulfate or sodium hydrogen sulfate, and it is particularly effective at low temperature annealing.
  • FIG. 1 is a diagram schematically illustrating nucleic acid synthesis according to an embodiment of the present invention.
  • A shows a state of nucleic acid synthesis in low-temperature annealing of the present invention
  • B shows a state of nucleic acid synthesis in high-temperature annealing. It is shown.
  • FIG. 2 is a diagram schematically illustrating annealing between a primer and a type III nucleic acid in low-temperature annealing.
  • FIG. 3 is a diagram schematically illustrating a mutual interference product of a nucleic acid synthesis product according to the embodiment of the present invention.
  • FIG. 4 is a dissociation curve waveform pattern obtained by the protocol of the present invention shown in Table 2.
  • Figure 5 shows the dissociation curve waveform patterns obtained with the comparative protocols (A) and (B) shown in Table 2.
  • FIG. 6 is an electrophoresis photograph of the present invention and a comparative protocol.
  • FIG. 7 shows a dissociation curve waveform pattern when a synthetic nucleic acid was added.
  • FIG. 8 is a dissociation curve waveform pattern when no synthetic nucleic acid was added.
  • rRNA DNA sequence encoding bacterial 16S ribosomal RNA
  • select 12 bases that are conserved in about 3000 bacterial species and have a low frequency of occurrence in non-bacterial DNA, and use this as a BSS primer.
  • the protocol of the present invention in which the annealing temperature was changed and the protocol in which the annealing was not changed were compared, and the effect of improving the sensitivity and specificity was performed as an example.
  • a sequence having homology to 163 rRNA, such as 23 S rRNA and 8 S is 1 and so on. Amplified at the same time.
  • the mutual interference products composed of synthetic single-stranded nucleic acids can be obtained in a sufficient physical quantity in the detection process, but have low relative specificity in many regions. Unnecessary presence of the single-stranded nucleic acid reduces specificity. Under high-temperature annealing conditions alone, the mutual interference products composed of synthetic single-stranded nucleic acids have high specificity because only single-stranded nucleic acids with high relative specificity to the target gene region are synthesized. However, it is difficult to ensure a sufficient physical quantity in the detection process.
  • a plurality of single-stranded nucleic acids having low relative specificity in multiple regions are simultaneously and simultaneously synthesized from regions other than these target gene regions under low-temperature annealing conditions.
  • Single-stranded nucleic acids with low relative specificity from regions other than the target gene region are not synthesized, and only single-stranded nucleic acids with high relative specificity to the target gene region are synthesized.
  • the mutual interference products composed of these synthetic single-stranded nucleic acids are expected to secure sufficient physical quantity in the detection step and also obtain specificity.
  • the 10X PCR buffer was supplied with Taq DNA polymerase or Taq-REX (TAKARA), and the 5X Cyber Green dilution was prepared by diluting the stock solution with sterile distilled water.
  • the one-fold dilution was used as a 5X Genopattern buffer, a product manufactured by Adgene, and a Taq DNA polymerase was used, Taq-REX (TAKARA).
  • the BSS primer is composed of 5, 1 ATCGCTATGTGC 13 ′ (base sequence 1),
  • Table 2 shows the protocol of the present invention and the comparative protocol.
  • each peak of the waveform pattern is sharp and clear, the difference in Dip i cate is small, and a sufficient amount of fluorescence is obtained.
  • the comparative protocol A in Fig. 5 (A) that is, the protocol under only low-temperature annealing conditions, was used, the same amount of fluorescence was obtained as compared to Fig. 4, but the sharpness of each peak in the waveform pattern was clear.
  • the synthetic nucleic acid can secure a sufficient amount of fluorescence in the detection step, the specificity is reduced by the presence of unnecessarily large single-stranded nucleic acid with low relative specificity in multiple regions. Show.
  • the present protocol makes it possible to obtain a sufficient amount of detected fluorescence while increasing the specificity required for nucleic acid identification with only one primer for synthesizing a single-stranded nucleic acid.
  • This protocol By using this protocol, highly sensitive nucleic acid detection that focuses on the presence of the target nucleotide sequence of nucleic acids, and various waveform patterns can be stably observed. Nucleic acid identification is achieved.
  • FIG. 6 shows electrophoresis photographs of the protocol of the present invention and the comparative protocol.
  • Lane 1 is the protocol of the present invention
  • lane 2 is comparative protocol A
  • lane 3 is comparative protocol B.
  • sufficient nucleic acid synthesis is performed by the protocol of the present invention and comparative protocol A, that is, the protocol using only low-temperature annealing conditions, but the detailed identification of nucleic acids must be performed by electrophoresis. And a step of detecting the dissociation curve waveform pattern is required.
  • the nucleic acid was not sufficiently synthesized, and unreacted primers were observed.
  • rRNA bacterial 16S ribosome RNA
  • coli was selected as a bacterium to be used, and DNA was extracted using ISOGEN-LS (manufactured by Nippon Gene Co., Ltd.) according to the manual. Each sample D NA was adjusted to 3 0 0 ng / 5 ⁇ 1 ( ⁇ 2 0), as shown in Table 1 below, 5 0 mu This was added to the reaction solution of No. 1 and performed as a composition solution having a total amount of 551. In addition, the examples were each performed in duplicate experiments. Table 3 shows examples of formulation of the composition for implementing the present invention.
  • Table 4 shows the protocol for implementing the present invention.
  • FIGS. 7 (A), (B), (C) and FIG. 8 when the synthetic nucleic acid of the present invention is added, it becomes the basis of the fluorescence intensity which was insufficient under the high temperature annealing condition. It was possible to capture the hydrogen bonding site, and the effect of improving the nucleic acid detection sensitivity, which was lacking when no addition was performed, was recognized.
  • this example suggests that by devising the base sequence of the added synthetic nucleic acid, it is possible to adjust the dissociation curve waveform pattern of the detected nucleic acid, and it is easy to detect and identify various nucleic acids according to the purpose. Industrial applicability shown to be feasible
  • any base sequence of a target nucleic acid can be specifically detected with high sensitivity and easily detected and identified.
  • the advantage of using one type of primer is that by synthesizing any nucleotide sequence of the target nucleic acid widely including its related region, the information is reflected in the dissociation curve waveform pattern, which is extremely useful. Can be observed. Therefore, after using one type of primer, Combining the present invention as an improved method of low sensitivity due to the low synthesis efficiency of the problem can be used as an easy nucleic acid detection and identification method.
  • the present invention makes it possible to detect and identify any base sequence of a target nucleic acid with high sensitivity and high specificity.
  • the advantage of using one type of primer is that By widely synthesizing the nucleotide sequence including its related region, it is extremely useful to reflect the information derived from the nucleotide sequence on the waveform pattern of the dissociation curve, so that the properties of the nucleic acid can be widely observed. Therefore, it can be applied not only to identification of bacteria, but also to genetic analysis of humans, such as genetic diagnosis of hereditary diseases, cancerous diseases or infectious diseases, drug metabolism, and gender discrimination.

