Nothing Special   »   [go: up one dir, main page]

WO2003004642A1 - Procede d'amplification d'un fragment d'acide nucleique non cyclique - Google Patents

Procede d'amplification d'un fragment d'acide nucleique non cyclique Download PDF

Info

Publication number
WO2003004642A1
WO2003004642A1 PCT/JP2002/006911 JP0206911W WO03004642A1 WO 2003004642 A1 WO2003004642 A1 WO 2003004642A1 JP 0206911 W JP0206911 W JP 0206911W WO 03004642 A1 WO03004642 A1 WO 03004642A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid fragment
primer
sequence
complementary
Prior art date
Application number
PCT/JP2002/006911
Other languages
English (en)
Japanese (ja)
Inventor
Akio Yamane
Original Assignee
Wakunaga Pharmaceutical 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 Wakunaga Pharmaceutical Co., Ltd. filed Critical Wakunaga Pharmaceutical Co., Ltd.
Publication of WO2003004642A1 publication Critical patent/WO2003004642A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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

Definitions

  • the present invention relates to a method for amplifying a nucleic acid fragment having a non-circular single-stranded nucleic acid fragment as type II.
  • a nucleic acid fragment having a non-circular single-stranded nucleic acid fragment as type II has been clarified, and SNP analysis has also been advanced in a huge project.
  • the simplification or low cost of gene detection or SNP detection has become an increasingly important issue for practical use of genetic diagnosis.
  • a small amount of the gene to be detected must be amplified by some method.
  • Such amplification methods generally include a method of amplifying a signal for detection and a method of amplifying a gene itself. At present, the latter are more practical.
  • PCR method As a method for amplifying a gene, the so-called PCR method (Polfs, A. et al., Clinical Diagnostics and Research, PCR: Springer-Verlag (1992)) is the most widespread, and is indispensable for the analysis of the human genome sequence. Technology.
  • Representative methods for isothermal amplification include, for example, the NA SBA method (Gabrielle, ME et al., J. General Microbiol. 139, 2423-2429 (1993)), the TMA method (acian, DL, U.S. Pat. No. 5,399,491) and the SDA method (Walker GT et al., Nucleic Acids Res. 20, 1691-1696 (1992)). These have also been put to practical use as genetic diagnosis methods. However, these methods use multiple enzymes, which complicates the reaction system and increases the cost.
  • the RCA method As another method of the isothermal amplification method, for example, the RCA method (Lizardi, PM et al., Nature Genetics 19, 225-232 (1998)) and the LAMP method (Notomi, T. et al., Nuclei c Acids Res. 28, e63 (2000)).
  • the RCA method is a method for amplifying a linear DNA probe as type II, and is considered to be an effective method for SNP detection by combining it with a ligase reaction.
  • the LAMP method is a method capable of isothermal amplification with a single enzyme, as far as the present inventor knows, it has not been widely used so far and its exact characteristics are not clear.
  • the extension reaction proceeds by adding a base corresponding to type I.
  • the target nucleic acid fragment can be isolated under isothermal conditions even for non-cyclic single-stranded nucleic acids.
  • the inventor has found that it can be repeatedly amplified. That is, the target non-circular single-stranded nucleic acid fragment (hereinafter, sometimes referred to as the target nucleic acid fragment) is type III and type II.
  • the target nucleic acid fragment could be continuously and repeatedly amplified by adjusting the fragment to have a circular or nearly circular structure and performing a strand displacement Bramer extension reaction.
  • the present invention is based on these findings.
  • an object of the present invention is to provide a method for amplifying a nucleic acid fragment complementary to a target nucleic acid fragment by a strand displacement primer single extension reaction using an acyclic single-stranded target nucleic acid fragment as type II.
  • the method for amplifying a nucleic acid fragment according to the present invention comprises the steps of: performing a strand-displacement primer extension reaction using a non-circular single-stranded target nucleic acid fragment as a ⁇ type and a first primer having a sequence complementary to the target nucleic acid fragment. Performing a nucleic acid fragment complementary to the target nucleic acid fragment by performing the method, the method for amplifying a nucleic acid fragment,
