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WO2013069773A1 - Procédé de marquage supportant une base, procédé d'acquisition d'informations de séquence de base, et acide nucléique à simple brin marqué supportant une base - Google Patents

Procédé de marquage supportant une base, procédé d'acquisition d'informations de séquence de base, et acide nucléique à simple brin marqué supportant une base Download PDF

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Publication number
WO2013069773A1
WO2013069773A1 PCT/JP2012/079141 JP2012079141W WO2013069773A1 WO 2013069773 A1 WO2013069773 A1 WO 2013069773A1 JP 2012079141 W JP2012079141 W JP 2012079141W WO 2013069773 A1 WO2013069773 A1 WO 2013069773A1
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base
labeled
nucleic acid
labeling
oligonucleotide
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English (en)
Japanese (ja)
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國昭 永山
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Nagayama IP Holdings LLC
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Nagayama IP Holdings LLC
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    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • the present invention relates to a base-corresponding labeling method, a base sequence information acquisition method, and a base-corresponding labeled single-stranded nucleic acid. More specifically, the present invention relates to a base-corresponding labeling method for performing indirect labeling suitable for identification under an electron microscope on a base in a nucleic acid.
  • each base of adenine (A), thymine (T), guanine (G), cytosine (C), uracil (U) is utilized by utilizing the resolution and high resolution of an electron microscope. Techniques for identifying are being developed. In this technique, each base is directly labeled with a different heavy atom or heavy atom cluster, and the base is identified based on the labeled heavy atom under an electron microscope.
  • Patent Document 1 discloses a base modification method that enables base identification under an electron microscope. According to this base modification method, the base in the nucleic acid can be directly labeled for each base species by chemical means while maintaining the base sequence information of the nucleic acid.
  • Patent Document 2 and Patent Document 3 disclose techniques capable of reading sequence information at high speed.
  • This technology is a next-generation base sequence determination technology (sequencing technology) called “Optipore”, which is currently being developed, and specifically includes the following procedures.
  • a single-stranded nucleic acid to be sequence-read hereinafter described as DNA and referred to as target ssDNA
  • An oligonucleotide having a predetermined number of bases is inserted and added to the 3 ′ end or 5 ′ end of the nucleotide.
  • This oligomer is specifically designed for each base of A, G, C, and T, and is inserted into the target ssDNA while retaining the base sequence information of the nucleotide at the insertion site.
  • a single-stranded DNA (beacon) having a base sequence complementary to the oligonucleotide and labeled with a fluorescent reagent for base discrimination is bound (hybridized) to the target ssDNA to which the oligonucleotide is inserted and added, Double stranded DNA is formed.
  • the formed double-stranded DNA is passed through the solid state nanopore.
  • the beacon is released from the target ssDNA, and the labeled fluorescent reagent that has been quenched on the double-stranded DNA due to the adjacent group effect emits light.
  • the base sequence of the target ssDNA is read.
  • the base in the target nucleic acid is labeled at a sufficiently high rate in order to improve sequence reading accuracy.
  • a base labeling method that makes all bases distinguishable is essential.
  • the main object of the present invention is to provide a technique for indirectly labeling a base in a nucleic acid at a high labeling rate while maintaining the base sequence information of the nucleic acid.
  • the present invention is a method for labeling a base in a single-stranded nucleic acid while maintaining the base sequence information of the nucleic acid.
  • a base-corresponding labeling method including a procedure for inserting an oligonucleotide that is specifically and highly labeled according to the type of base constituting the nucleotide on the end side or 5 ′ end side. According to this base-corresponding labeling method, each base in a single-stranded nucleic acid can be reliably labeled with the inserted oligonucleotide.
  • the oligonucleotide can be inserted by a circular DNA conversion method.
  • the oligonucleotide is an oligonucleotide labeled with a metal cluster having an average particle diameter of 1-1000 nanometers and / or inorganic phosphor particles having an average particle diameter of 0.5-1000 nanometers. Can be used. It is preferable to use four types of metal clusters having different average particle diameters. In addition, gold colloid can be used for the metal cluster.
  • inorganic phosphor particles having different average particle diameters and / or emission wavelengths.
  • this base-corresponding labeling method it is preferable to use the inorganic phosphor particles and / or oligonucleotides separated and purified after labeling the inorganic phosphor particles as the oligonucleotides.
