WO1998038337A1 - Procede de clivage d'adn au moyen d'integrases nucleotidiques - Google Patents

Procede de clivage d'adn au moyen d'integrases nucleotidiques Download PDF

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WO1998038337A1
WO1998038337A1 PCT/US1998/003990 US9803990W WO9838337A1 WO 1998038337 A1 WO1998038337 A1 WO 1998038337A1 US 9803990 W US9803990 W US 9803990W WO 9838337 A1 WO9838337 A1 WO 9838337A1
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intron
rna
substrate
group
sequence
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PCT/US1998/003990
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English (en)
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Alan M. Lambowitz
Steve Zimmerly
Huatao Guo
Georg Mohr
Clifford J. Beal
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Ohio State Research Foundation
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Priority to CA002282737A priority Critical patent/CA2282737A1/fr
Priority to AU66762/98A priority patent/AU748237B2/en
Priority to JP53793498A priority patent/JP2002515759A/ja
Priority to EP98908823A priority patent/EP0996742A4/fr
Publication of WO1998038337A1 publication Critical patent/WO1998038337A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]

Definitions

  • nucleotides that are located upstream of the cleavage site on the top strand have a (-) position relative to the cleavage site, and nucleotides that are located downstream of the cleavage site have a (+) position relative to the cleavage site.
  • the cleavage site is located between nucleotides -1 and +1 on the top strand of the double-stranded DNA substrate.
  • the top strand of the substrate comprises a first intron RNA binding sequence, hereinafter referred to as the "IBS1" sequence and a second intron RNA binding sequence, hereinafter referred to as the "IBS2" sequence.
  • the nucleotide immediately preceding the first nucleotide of the EBS1 sequence on the group II intron RNA is complementary to the nucleotide at +1 on the top strand of the substrate, hereinafter referred to as the ⁇ ' nucleotide.
  • the EBS1 sequence of the group II intron RNA comprises from about 5 to 7 nucleotides and has substantial complementarity with the nucleotides at positions -1 to about -5 or about -7 on the top strand of the DNA substrate.
  • Another method provided by the present invention employs a nucleotide integrase for cleaving a single-stranded nucleic acid substrate and for inserting the group II intron RNA of the nucleotide integrase into the cleavage site.
  • the method comprises the steps of: providing a nucleotide integrase having two hybridizing sequences.
  • EBS1 and EBS2 that are capable of hybridizing with two intron RNA-binding sequences, IBS1 and IBS2, on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate for a time and at a temperature sufficient to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.
  • the EBS1 sequence of the group II intron RNA comprises from about 5 to 7 nucleotides that have substantial complementarity with the nucleotides at positions -1 to about -5 or about -7 relative to the putative cleavage site.
  • the EBS2 sequence comprises from about 4 to 7 nucleotides that have substantial complementarity with the nucleotides at positions from about -6 to about -14 relative to the putative cleavage site.
  • the ⁇ nucleotide of the group II intron RNA is complementary to the ⁇ ' nucleotide on the top strand of the substrate.
  • Figure 4 is a graph showing the relative extent of cleavage of the substrates having mutations in the first sequence element by a nucleotide integrase comprising a wild-type aI2 intron RNA and the protein encoded thereby.
  • Figure 5 is a graph showing the relative extent of cleavage of the substrates having mutations in the second sequence element by a nucleotide integrase comprising a wild-type aI2 intron RNA and the protein encoded by the aI2 intron RNA.
  • Figure 6 is a chart depicting the sequence of a DNA substrate cleaved by a nucleotide integrase comprising a wild-type all intron RNA, and the protein encoded by the all intron RNA and the position of the mutations made in this sequence.
  • Figure 7 is a graph showing the relative extent of cleavage of the substrates having mutations upstream of the cleavage site by a nucleotide integrase comprising a wild-type all intron RNA and the protein encoded thereby.
  • Figure 9 is a graph showing the relative extent of cleavage of the substrates having mutations in the first sequence element by a nucleotide integrase comprising a wild-type Ll.ltrB intron RNA and the ItrA protein.