  • This is a method for amplifying a nucleic acid fragment by continuously forming a fragment complementary to a nucleic acid sequence containing a discontinuous sequence formed by the target nucleic acid fragment as a repetitive sequence.
  • FIG. 1 is a diagram schematically showing a strand displacement elongation reaction in the case where the type III chain has a nick-like structure.
  • FIG. 2 is a diagram showing an outline of the method for amplifying a nucleic acid fragment of the first preferred embodiment of the present invention.
  • FIG. 3 is a diagram showing an outline of a method for amplifying a nucleic acid fragment according to the second preferred embodiment of the present invention.
  • FIG. 4 is a diagram schematically showing a nucleic acid fragment amplification method according to another preferred embodiment of the present invention.
  • FIG. 5 is a diagram schematically illustrating a method for amplifying a nucleic acid fragment according to another more preferred embodiment of the present invention.
  • FIG. 6 is a diagram outlining a method for amplifying a nucleic acid fragment according to one preferred embodiment of the present invention.
  • FIG. 7 shows the analysis results of 2% agarose electrophoresis of Test A in the examples.
  • M indicates the size of the size (HapII digest of pUC19), and each lane number corresponds to the test number in each Example.
  • FIG. 8 shows the analysis results of 10% polyacrylamide electrophoresis of Test B in the example.
  • M indicates one size marker (10 base ladder: GibcoBRL # 1082 015), and each lane number corresponds to a test number in each embodiment. “” Corresponds to 100 bases.
  • the “non-contiguous sequence formed by the target nucleic acid fragment” refers to a ⁇ -shaped sequence that forms a complementary strand as described above, which is continuously arranged as a nick-like structure, that is, a base sequence. However, it refers to a sequence containing a structure in which a phosphate ester bond is not formed between some adjacent bases. Specifically, for example, when the 5 ′ end and the 3 ′ end of the non-cyclic single-stranded target nucleic acid fragment are closely adjacent to each other, the nucleic acid fragment forms a circular structure. An example is a sequence composed of adjacent portions at the ends.
  • a non-circular single-stranded nucleic acid fragment of interest has complementary sequences at both ends thereof, and a stem structure is formed by binding of the complementary sequences, thereby forming a loop and a stem structure.
  • a discontinuous portion is generated as a loop structure arrangement at the boundary with the stem structure part. The arrangement of such a portion is also included.
  • "j which is continuously formed as a repetitive sequence” means that a non-cyclic single-stranded target nucleic acid fragment is generated as a tandem repetitive sequence and the same nucleic acid fragment is continuously formed.
  • the 5 ′ end and 3 ′ end of the acyclic single-stranded target nucleic acid fragment are close to each other.
  • a sequence similar to the circular sequence is formed, and a sequence complementary thereto is formed by the strand displacement extension reaction.
  • the formed nucleic acid fragments are first formed one by one with respect to the sequence similar to the circular sequence, and subsequently, the extension reaction acts to trace the sequence similar to the circular sequence, and the target sequence is successively determined. It is believed that a fragment complementary to the nucleic acid fragment is formed.
  • the nucleic acid fragments thus formed can be obtained continuously in such a manner as to repeatedly have one unit of fragments.
  • the target nucleic acid fragment has a region having the same sequence at both ends. When a fragment is amplified, only one of the regions will be short-circuited and the remaining sequence will be used repeatedly for amplification.
  • the acyclic single-stranded target nucleic acid fragment that can be used is not particularly limited as long as its base sequence is known, and may be either RNA or DNA. . Also, the target nucleic acid fragment is restricted by its origin. Therefore, the present invention is also applicable to nucleic acids derived from eukaryotes, prokaryotes, and viruses, and also to those synthesized.
  • target nucleic acid fragment used herein includes, in addition to the target sequence itself to be amplified, if necessary, a sequence region complementary to each other and a region identical to each other. It shall be.
  • RNA When RNA is used as a target for direct amplification, a reverse transcriptase that synthesizes a DNA chain using RNA as type II or a DNA polymerase that can synthesize DNA using certain RNAs as type II Can be used.