  • the present invention acquires the base sequence information by identifying the label of the oligonucleotide under an electron microscope for the single-stranded nucleic acid labeled by any one of the above-described base correspondence labeling methods Provide a method.
  • the present invention provides a single-stranded nucleic acid in which an oligonucleotide having a specific label according to the type of the base is inserted at the 3 ′ end or 5 ′ end of the nucleotide containing the base to be read.
  • the label may be a metal cluster having an average particle diameter of 1 to 1000 nanometers and / or inorganic phosphor particles having an average particle diameter of 0.5 to 1000 nanometers.
  • a technique for labeling and identifying a base in a nucleic acid with a high labeling rate while maintaining the base sequence information of the nucleic acid is provided.
  • the base-corresponding labeling method In the base-corresponding labeling method according to the present invention, specific and high-precision labeling is performed in advance on the 3 ′ end side or 5 ′ end side of the nucleotide in the single-stranded nucleic acid according to the type of base constituting the nucleotide.
  • an oligonucleotide hereinafter referred to as “labeled oligomer”
  • the base is labeled while maintaining the base sequence information of the nucleic acid.
  • the insertion of the labeled oligomer can be carried out using a circular DNA conversion method.
  • the CDC method is a method in which different types of oligonucleotides can be inserted for each type of base of nucleotides at the 3 ′ end or 5 ′ end of nucleotides in a single-stranded nucleic acid, and there are several variations. .
  • a method for obtaining base sequence information of a single-stranded nucleic acid by indirectly labeling a base in the single-stranded nucleic acid in a base-corresponding manner by inserting a labeled oligomer according to the procedure of the example will be described.
  • Double-stranded library for conversion [Conversion double-stranded structure] (See Fig. 1) A plurality of “double strands for conversion” that function to insert labeled oligomers into a single-stranded nucleic acid (target ssDNA) to be sequence-readed are prepared and constructed as a library.
  • the conversion duplex contains a labeled oligomer in one of the duplexes.
  • FIG. 1 shows the structure of the double strand for conversion.
  • the conversion duplex has a double-stranded portion, a first overhang and a second overhang.
  • the labeled oligomer L has a base sequence Xx and a base sequence R that serves as a recognition site for a type II restriction enzyme.
  • the labeled oligomer includes a base (X) to be identified as a reading target in the target ssDNA (in FIG. 5, for example, the first X 1 in the sequence of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 ) Is a specific label depending on the type.
  • the symbol X ′ shown in the second overhang indicates a base complementary to this labeled base (X).
  • the base sequence Xx includes a known base sequence (5′-S 1 -S 2 -S 3 -S 4 -S 5 -3 ′ ) at the 5 ′ end .
  • This base sequence (hereinafter referred to as “adapter sequence”) is the same as the sequence added to the 5 ′ end of the target ssDNA in the procedure described later.
  • the other of the double strands has a base sequence X′x and a base sequence R ′ that are complementary to the base sequence Xx and the base sequence R, respectively, and further the first on the 5 ′ end side of the base sequence R ′.
  • the second overhang is located on the 3 ′ end side of the base sequence X′x (see FIG. 5).
  • the first overhang includes a nucleotide having at least one random base (n) at a position adjacent to the 5 ′ end of the nucleotide of the base sequence R ′ (n in FIG. 1 is 1).
  • the base (n) is any one of A, G, C, and T.
  • the first overhang is a base sequence complementary to the adapter sequence (5′-S 1 -S 2 -S 3 -S 4 -S 5 -3 ′ ) added to the 5 ′ end of the target ssDNA. having (5'-S 5 '-S 4'-S 3 '-S 2'-S 1 '-3').
  • the second overhang is a nucleotide having a base (X ′ 1 ) complementary to the base (X 1 ) to be identified in the target ssDNA at a position adjacent to the 3 ′ end of the nucleotide of the base sequence X′x.
  • the base (X ′ 1 ) is any one of A, G, C, and T.
  • the second overhang contains nucleotides with at least 3 random bases (n) (n is 5 in FIG. 1).
  • the base length of the first overhang and the second overhang is 3 to 12 mer.
  • the base length of the base sequence Xx of the labeled oligomer L (including the adapter sequence) is appropriately set in the range of 4 to 25 mer depending on the type of type II restriction enzyme used.
  • the base sequence Xx the base sequence other than the adapter sequence and the length thereof are not particularly limited as long as the label described later is possible.
  • the base sequence of the base sequence R of the restriction enzyme recognition site of the labeled oligomer L and its length can be appropriately designed according to the type of type II restriction enzyme used.
  • the length of the base sequence Xx is such that the enzyme cleavage site is 3 ′ of the target ssDNA in the procedure of treating the complex of the circularized target ssDNA and the double strand for conversion with a type II restriction enzyme (described in detail later).