Abstract

Le procédé de cette invention permet de synthétiser des acides nucléiques à simple brin avec la détection d'une 1-amorce et d'un acide nucléique et leur identification d'après le modèle de forme d'onde courbe de dissociation d'un produit d'interférence mutuelle, ce qui représente un avantage mais a une contrepartie, à savoir, la sensibilité de détection en raison d'une faible efficacité de synthèse de l'acide nucléique synthétique à simple brin. Afin de résoudre le problème, on met en oeuvre un procédé consistant à synthétiser par recuisson à température basse un acide nucléique à simple brin à faible spécificité afin d'améliorer la sensibilité de détection ainsi qu'un acide nucléique à simple brin cible de haute spécificité ; à synthétiser par recuisson à température élevée uniquement un acide nucléique cible à simple brin à forte spécificité et à former un produit d'interférence mutuelle d'acide nucléique à simple brin en présence des deux ; et à mettre en oeuvre la détection et l'identification d'un acide nucléique spécifique à forte sensibilité d'après son modèle de forme d'onde courte de dissociation. En outre, on met en oeuvre un procédé consistant à ajouter un acide nucléique synthétique à simple brin en vue d'améliorer la sensibilité de détection ; à former un produit d'interférence mutuelle d'acide nucléique à simple brin en présence des deux et un acide nucléique cible à simple brin à forte spécificité synthétisé par recuisson à température élevée ; et à mettre en oeuvre la détection et l'identification d'un acide nucléique spécifique à forte sensibilité.
PCT/JP2004/010170 2003-07-15 2004-07-09 Procede de detection et procede d'identification d'un acide nucleique WO2005005664A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005511598A JPWO2005005664A1 (ja) 2003-07-15 2004-07-09 核酸の検出方法および同定方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003-294721 2003-07-15
JP2003294721 2003-07-15

Publications (1)

Publication Number Publication Date
WO2005005664A1 true WO2005005664A1 (fr) 2005-01-20

Family

ID=34056216

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/010170 WO2005005664A1 (fr) 2003-07-15 2004-07-09 Procede de detection et procede d'identification d'un acide nucleique

Country Status (2)