  • Figure 10 shows the Ll.ltrB intron DNA sequence and portions of the nucleotide sequence of the flanking exons ItrBEl and ltrBE2, SEQ.ID.N0.5, the nucleotide sequence of the open reading frame, of the Ll.ltrB intron SEQ. ID. NO. 6 and the amino acid sequence of the ItrA protein, SEQ.ID.NO. 7.
  • the nucleotide integrase employed in this method comprises a group II intron-encoded protein bound to an excised group II intron RNA.
  • the EBS1 sequence and EBS2 sequence of the group II intron RNA have at least 80%, preferably 90%, more preferably full complementarity with the IBSl sequence and IBS2 sequence, respectively, that are on the top strand of the substrate.
  • the group II intron-encoded protein comprises an RT domain, an X domain, and the non-conserved portion of the Zn domain.
  • EBS1 is located in domain I of the group II intron RNA and comprises from about 5 to 7 nucleotides that are capable of hybridizing to the nucleotides of the IBSl sequence of the substrate.
  • EBS2 is located in domain I of the group II intron RNA upstream of EBS 1 and comprises from about 4 to 7 nucleotides that are capable of hybridizing to the nucleotides of IBS2 sequence of the substrate. If the nucleotides of the EBS1 and EBS2 sequences of the group II intron RNA are not at least 80% complementary to the nucleotides of the IBSl or IBS2 sequences, respectively, then the group II intron RNA is modified to increase the complementarity between the EBS and IBS sequences. As shown in Fig. 1 the IBSl sequence of the substrate is upstream of the cleavage site and the IBS2 sequence of the substrate is upstream of the IBSl sequence.
  • Suitable nucleotide integrases for use in this method include, for example the aI2 nucleotide integrase, the all nucleotide integrase, and the ItrA nucleotide integrase.
  • EBS2 sequence of the aI2 intron RNA does not have substantial complementarity to the IBS2 sequence, i.e., the sequence of 6 nucleotides that lie immediately downstream from one of these target sequences, and/or if EBS 1 sequence of the aI2 intron RNA does not have substantial complementarity to the IBS l sequence, i.e., the sequence of six nucleotides that lie immediately downstream of the IBS2 sequence, then EBS 1 and EBS2 are modified to have substantial complementarity, as hereinafter explained.
  • the EBS2 sequence of the all intron RNA does not have substantial complementarity to the IBS2 sequence, i.e., the sequence of 6 nucleotides that lie immediately downstream from the C nucleotide at -13, and/or if EBSI sequence of the all intron RNA does not have substantial complementarity to the IBSl sequence, i.e., the sequence of six nucleotides that lie immediately downstream of the IBS2 sequence and immediately upstream of the cleavage site, then the EBS I sequence and the EBS2 sequence of the group II intron RNA are modified to have substantial complementarity, as hereinafter explained.
  • the ItrA nucleotide integrase comprises an excised, wild-type or modified excised group Ll.ltrB group II intron RNA of the Lactococcus lactis ltrB gene, hereinafter referred to as the "Ll.ltrB intron” RNA, and a wild-type or modified Ll.ltrB intron-encoded protein, hereinafter referred to as the ItrA protein.
  • the sequence of the Ll.ltrB intron RNA is shown in Fig. 10 and SEQ. ID. NO. 5.
  • the sequence of the ItrA protein is set forth in SEQ. ID. NO.7.
  • the EBS I sequence of the Ll.ltrB intron RNA comprises 7 nucleotides and is located at positions 457 to 463.
  • the EBS I sequence of the wild-type Ll.ltrB intron RNA has the sequence 5'-GUUGUGG.
  • the EBS2 of the Ll.ltrB intron RNA comprises 6 nucleotides and is located at positions 401 to and including 406.
  • the EBS2 sequence of the wild-type Ll.ltrB intron RNA has the Sequence 5'AUGUGU.
  • the ItrA nucleotide integrase is used to cleave the top strand of a double-stranded DNA substrate when the top strand has a G at -21 and an A at -20 relative to the cleavage site.
  • the ItrA nucleotide integrase cuts the top strand more efficiently when there is a G at -21, an A at -20, a T at -19, a G at -17, and a G at -15.
  • the non-complementary nucleotides are modified, preferably, by recombinant techniques.
  • the ⁇ nucleotide of the group II intron RNA is complementary to the nucleotide at +1 in the top strand. If the ⁇ nucleotide is not complementary to the nucleotide at +1 , preferably the ⁇ nucleotide is modified to be complementary.