  • a DNA polymerase when a complementary DNA is synthesized from the target RNA, or when DNA is used as an overnight getter for amplification from the beginning, a DNA polymerase can be used.
  • a strand displacement primer extension reaction is performed.
  • the polymerase used in the strand displacement primer extension reaction is not particularly limited as long as it has at least strand displacement activity, and any enzyme can be used as long as it has such activity. .
  • M-MuLV reverse transcriptase which is a reverse transcriptase having strand displacement activity
  • DNA polymerase having a strand displacement activity such as Klenow Fragment, Vent DNA polymerase, ⁇ 29 DNA polymerase ⁇ Bst DNA polymerase, etc.
  • Klenow Fragment a strand displacement activity
  • Vent DNA polymerase a strand displacement activity
  • ⁇ 29 DNA polymerase ⁇ Bst DNA polymerase, etc.
  • those lacking exonuclease activity are preferred. The activity is described in the catalog of the enzyme sales company. It is considered that the reaction of the present invention for synthesizing a continuous complementary strand to the discontinuous type ⁇ is inconsistent with the reaction by strand displacement activity (Canceill, D. et al., J.
  • the strand displacement activity can be controlled by the reaction temperature or the salt concentration in the reaction solution. If necessary, a plurality of polymerases having different strand displacement activities may be used simultaneously.
  • the reaction temperature of the strand displacement Bramer extension reaction can be appropriately selected based on the optimum temperature according to the enzyme used.
  • the reaction temperature is usually the temperature of the primer extension reaction. Relatively high temperatures are preferred to increase specificity. In such cases, it is desirable to use a heat-resistant DNA polymerase suitable for that temperature.
  • primer used in the present invention any known primer used in general primer extension reaction, for example, polymerase chain reaction, can be used.
  • the chain length of the primer to be used can be appropriately selected according to general usage.
  • the length of the first primer used in the strand displacement primer extension reaction is typically 4 to 100 bases, and preferably 10 to 30 bases.
  • the primer has an action of bringing the 5 'end and the 3' end of the discontinuous target nucleic acid fragment closer to each other, it is desirable that the primer be capable of maintaining its structure stably under the reaction conditions.
  • both a primer having a sequence selected from the sequence complementary to the target nucleic acid fragment and a primer having a sequence selected from the sequence on the target nucleic acid fragment are used, both of these are not necessarily required. It does not need to be present at another position of the target nucleic acid fragment, and may be complementary to each other.
  • an oligonucleotide having a region complementary to both the 5, 5 terminal and the 35 terminal of the target nucleic acid fragment to be amplified is prepared as a first primer.
  • the extension reaction is performed in the presence of a polymerase having strand displacement activity and a substrate for the extension reaction.
  • the first primer At the 5 terminal end, there is a region of a sequence complementary to the 5 'end of the non-cyclic single-stranded target nucleic acid fragment, and at the 3 5 terminal end, the 3' end of the non-cyclic single-stranded target nucleic acid fragment It is designed to have a region of a sequence complementary to the side.
  • the non-consecutive 5, 5 and 3 'ends of the target nucleic acid fragments attracted to each other by the primers used become unstable depending on the conditions of the strand displacement extension reaction, and a continuous complementary strand is formed. May dissociate before. For this reason, if necessary, mutually complementary sequences may be introduced into both ends of the target nucleic acid fragment to form a loop and a stem structure (FIG. 3).
  • the non-circular single-stranded nucleic acid fragment of interest has, at both ends thereof, sequence regions complementary to each other so that a loop and a stem structure can be formed.
  • the first primer has a sequence region complementary to the 5 ′ end of the loop-forming portion at the 5 ′ end of the primer, and further, the 3 ′ end of the primer On the side, it is designed so as to have a sequence region complementary to the 3, terminal side of the loop structure.