  • the length is designed so that at least one nucleotide can be cut off from the terminal.
  • the size of the library depends on the number of nucleotides with random base n.
  • the conversion duplex contained in the library is the seventh power of 4 (4 types of A, G, C, and T) (the number of bases n 6 and the base X
  • the total number of '1') is 16,384.
  • the label of the labeled oligomer L is specific depending on the type of the base X ′ located in the second overhang (that is, the type of the base X to be labeled in the target ssDNA complementary thereto). .
  • the label of the labeled oligomer L is obtained by chemically treating the above-described oligonucleotide having a base length with a metal cluster having an average particle diameter of 1-1000 nanometers and / or inorganic phosphor particles having an average particle diameter of 0.5-1000 nanometers. This can be done by bonding to The label oligomer L can be labeled with one or both of the metal cluster and the inorganic phosphor particles.
  • Labeling with metal clusters is preferably performed by assigning four types of metal clusters having different average particle diameters to the respective labeled oligomers for labeling A, G, C, and T.
  • An example of the label is shown below. According to this labeling, it is possible to identify the type of base corresponding to the labeled oligomer by reading the particle size of the metal cluster labeled on the labeled oligomer under an electron microscope.
  • Labeled nucleotide A for labeling oligomers L A: labeled oligomers for metal clusters labeled nucleotide G of a particle diameter 5 nm L G: labeled oligomers for particle size 10nm metal clusters labeled nucleotide C L C: particle size 15nm of metal clusters labeled bases Labeled oligomer L T for T : metal cluster with a particle size of 20 nm
  • labeling with metal clusters can also be performed by labeling two types of metal clusters having different average particle diameters on the respective label oligomers for A, G, C, and T in different combinations.
  • labeling is performed as follows (see also FIG. 2). Also in this case, it is possible to identify the type of base corresponding to the labeled oligomer by reading the particle diameter of the gold colloid labeled on the labeled oligomer under an electron microscope.
  • labeling with inorganic phosphor particles can be performed using inorganic phosphor particles having different emission wavelengths.
  • the inorganic phosphor particles include particles composed of rare earth element activated metal oxides, metal sulfides or metal sulfates, halophosphate compounds having emission wavelengths in the red, green, blue, or violet-near ultraviolet region.
  • particles made of the following phosphors can be used.
  • the inorganic phosphor particles those obtained by coating semiconductor atoms (cadmium mixed with selenium or tellurium) with a semiconductor shell (zinc sulfide), which is commercially available under the registered trademark “Qdot”, can also be used.
  • Red phosphor Y 2 O 3 : Eu Y 2 O 2 S: Eu Gd 2 O 2 S: Eu YVO 4 : Eu Y 2 O 2 S: Eu, Sm SrTiO 3 : Pr BaSi 2 Al 2 O 8 : Eu BaMg 2 Al 16 O 27 : Eu Y 0.65 Gd 0.35 BO 3 : Eu La 2 O 2 S: Eu 3+ , Sm
  • Green phosphor Y 2 O 3 : Tb Ba 2 SiO 4 : Eu 2+ Zn (Ga, Al) 2 O 4 : Mn Y 3 (Al, Ga) 5 O 12 : Tb Y 2 SiO 5 : Tb ZnS: Cu, Zn 2 SiO 4 : Mn
  • the labeling is performed as follows (see FIG. 3). According to this label, it is possible to identify the type of the base labeled by the labeled oligomer by reading the cathodoluminescence emission wavelength of the inorganic phosphor particles labeled with the labeled oligomer under an electron microscope. If phosphor particles having different particle diameters are used, the particle diameter can also be used as information for identifying the type of base, as in the case of the metal cluster described above.
  • the binding procedure of the metal clusters or inorganic phosphor particles to the labeled oligomer L is first made from an amino compound having a primary or secondary amino group (for example, monoamino undecagold). Then, the base in the labeled oligomer L is liberated by the action of an appropriate base releasing means to form a hemiacetal-type ribose residue, and then the base is subjected to a reductive amination reaction with an amino compound with respect to the hydroxyl group at the base releasing site. An amino compound is introduced at the position where is bound (see Patent Document 1). After the introduction of the amino compound, the labeled oligomer L is preferably purified to 99.9% or more by various high purity separation and purification techniques.
  • an amino compound having a primary or secondary amino group for example, monoamino undecagold.
  • an adapter sequence is added to the 5 ′ end of the target ssDNA and immobilized on the surface of the solid phase (see FIG. 4).
  • an example will be described in which the base sequence of the target ssDNA is determined from the 3 ′ end, but when it is desired to determine from the 5 ′ end, the adapter sequence is added to the 3 ′ end of the target ssDNA.
  • genomic DNA or artificial double-stranded DNA can be used, and cDNA can also be used.