  • the group II intron-encoded protein comprises an RT domain, an X domain, and the conserved and non-conserved regions of a Zn domain. To insert a cDNA into the cleavage site on the bottom strand of the substrate, the group II intron-encoded protein also comprises a reverse transcriptase domain.
  • the method of cleaving both strands of a double-stranded DNA sequence having a recognition site comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two sequences, EBS I and EBS2, that are capable of hybridizing with two intron RNA-binding sequences, IBS l and IBS2, on the top strand of the DNA substrate, and a group Il-intron encoded protein that binds to a first sequence element and to a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double- stranded DNA substrate for a time and at a temperature sufficient to permit the nucleotide integrase to cleave both strands of the DNA substrate and to insert the group II intron RNA into the cleavage site of the top strand.
  • the first sequence element of the recognition site is upstream of the putative cleavage site, the IBSl sequence and the IBS2 sequence.
  • the first sequence element comprises from about 10 to about 12 pairs of nucleotides.
  • the second sequence element comprises from about 10 to about 12 nucleotides and lies downstream of the cleavage site, i.e., from position + 1 to about position +10, +1 1 , or +12.
  • Nucleotide integrases that may be employed to cleave both strands of a DNA substrate include, but are not limited to an aI2 nucleotide integrase, an all nucleotide integrase, and an ItrA nucleotide integrase.
  • the preferred recognition site for the aI2 nucleotide integrase comprises on the top strand thereof a C at -18, a T at -15, a T at -13, a G at -16 or -19, a T at +1, a T at +4, and a G at +6 relative to the cleavage site.
  • the aI2 nucleotide integrase to cleave both strands of the DNA substrate, one first examines the substrate sequence to determine if one strand thereof contains this set of nucleotides. Then, if the EBS2 sequence of the aI2 intron RNA does not have substantial complementarity to the IBS2 sequence of the substrate, i.e., the sequence of 6 nucleotides that lies immediately downstream from the T at -13, and/or if EBSI sequence of the aI2 intron RNA does not have substantial complementarity to the IBSl sequence, i.e., the sequence of six nucleotides that lie immediately downstream of the IBS2 sequence and immediately upstream of the T at +1 , then the EBS I sequence and EBS2 sequence of the group II intron RNA are modified to have substantial complementarity, as hereinafter explained.
  • the aI2 nucleotide integrase cleaves both strands of the substrate with greater efficiency if the top strand of the substrate has an A at -21 , a G at -19, a C at -18, a G at - 16, a T at - 15, a T at -13, a T at +1, a T at +4, and a G at +6.
  • the aI2 cleaves both strands of the substrate with even greater efficiency if the top strand has an A at -21, a T at -20, a G at -19, a C at -18, -a T at -17, a G at -16, a T at -15, a T at -13 a T at +1, a T at +4, and a G at +6. If the top strand of the substrate additionally has a C at +2, a T at +3, a T at +7, an A at +8, an A at +9, and a T at +10, cleavage will be even greater.
  • the all integrase is used to cleave both strands of a DNA substrate that has on the top strand thereof a C residue at position -13 relative to the cleavage site a T at +1, a T at +2, a T at +3, a T at +4, an A at +5, a G at +6, a T at +7, and an A at +8 relative to the cleavage site.
  • the top strand of the double-stranded substrate has a C at -13, a G at -22, a G at -21, an A at -19 a T at +1, a T at +2, a T at +3, a T at +4, an A at +5, a G at +6, a T at -7, and an A at +8.
  • the ItrA nucleotide integrase is used to cleave both strands of a double-stranded DNA substrate, when the substrate has on the top strand thereof a G at -21, an A at -20 a C +1, an A at +2, a T at +3. an A at +4, a T at -5, a C at -H.. an A at +7, and a T at +8.
  • the ItrA nucleotide integrase cleaves both strands of the substrate more efficiently if the top strand has a G at -21, an A at -20, a T at -19, a G at - 17, and G at - 15, a C +1 , an A at +2, a T at -3, an A at +4, a T at +5, a C at +6, an A at +7, and a T at +8. If the top strand additionally has a C at -22, a C at -18. a T at - 16. an A at - 14, an A at - 13, a T at +9 and a T at +10, cleavage will be even greater.