  • the stability of the stem structure can be enhanced by making the sequence constituting the structure rich in guanine-cytosine base.
  • a sequence complementary to each other in the target nucleic acid fragment is found at both ends and used, or used as a primer when synthesizing the target nucleic acid fragment.
  • Such a sequence can be introduced in advance and used.
  • a kit for nucleic acid amplification comprising at least a first primer, a second primer, and a DNA polymerase.
  • the first primer has a region of a sequence complementary to the 5 ′ end of the non-circular single-stranded target nucleic acid fragment at its 5 'end, and at its 3' end,
  • the second primer comprises an arbitrary sequence selected from the non-circular single-stranded nucleic acid fragment, and the DNA polymerase Is characterized by having strand displacement activity.
  • the non-circular single-stranded target nucleic acid fragment has, at both ends thereof, regions whose sequences are identical to each other; Consists of any sequence that is complementary to the circular single-stranded nucleic acid fragment. That is, a sequence region having the same sequence as that of the target nucleic acid fragment is introduced into the 5 ′ terminal side and the 3 ′ terminal side of the target nucleic acid fragment, and any sequence complementary to the target nucleic acid fragment (preferably an oligonucleotide ) Can be used as a primer to continuously and repeatedly amplify a nucleic acid fragment having a sequence complementary to the target nucleic acid fragment.
  • the primer extension reaction proceeds, the primer is once stopped at the 5 ′ end of the target nucleic acid fragment, and the sequence complementary to the 5 ′ end of the synthesized target nucleic acid fragment regions, it is thought that there may be the same sequence area and the High Priestess soybean present in the 3 5-terminal side rather than the 5 'end of the desired nucleic acid fragment.
  • the extension reaction is restarted.
  • the target nucleic acid fragment can be continuously and repeatedly amplified (FIG. 4) (note that the specific description is only for describing the present invention. And does not limit the scope of the present invention).
  • one of the regions having the same sequence needs to be located near the 5 ′ end of the target nucleic acid fragment, and the other needs to be located near the 3 ′ end.
  • regions of identical sequence are located at both ends of the nucleic acid fragment of interest.
  • the chain length of the sequence region may be about 2 to 100 bases, but is preferably 4 to 100 bases, and more preferably 8 to 30 bases.
  • the oligonucleotide serving as a primer is not necessarily complementary to both the 5 ′ terminal and 3 ′ terminal sequences of the target nucleic acid fragment. It is not necessary to have a sequence. 5 'to the contiguous area other than the sequence of the distal and 35 terminal but it may also be a sequence complementary.
  • the non-circular single-stranded nucleic acid fragment of interest has at each end thereof: (i) a sequence region complementary to each other so as to form a loop and a stem structure; ii) a region having the same sequence as that of the target nucleic acid fragment in order from the end of the target nucleic acid fragment, and the first primer is complementary to a portion of the non-circular single-stranded nucleic acid fragment to be the loop structure. Consists of an arbitrary sequence.
  • a region having the same sequence is provided at both ends of the target nucleic acid fragment, and sequences complementary to each other are provided at both ends so as to form a loop and a stem structure with the regions interposed therebetween.
  • sequences complementary to each other are provided at both ends so as to form a loop and a stem structure with the regions interposed therebetween.
  • the target nucleic acid fragment and the nucleic acid fragment complementary thereto can be continuously and repeatedly amplified by the strand displacement primer extension reaction using the first primer, with the target nucleic acid fragment being type III.
  • an oligonucleotide selected from the same sequence as the target nucleic acid fragment in the reaction can be used as the second primer.
  • the amplification efficiency can be dramatically increased.
  • RecA protein or the like related to homologous recombination may be added.