  • Genomic DNA or artificial double-stranded DNA is fragmented (divided) by DNase treatment, ultrasonic treatment, stirring treatment, etc., and then heated to about 95 ° C. to be denatured into single strands.
  • the target single-stranded nucleic acid includes not only DNA but also mRNA, tRNA, rRNA, siRNA, miRNA, and shRNA. .
  • the solid-phase immobilization of the target ssDNA for example, a method of biotinylating the target ssDNA and binding it to the slide glass surface using an avidin-biotin bond can be employed.
  • the solid phase may be microbeads, membranes or filters.
  • a complex of the circularized target ssDNA and the double strand for conversion is treated with a type II restriction enzyme (see FIG. 7).
  • the restriction enzyme binds to the recognition site (base sequence R) in the double strand for conversion, and the nucleotide sequence site (X 1 -X 2 -X 3 -X 4 -X 5 in FIG. 7) to be identified in the target ssDNA.
  • An enzyme having a cleavage site at a position where at least one nucleotide can be excised from the 3 ′ end of ⁇ X 6 ) is used. In the figure, an example is shown in which only the first labeled base X1 present at the 3 ′ end of the target ssDNA is cut out.
  • FIG. 9 and 10 schematically show an example of the generated converted ssDNA.
  • L A , L G , L C , and L T indicate labeled oligomers in which specific labels for each base of A, G, C, and T are formed by a gold colloid.
  • FIG. 9 "labeled oligomer L A and base A"3'-5'direction,”labeled oligomer L G and base G", “labeled oligomer L C and base C”, “labeled oligomer L T and bases T "Is shown.
  • the sequence order of each base of A, G, C, and T in the repetitive sequence is the same as that of the target ssDNA, and the converted ssDNA maintains the base sequence information of the target ssDNA.
  • the correspondence between the fluorescently labeled oligomer labeled with the phosphor of FIG. 10 and the base is the same.
  • the reading accuracy of the base sequence of the target ssDNA depends on the labeling rate of the base in the target ssDNA, and the labeling rate of the base in the target ssDNA is equal to the labeling rate of the labeled oligomer L. For this reason, it is preferable to use the labeled oligomer L having a labeling rate of 99.9% or more using a high-purity separation / purification technique after labeling the inorganic phosphor particles and / or inorganic phosphor particles.
  • the converted ssDNA is obtained by transferring the base information of each base to the labeled oligomer L in a base-specific and high-precision manner while maintaining the base sequence information of the target ssDNA. Accordingly, by identifying the label of the labeled oligomer in each repeating unit in the converted ssDNA, the type of base in the same unit can be determined, and the base sequence of the target ssDNA can be determined.
  • the base sequence is determined by the particle size of the colloidal gold labeled with the labeled oligomer L or the particle size of the inorganic phosphor particles labeled with the labeled oligomer L and / or the high resolution and high resolution of the electron microscope. This can be done by directly reading the emission wavelength.
  • a labeled oligomer L specifically labeled according to the type of base can be inserted and added to each nucleotide of the target ssDNA, and the label is labeled under an electron microscope. By identifying, it is possible to obtain base sequence information of the target ssDNA.
  • the base constituting the nucleotide can be indirectly labeled by reliably inserting the labeled oligomer L at the 3 ′ end or 5 ′ end of each nucleotide of the target ssDNA.
  • the labeled oligomer L can be highly purified in advance, the base at a higher labeling rate than the conventional method (Patent Document 1), which has a limit in the labeling rate for chemically directly labeling the base in the target ssDNA. Can be labeled.
  • the conventional direct method it is difficult for the labeling rate to exceed 95% in terms of chemical reaction yield.
  • the labeled oligomer L can be separated and purified in advance, so that the labeling rate is 99. .9% or more.
  • the labeling rate decreased.
  • the base-corresponding labeling method according to the present invention in order to reliably insert the labeled oligomer L previously labeled with a metal cluster or inorganic phosphor particles between the nucleotides of the target ssDNA, Steric hindrance does not occur.
  • the label oligomer L itself inserted at the 3 ′ end side or the 5 ′ end side of each nucleotide of the target ssDNA is directly labeled for base identification.
  • Patent Document 4 in which a beacon labeled with a fluorescent reagent for base discrimination is further hybridized to the oligonucleotide thus prepared, there is no reaction error associated with hybridization, so that the base can be detected at a higher labeling rate. Labeling can be performed.
  • bases in nucleic acids can be indirectly labeled at a high labeling rate while maintaining the base sequence information possessed by the nucleic acids. Therefore, the present invention can be used in a sequencing technique for identifying a base through a label under an electron microscope and acquiring nucleic acid base sequence information, and can contribute to simplification and speed-up of the technique.