  • IBSl and IBS2 respectively on the substrate, and a group II intron encoded protein having an RT domain, an X domain and the non-conserved portions of the Zn domain; and reacting the substrate with the nucleotide integrase.
  • the EBS2 sequence of the group II intron RNA comprises 4 to 7 nucleotides and has at least 80%o, preferably 90%, more preferably full complementarity with the nucleotides at positions from about -6 to about -14.
  • the nucleotide immediately preceding the first nucleotide of EBS I is complementary to the nucleotide at +1 in the sense strand.
  • the present invention also provides a method of determining whether a nucleic acid substrate comprises a particular recognition site.
  • the method comprises the steps of providing a nucleotide integrase capable of cleaving a nucleic acid substrate with a particular recognition site; reacting the nucleotide integrase with the nucleic acid substrate: and assaying for cleavage of the substrate. Cleavage of the substrate indicates that the substrate comprises the particular recognition site.
  • cleavage may be detected by assaying for incorporation into or attachment of the group II intron RNA to one strand of the nucleic acid substrate.
  • a suitable reaction medium contains a monovalent cations such as Na + or K + , at a concentration from about 0 to about 300 mM: preferably from about 10 to about 200 mM KC1, and a divalent cation, preferably a magnesium or manganese ion, more preferably a magnesium ion, at a concentration that is less than 100 mM and greater than 1 M.
  • the divalent cation is at a concentration of about 5 to about 20mM, more preferably about 10 to about 20 mM.
  • the preferred pH for the medium is from about 6.0-8.5, more preferably about 7.5-8.0.
  • the single stranded nucleic acid substrates and the top strand of the double-stranded DNA substrate are cut by the excised group II intron RNA.
  • the cleavage that is catalyzed by the excised group II intron RNA is a reverse splicing reaction that results in the insertion, either partially or completely, of the excised group II intron RNA into the cleavage site, i.e. between nucleotides - 1 and +1 in the top strand.
  • the group II intron RNA is covalently attached to the +1 nucleotide on the top strand of the cleavage site.
  • the bottom strand or antisense strand of the double-stranded DNA substrate is cut by the group II intron-encoded protein
  • the bottom strand of the double-stranded DNA substrate is cut at a position from about 9 to about 1 1 base pairs downstream of the cleavage site in the top strand, I e .
  • nucleotide integrase as an endonuclease to cleave a substrate DNA are useful analytical tools for determining the presence and location of a particular recognition site in a DNA substrate
  • simultaneous insertion of a nucleic acid molecule into the DNA substrate which occurs when either single-stranded DNA or double-stranded DNA is cleaved with a nucleotide integrase, permits tagging of the cleavage site of the DNA substrate with a radiolabeled molecule, a feature which facilitates in identifying DNA substrates that contain a particular recognition site
  • the automatic attachment of an RNA molecule onto one strand of a double-stranded DNA substrate permits identification of the cleavage site through hybridization studies that use a probe that is complementary to the attached RNA molecule An attached RNA molecule that is tagged with a molecule such as biotin also enables
  • Group II intron encoded protein as used herein, is a protein encoded by a group II intron open reading frame
  • Group II mtrons are a specific type of intron which is present in the DNA of bacteria and in the DNA of organelles, particularly the mitochondria of fungi, yeast and plants and the chloroplast of plants
  • the group II intron RNA molecules that is, the RNA molecules which are encoded by the group II introns, share a similar secondary and tertiary structure
  • Figure 2 depicts the secondary structure of the al l and aI2 intron RNA and part of the nucleotide sequence of the wild-type al l and aI2 intron RNA
  • the group II intron RNA molecules typically have six domains Domain IV of the group II intron RNA contains the nucleotide sequence which encodes the ' group II intron encoded protein "
  • the group II intron-encoded protein of the nucleotide integrase comprises an X domain and a Zn domain.
  • the X domain of the protein has a maturase activity.
  • the Zn domain of the protein has Zn 2+ finger-like motifs.
  • the group II intron-encoded protein further comprises a reverse transcriptase domain.