  • the non-circular single-stranded nucleic acid fragment When the non-circular single-stranded nucleic acid fragment has complementary sequences at both ends to form a loop and a stem structure, it is selected from the loop-forming portion of the non-cyclic single-stranded nucleic acid fragment. It is preferable to use any arbitrary sequence as the second primer in the strand displacement primer extension reaction. When the non-cyclic single-stranded nucleic acid fragment does not form a loop and a stem structure, an arbitrary sequence selected from the non-cyclic single-stranded nucleic acid fragment may be replaced with a second sequence in the strand displacement primer-elongation reaction. It is preferably used as a primer. According to one more preferred embodiment of the present invention, a nucleic acid fragment can be amplified by applying the method of the present invention using four types of primers (primers 1 to 4) as described below (FIG. 6).
  • a primer for first introducing a sequence complementary to each other in the target nucleic acids fragments prepared primer one 2, 5 5-terminal side and the 3 'end of the objective nucleic acid fragment by performing an extension reaction using the primers To introduce sequences complementary to each other. Subsequently, a strand displacement reaction occurs by the primer 11 located upstream of the primer 12, and the extension product of the primer 12 becomes single-stranded, and then the loop is formed by a sequence complementary to each other introduced into the strand. And take the stem structure. Further, a strand displacement reaction is performed using a primer 3 complementary to the 5 ′ end and 3 ′ end of the loop structure and a primer 14 having a sequence selected from the sequence of the target nucleic acid fragment.
  • the target nucleic acid fragment to be amplified can be continuously and repeatedly amplified.
  • the extension from the primer 12 is similarly performed.
  • the product is released into single-stranded nucleic acid fragments by an extension reaction from the primer 11.
  • the released single-stranded nucleic acid fragments have complementary sequences on the terminal side and spontaneously form a stem structure. Excess bra one more 3 free state exists, the extension reaction was hybridized to both the connection portion of the loop-stem structure of single-stranded nucleic acid fragments to form a stem structure from 3 5-terminal side of the loop structure Start.
  • extension reaction When the extension reaction reaches the 5 'end of the primer, it continues the extension reaction while displacing the existing complementary strand (strand displacement). When the extension reaction reaches the stem portion, the extension reaction does not go to the stem portion but jumps over the stem portion (to the base immediately next to the 5 'end of the complementary strand forming the stem). Add complementary bases. Thereafter, the same reaction is repeated to continuously produce the target nucleic acid fragment.
  • the generated target nucleic acid fragment is mostly single-stranded, and the primer 4 hybridizes to the target nucleic acid fragment.
  • Primer 4 is complementary to all target nucleic acid fragments of the single-stranded nucleic acid fragment containing the target nucleic acid fragment repeatedly.
  • primer 3 is also complementary to these and again An extension reaction involving exchange is started, and as a result, an extension reaction containing the target sequence occurs explosively.
  • the first primer is composed of any sequence complementary to the non-linear single-stranded target nucleic acid fragment
  • the second primer is located in a region downstream of the target nucleic acid fragment from the first primer. It has a sequence that is complementary to the 5 'end of the second primer and is complementary to the 3' end sequence so that the complementary strand generated by primer extension can form a loop and stem structure.
  • the third primer has a sequence region complementary to the 5 ′ end of the loop structure formed by the complementary strand at the 5 ′ end of the primer, and a primer at the 5 ′ end of the primer.
  • a sequence region complementary to the 3rd terminal side of the loop structure portion, and the fourth primer is an arbitrary sequence selected from the loop structure portion .
  • Detection of the target nucleic acid fragment amplified by the present invention can be applied by any method as long as it is a general technique used for nucleic acid fragment detection (Keller 3 GH, Manak, MM DNA Probes Stockton Press (1993)).
  • a method of detecting the amplified target nucleic acid fragment for example, a method of subjecting a part of the amplification reaction solution to electrophoresis and detecting the nucleic acid fragment amplified with a stain can be mentioned.
  • a target nucleic acid fragment strand has a recognition site for a restriction enzyme