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Abstract

L'invention concerne une technique qui permet à des bases dans un acide nucléique d'être indirectement marquées à une vitesse de marquage élevée, tout en conservant des informations de séquence de base pour l'acide nucléique. L'invention concerne un procédé de marquage supportant une base qui consiste en le marquage indirect des bases dans un acide nucléique à simple brin, tout en conservant des informations de séquence de base pour ledit acide nucléique et comprend une procédure d'introduction d'un oligonucléotide (L) qui a été marqué d'une manière spécifique et hautement précise selon le type de base constituant un nucléotide, dans l'extrémité 3'-terminale ou 5'-terminale du nucléotide. Selon le procédé de marquage supportant une base, chaque base dans l'acide nucléique à simple brin peut être identifiée avec une précision élevée selon l'oligonucléotide (L) introduit. Par conséquent, par l'identification du marqueur oligonucléotidique (L) au microscope électronique, par exemple, des informations de séquence de base hautement précises à propos de l'acide nucléique à simple brin peuvent être acquises.
PCT/JP2012/079141 2011-11-11 2012-11-09 Procédé de marquage supportant une base, procédé d'acquisition d'informations de séquence de base, et acide nucléique à simple brin marqué supportant une base Ceased WO2013069773A1 (fr)

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JP2011247302A JP2014176297A (ja) 2011-11-11 2011-11-11 塩基対応標識方法、塩基配列情報取得方法及び塩基対応標識化一本鎖核酸
JP2011-247302 2011-11-11

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WO2013069773A1 true WO2013069773A1 (fr) 2013-05-16

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002153271A (ja) * 2000-11-17 2002-05-28 Jeol Ltd Ｄｎａあるいはｒｎａの塩基配列決定方法およびｄｎａシーケンサー
WO2007097443A1 (fr) * 2006-02-20 2007-08-30 National University Corporation Hokkaido University Procede de determination de la sequence des bases d'un adn
WO2010053820A1 (fr) * 2008-10-29 2010-05-14 Trustees Of Boston University Conversion d'adn avec conservation de séquence
JP2010539991A (ja) * 2007-10-04 2010-12-24 ハルシオン モレキュラー 電子顕微鏡を用いた核酸ポリマーの配列決定

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002153271A (ja) * 2000-11-17 2002-05-28 Jeol Ltd Ｄｎａあるいはｒｎａの塩基配列決定方法およびｄｎａシーケンサー
WO2007097443A1 (fr) * 2006-02-20 2007-08-30 National University Corporation Hokkaido University Procede de determination de la sequence des bases d'un adn
JP2010539991A (ja) * 2007-10-04 2010-12-24 ハルシオン モレキュラー 電子顕微鏡を用いた核酸ポリマーの配列決定
WO2010053820A1 (fr) * 2008-10-29 2010-05-14 Trustees Of Boston University Conversion d'adn avec conservation de séquence

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KUNIAKI NAGAYAMA: "Jitsuyoka ni Mukau 1 Bunshi Keisoku Dai 4 Sedai Sequencer: Chosa no Yomitori ni Kitai Denshi Kenbikyo ni yoru 1 Bunshi DNA Sequencer", GENDAI KAGAKU, 1 November 2011 (2011-11-01), pages 36 - 37 *

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