  • a group II intron-encoded protein includes modified group II intron-encoded proteins that have additional amino acids at the N terminus, or C terminus, or alterations in the internal regions of the protein as well as wild-type group II intron-encoded proteins. It is believed that the group II intron-encoded protein is bound to 3' region of the group II intron RNA.
  • the nucleotide integrase may be used in a partially-purified preparation which contains the RNP particles and reconstituted particles that have nucleotide integrase activity as well as other RNP particles, such as for example ribosomes.
  • This partially-purified preparation is free of organelles.
  • the organelle and bacterium lysates are then centrifuged through a sucrose cushion to obtain the ribonucleoprotein (RNP) particle preparation.
  • the RNP particles may be further purified by separation on a sucrose gradient, or a gel filtration column, or by other types of chromatography.
  • the nucleotide integrase is also isolated from reconstituted RNP particle preparations that are prepared by combining an RNA-protein complex preparation with an exogenous, excised group II intron RNA.
  • the RNA-protein complex preparation is preferably isolated from a yeast, fungi, or bacterium using the protocol for RNP particles described above.
  • the RNA-protein complex preparation comprises group II intron-encoded protein molecules complexed with RNA molecules that are not an excised group II intron RNA having a sequence that encodes the group II intron-encoded protein.
  • the group II intron-encoded protein of the RNA-protein complex preparation is associated with either a ribosomal RNA molecule, an mRNA molecule, or an excised group II intron RNA that does not encode the group Il-intron encoded protein.
  • the RNP particles are further purified from the RNP particle preparation or from the soluble fraction by, for example, separation on a sucrose gradient, or a gel filtration column, or by other types of chromatography.
  • the RNP particle may be further purified from the RNP particle preparation by affinity chromatography on a matrix which recognizes and binds to the tag.
  • NiNTA Superflow from Qiagen, Chatsworth CA is suitable for isolating RNP particles in which the group II intron-encoded protein has a His 6 tag.
  • the following methods for preparing nucleotide integrases are included for purposes of illustration and are not intended to limit the scope of the invention.
  • the RNP particle preparations of the following formulations 1-10, and the RNA-protein complex of the formulation 12 were isolated from the mitochondria of the wild-type Saccharomyces cerevisiae yeast strain ID41-6/161 MATa adel lysl, hereinafter designated " 161", and derivatives thereof.
  • the mitochondria of the wild-type yeast strain 161 contains a COXl gene that includes the group II intron all and the group II intron aI2.
  • the COXl gene in the mutant yeast strains either lacks one of the group II introns or has a mutation in one of the group II introns.
  • the excised group II intron RNA molecules and the group II intron encoded proteins are derived from the group II introns all and aI2 that are present in the wild-type and mutant yeast strains.
  • the intron composition of the COXl gene in the different yeast strains is denoted by a convention in which a superscript "+" indicates the presence of the all intron or the aI2 intron, a superscript "0" indicates the absence of the al l or aI2 intron, and other superscripts refer to specific alleles or mutations in the aI2 intron.
  • the mitochondria were layered on a flotation gradient consisting of a 44% sucrose solution layer, a 53% sucrose solution layer, and a 65% sucrose solution layer and centrifuged in a Beckman SW28 rotor at 27,000 rpm for 2 hours and 10 minutes.
  • the mitochondria were collected from the 53%/44% interface and suspended in buffer containing 0.5M KCl, 50 mM CaCl 2 , 25 mM Tris-HCl, pH 7.5, 5 mM DTT and lysed by the addition of Nonidet P-40 to a final concentration of 1%.
  • the I°2 + ' mutant strain was constructed as follows: (i) the aI2 intron from strain 161 was cloned as a Clal-to-BamHl fragment into pBluescript KS + obtained from Stratagene to yield pJVM4; (ii) pJVM4 was cleaved with Clal and Ndel to remove the 5' end of the insert; and (iii) an MspI-to-Nde! fragment that contains exons 1 and 2 of the mitochondriae COXl gene plus the 5' end of aI2 from yeast strain C10361 ⁇ one was inserted to yield plasmid pJVM164.