  • the fragment may be digested with the restriction enzyme and detected by electrophoresis in the same manner. Furthermore, it may be confirmed whether or not the nucleic acid fragment amplified by the probe method after electrophoresis contains the target sequence.
  • the amplification product may be mixed in the next reaction, leading to an erroneous result. Therefore, it is preferable to inspect the reaction product in a sealed state.
  • a method of detecting the amplification reaction solution as it is is often used.
  • a nucleic acid stain is allowed to coexist in the amplification reaction solution, There is a method based on the principle that a staining agent develops color when an amplification reaction occurs (Wittwer, CT et al., BioTechniques 22, 176-181 (1997)).
  • sequence-specific detection such as the molecular beacon method
  • the production of an amplified product may be detected as fluorescence emission by coexisting a probe with a special label in advance (Tyagi , S. et al., Nature Biotechnology 14, 303-308 (1996)).
  • a method for detecting a target base sequence in a sample comprising performing the above-described amplification method and observing whether or not an amplification reaction product has been generated.
  • the oligonucleotide used in this example was purchased from Nippon Bio Services.
  • the Bst DNA polymerase was purchased from New England Biolabs, Inc., and the attached reaction buffer was used for the reaction.
  • the nucleotide sequence was determined using a DNA sequencing kit (PE Applied Biosystems # 43031521) and reacted with ABI PRISM 310.
  • Type II nucleic acid fragments and primers used in the experiment were as follows.
  • Primer A 1 (complementary to type I): XPA1
  • Primer A2 (complementary to type II): XPA2
  • Primer S I (same chain as type II): XPS1
  • Primer S 2 (same chain as type II): XPS2
  • CCTGAGAACTTCAGGGTGAG type I contains an inverted repeat consisting of 13 bases at both ends, that is, a region whose sequences are complementary to each other.
  • Type II is obtained by removing the inverted repeat sequence at the 3 'end from Type 1.
  • ⁇ type 3 is obtained by removing the inverted repeats at both ends from ⁇ type 1 and consists only of the sequence to be amplified.
  • Primer A1 and primer A2 are sequences complementary to type I. Of these, the primer A1 has a sequence complementary to the type 5 nucleic acid fragment to be amplified at the 5, 5 and 3, terminal sides, respectively.
  • Primer A2 has a sequence complementary to a portion other than the type II inverted repeat sequence. Each of the primer S1 and the primer S2 has the same sequence as a part of the portion other than the type II inverted repeat sequence. Evaluation test
  • test sample No. I prepared ⁇ 12 each.
  • type ((1 pmol) and primer (16 pmol) were selected and mixed, and the test sample No. I prepared ⁇ 12 each.
  • four kinds of deoxynucleotide triphosphates 200 ⁇ mol
  • a reaction buffer were added, and then denatured at 94 ° C. for 30 seconds.
  • each sample was annealed at 60 ° C. for 1 minute. At this time, the total amount of the reaction solution was 60 / L.
  • the reaction was carried out at 60 ° C for 2 hours.
  • FIG. 7 shows the result of staining with ethidium bromide.
  • nucleotide sequence of the band considered to be the target amplified product was confirmed, it was consistent with the type III sequence.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne un procédé d'amplification d'un fragment d'acide nucléique qui consiste à utiliser un fragment d'acide nucléique cible simple brin non cyclique comme matrice, puis à effectuer une réaction d'élongation de l'amorce par déplacement de brin au moyen de l'utilisation d'une première amorce dont la séquence est complémentaire au fragment d'acide nucléique cible afin qu'un fragment d'acide nucléique complémentaire au fragment d'acide nucléique cible soit formé. De ce fait, le fragment d'acide nucléique est amplifié par la formation en continu, comme séquence répétée, de fragments complémentaires à la séquence d'acide nucléique contenant des séquences interrompues formées par le fragment d'acide nucléique cible. Ainsi, un fragment d'acide nucléique cible et un fragment d'acide nucléique complémentaire à ce dernier peuvent être efficacement amplifiés par l'utilisation d'un fragment d'acide nucléique cible simple brin servant de matrice.
PCT/JP2002/006911 2001-07-06 2002-07-08 Procede d'amplification d'un fragment d'acide nucleique non cyclique WO2003004642A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001206389A JP2005052002A (ja) 2001-07-06 2001-07-06 非環状核酸断片の増幅方法
JP2001-206389 2001-07-06