Country Link
JP (1) JPWO2005005664A1 (fr)
WO (1) WO2005005664A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7547514B2 (en) 2004-07-28 2009-06-16 Canon U.S. Life Sciences, Inc. Methods for monitoring genomic DNA of organisms
US7604938B2 (en) 2005-02-18 2009-10-20 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic DNA of organisms
CN110964782A (zh) * 2019-12-09 2020-04-07 上海鹍远健康科技有限公司 单链核酸连接效率检测方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0670799A (ja) * 1992-08-26 1994-03-15 Toshiba Corp ハイブリダイゼーション法
JP2003180374A (ja) * 2001-10-12 2003-07-02 Adgene Co Ltd 核酸溶解曲線を応用した核酸変異検出法
JP2003274959A (ja) * 2002-03-22 2003-09-30 Adgene Co Ltd ポリメラーゼ連鎖反応を応用した非特異核酸増幅法
JP2003334082A (ja) * 2002-05-17 2003-11-25 Adgene Co Ltd 波形生成プライマー、該プライマーを使用した核酸増幅方法及び核酸同定方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0670799A (ja) * 1992-08-26 1994-03-15 Toshiba Corp ハイブリダイゼーション法
JP2003180374A (ja) * 2001-10-12 2003-07-02 Adgene Co Ltd 核酸溶解曲線を応用した核酸変異検出法
JP2003274959A (ja) * 2002-03-22 2003-09-30 Adgene Co Ltd ポリメラーゼ連鎖反応を応用した非特異核酸増幅法
JP2003334082A (ja) * 2002-05-17 2003-11-25 Adgene Co Ltd 波形生成プライマー、該プライマーを使用した核酸増幅方法及び核酸同定方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7547514B2 (en) 2004-07-28 2009-06-16 Canon U.S. Life Sciences, Inc. Methods for monitoring genomic DNA of organisms
US7604938B2 (en) 2005-02-18 2009-10-20 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic DNA of organisms
US8841093B2 (en) 2005-02-18 2014-09-23 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic DNA of organisms
CN110964782A (zh) * 2019-12-09 2020-04-07 上海鹍远健康科技有限公司 单链核酸连接效率检测方法

Also Published As

Publication number Publication date
JPWO2005005664A1 (ja) 2006-08-24

Similar Documents

Publication Publication Date Title
JP6480511B2 (ja) 試料中の核酸配列を定量するための組成物および方法
CN107488710B (zh) 一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
US20220307080A1 (en) Methods and reagents for nucleic acid amplification and/or detection
JP2013520980A (ja) 増強マルチプレックスｆｉｓｈ
KR20160096633A (ko) 핵산 프로브 및 게놈 단편을 검출하는 방법
EP3292217B1 (fr) Formation de structures en épingle à cheveux in situ à l'aide d'invasion de brin forcé
JP2005518216A5 (fr)
Gerasimova et al. Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors
JP7313645B2 (ja) Ｒｎａ検出方法
EP3802873B1 (fr) Procédé de détection d'un polymorphisme mononucléotidique (snp) à l'aide d'amorces lamp et de blocage
WO1995032306A1 (fr) Procede de detection d'un acide nucleique cible
US20080090238A1 (en) Increased sensitivity of proximity ligation assays
WO2003100095A1 (fr) Procede de detection d'un acide nucleique cible
CN104593483B (zh) 选择和扩增多核苷酸的方法
EP3748011A1 (fr) Compositions et procédés permettant d'améliorer et/ou de prédire l'amplification d'adn
WO2005005664A1 (fr) Procede de detection et procede d'identification d'un acide nucleique
US20130078676A1 (en) Method of regulating oligonucleotide functionality
JP2008182974A (ja) 核酸プローブセットおよびその使用方法
WO2006004365A1 (fr) Procede de selection de sondes hautement specifiques pour l'analyse du genotype du papillomavirus humain et sondes a cet effet
JP2014522659A (ja) 保存生成物および任意生成物の蛍光標識フラグメントを生成するための核酸の増幅方法
Zeng et al. Strand displacement amplification for multiplex detection of nucleic acids
US20230227896A1 (en) Compositions, Kits and Methods for Isolating Target Polynucleotides
KR20110024025A (ko) Ｐｃｒ 프라이머를 이용한 녹강병 및 백강병의 진단
JP2003334082A (ja) 波形生成プライマー、該プライマーを使用した核酸増幅方法及び核酸同定方法
US20240132876A1 (en) Self-priming and replicating hairpin adaptor for constructing ngs library, and method for constructing ngs library using same

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2005511598

Country of ref document: JP

122 Ep: pct application non-entry in european phase