  • the reactions and manipulations directed at cloning DNA such as ligations, restriction enzyme digestions, bacterial transformation, DNA sequencing etc. were carried out according to standard techniques, such as those described by Sambrook et al., Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y. Yeast mitochondrial transformations were also carried out according to standard techniques such as those described in Belcher et al., 1994, Biolistic transformation of mitochondria in Saccharomyces cerevisiae, 101 -1 15. /n N.-S. Yang and P. Christou (ed.) Particle Bombardment Technology for Gene Transfer. Oxford University Press, New York.
  • the RNP particle preparation was made from the mitochondria of mutant yeast strain 1°2 * ⁇ as in formulation 1. Formulation 3 RNP particle preparation from mutant yeast strain ⁇ +l 2°
  • Yeast strain 1 +1 2° is a derivative of the wild-type yeast strain 161.
  • the yeast strain V2° was obtained from Dr. Philip S. Perlman and was prepared as described in Kennell et al., 1993. Cell 73, 133-146.
  • Yeast strain P'2° contains a segment of the COXl gene of S. diastaticus, which lacks aI2, inserted into wild-type 161 mtDNA via mitochondrial transformation.
  • the construction started with plasmid pSH2, which contains al l from wild- type 161 and some flanking sequences cloned as a Hpall/EcoRI fragment in pBS(+) (Stratagene, La Jolla. CA).

Abstract

La présente invention se rapporte à des procédés permettant de cliver, au moyen d'une intégrase nucléotidique, des substrats d'acides nucléiques au niveau de sites spécifiques, et d'insérer une molécule d'acide nucléique dans le substrat clivé. Ledit procédé de clivage d'un brin d'un substrat d'ADN double brin consiste à utiliser une intégrase nucléotidique comportant un ARN d'intron du groupe II ayant deux séquences d'hybridation susceptibles de s'hybrider à deux séquences de liaison à l'ARN d'intron sur ledit brin du substrat et une protéine codée par l'intron du groupe II qui se lie à un élément de la première séquence du substrat. Le procédé de clivage des deux brins d'un substrat d'ADN double brin consiste à utiliser une intégrase nucléotidique comportant un ARN d'intron du groupe II ayant deux séquences d'hybridation susceptibles de s'hybrider à deux séquences de liaison à l'ARN d'intron sur un brin du substrat et une protéine codée par l'intron du groupe II susceptible de se lier à des éléments des première et seconde séquences au niveau du site de reconnaissance du substrat. Le procédé de clivage d'un substrat d'acide nucléique simple brin consiste à utiliser une intégrase ayant deux séquences d'hybridation susceptibles de s'hybrider à deux séquences de liaison à l'ARN d'intron sur le substrat et une protéine codée par un intron du groupe II.
PCT/US1998/003990 1997-02-28 1998-02-27 Procede de clivage d'adn au moyen d'integrases nucleotidiques WO1998038337A1 (fr)

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CA002282737A CA2282737A1 (fr) 1997-02-28 1998-02-27 Procede de clivage d'adn au moyen d'integrases nucleotidiques
AU66762/98A AU748237B2 (en) 1997-02-28 1998-02-27 Methods for cleaving DNA with nucleotide integrases
JP53793498A JP2002515759A (ja) 1997-02-28 1998-02-27 ヌクレオチドインテグラーゼを使用するｄｎａ切断方法
EP98908823A EP0996742A4 (fr) 1997-02-28 1998-02-27 Procede de clivage d'adn au moyen d'integrases nucleotidiques

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USRE44591E1 (en) 1998-05-04 2013-11-12 Electronics And Telecommunications Research Institute Apparatus for making a random access to the reverse common channel of a base station in CDMA and method therefor
USRE45237E1 (en) 1998-05-04 2014-11-11 Electronic And Telecommunications Research Institute Apparatus for making a random access to the reverse common channel of a base station in CDMA and method therefor
US6358712B1 (en) 1999-01-05 2002-03-19 Trustee Of Boston University Ordered gene assembly
US8090003B2 (en) 1999-05-31 2012-01-03 Electronics And Telecommunications Research Institute Apparatus and method for modulating data message by employing orthogonal variable spreading factor (OVSF) codes in mobile communication system
US7592161B2 (en) 1999-10-15 2009-09-22 The Ohio State University Research Foundation Methods for analyzing the insertion capabilities of modified group II introns
US10544422B2 (en) 2006-06-21 2020-01-28 Morvus Technology Limited DNA molecules and methods

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