Publications (1)

Publication Number Publication Date
WO2003004642A1 true WO2003004642A1 (fr) 2003-01-16

Family

ID=19042531

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/006911 WO2003004642A1 (fr) 2001-07-06 2002-07-08 Procede d'amplification d'un fragment d'acide nucleique non cyclique

Country Status (2)

Country Link
JP (1) JP2005052002A (fr)
WO (1) WO2003004642A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070298415A1 (en) * 2003-12-10 2007-12-27 Takara Bio Inc. Method of Amplifying Nucleic Acid

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04262799A (ja) * 1991-02-18 1992-09-18 Toyobo Co Ltd 核酸配列の増幅方法およびそのための試薬キット
WO1997019193A2 (fr) * 1995-11-21 1997-05-29 Yale University Amplication et detection de segments unimoleculaires
US5714320A (en) * 1993-04-15 1998-02-03 University Of Rochester Rolling circle synthesis of oligonucleotides and amplification of select randomized circular oligonucleotides
WO1999049079A1 (fr) * 1998-03-25 1999-09-30 Ulf Landegren Replication en cercle roulant destinee a des sondes cadenas
EP0971039A2 (fr) * 1998-06-24 2000-01-12 Enzo Diagnostics, Inc. Méthode utiles pour l'amplification et pour le séquençage d'acides nucléiques, et la production d'acides nucléiques présentant une stabilité thermodynamique réduite
EP1020534A1 (fr) * 1998-11-09 2000-07-19 Eiken Kagaku Kabushiki Kaisha Procede de synthese d'acide nucleique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04262799A (ja) * 1991-02-18 1992-09-18 Toyobo Co Ltd 核酸配列の増幅方法およびそのための試薬キット
US5714320A (en) * 1993-04-15 1998-02-03 University Of Rochester Rolling circle synthesis of oligonucleotides and amplification of select randomized circular oligonucleotides
WO1997019193A2 (fr) * 1995-11-21 1997-05-29 Yale University Amplication et detection de segments unimoleculaires
WO1999049079A1 (fr) * 1998-03-25 1999-09-30 Ulf Landegren Replication en cercle roulant destinee a des sondes cadenas
EP0971039A2 (fr) * 1998-06-24 2000-01-12 Enzo Diagnostics, Inc. Méthode utiles pour l'amplification et pour le séquençage d'acides nucléiques, et la production d'acides nucléiques présentant une stabilité thermodynamique réduite
EP1020534A1 (fr) * 1998-11-09 2000-07-19 Eiken Kagaku Kabushiki Kaisha Procede de synthese d'acide nucleique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BANER J. ET AL.: "Signal amplification of padlock probes by rolling circle replication", NUCLEIC ACIDS RES., vol. 26, no. 22, 15 November 1998 (1998-11-15), pages 5073 - 5078, XP002112357 *
LIZARDI P.M. ET AL.: "Mutation detection and single-molecule counting using isothermal rolling-circle amplification", NAT. GENET., vol. 19, no. 3, July 1998 (1998-07-01), pages 225 - 232, XP000856939 *

Also Published As

Publication number Publication date
JP2005052002A (ja) 2005-03-03

Similar Documents

Publication Publication Date Title
US11130984B2 (en) Compositions, methods, systems and kits for target nucleic acid enrichment
EP2253717B1 (fr) Méthode d'amplification d'acides nucléiques en utilisant comme matrice des acides nucléiques double-brin
US9657339B2 (en) Method for amplification of target nucleic acid
EP3470529B1 (fr) Procédé d'auto-amplification isothermique d'acide nucléique
EP3568493B1 (fr) Méthodes et compositions pour la réduction de codes-barres moléculaires redondants créés dans des réactions d'extension d'amorce
JPH022934A (ja) テンプレート−依存性核酸プロ−ブ再構成を用いたアッセイ
US20230257805A1 (en) Methods for ligation-coupled-pcr
CN111801427B (zh) 用于单分子的单链环状dna模板的产生
JP2020501565A (ja) 改変されたマルチプレックスおよびマルチステップ増幅反応ならびにそのための試薬
EP1333089A1 (fr) Methode de synthese d'acide nucleique simple brin
CN106701738B (zh) 一种等温解开双链dna及制备单链dna的方法
WO2002090538A1 (fr) Procede de synthese d'acide nucleique
WO2017183648A1 (fr) Procédé d'amplification de multiples éléments
JP2021505199A (ja) 鋳型切り換え機構を通じて核酸ライブラリを調製するためのシステムと方法
JP2007319096A (ja) エンドヌクレアーゼのニッキング活性を利用した核酸増幅法
WO2003004642A1 (fr) Procede d'amplification d'un fragment d'acide nucleique non cyclique
US20090305288A1 (en) Methods for amplifying nucleic acids and for analyzing nucleic acids therewith
US10590453B2 (en) Methods for amplification of nucleic acids utilizing a circularized template prepared from a target nucleic acid
US20060110745A1 (en) Method for amplifying nucleic acids
JP7191115B2 (ja) エンドヌクレアーゼ媒介移動平衡（ＥＭ－ＳＥｑ）による核酸の増幅のための方法
JP2008528021A (ja) 生化学試薬及びその使用
JP2005102502A (ja) 一本鎖目的核酸断片の増幅方法
JP2011010591A (ja) Ｄｎａの増幅方法

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 BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE 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 NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ 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 IE IT LU MC NL PT SE 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
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP