Processing

Please wait...

Settings

Settings

Goto Application

1. WO2019229485 - METHOD FOR DNA CLONING WITHOUT USING IN VITRO ENZYMATIC REACTION, AND THE SEQUENCES OF RELATED DNA VECTORS

Publication Number WO/2019/229485
Publication Date 05.12.2019
International Application No. PCT/HU2019/000016
International Filing Date 29.05.2019
IPC
C12N 15/63 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
C12N 15/70 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
70Vectors or expression systems specially adapted for E. coli
CPC
C12N 15/63
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
C12N 15/70
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
70Vectors or expression systems specially adapted for E. coli
C12N 15/902
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
90Stable introduction of foreign DNA into chromosome
902using homologous recombination
C12N 2310/20
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2310Structure or type of the nucleic acid
10Type of nucleic acid
20involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Applicants
  • VELLAI, Tibor [HU]/[HU]
  • STURM, Adam [HU]/[HU]
Inventors
  • VELLAI, Tibor
  • STURM, Adam
Common Representative
  • VELLAI, Tibor
Priority Data
P180018029.05.2018HU
Publication Language English (en)
Filing Language Hungarian (HU)
Designated States
Title
(EN) METHOD FOR DNA CLONING WITHOUT USING IN VITRO ENZYMATIC REACTION, AND THE SEQUENCES OF RELATED DNA VECTORS
(FR) PROCÉDÉ DE CLONAGE D'ADN SANS UTILISATION DE RÉACTION ENZYMATIQUE IN VITRO, ET SÉQUENCES DE VECTEURS D'ADN APPARENTÉS
Abstract
(EN) The subject of the present innovation is a novel molecular DNA cloning procedure, during which a given DNA fragment (DNA stretch subjected to cloning, also called "insert") aimed to be multiplied is introduced into a vector DNA ("vehicle") without using in vitro added enzymes such as restriction endonucleases, ligases, DNA polymerases and 5' exonucleases. This method is termed as DNA auto-cloning because it requires no in vitro enzymatic reaction for introducing a DNA fragment (insert) into a cloning vector to generate a construct DNA. Specifically, the innovation is a novel molecular DNA cloning method, where a given DNA fragment (insert) that contains two homologous arms specific to a target DNA sequence is introduced into a vector DNA (most frequently a plasmid DNA of bacterial origin) bearing two copies of the target DNA sequence and, in addition, four Cas9 recognition sites. The vector DNA is found in a competent bacterial (most frequently Escherichia coli) cell expressing Cas9 nuclease (enzyme) and Cas9 recognition site-specific guide RNA (gRNA). Thus, the cloning process requires only the insert DNA amplified previously by polymerase chain reaction (PCR) and competent bacteria carrying the vector DNA and expressing Cas9 and gRNA, but no in vitro enzymatic reaction. The insert DNA transformed into competent cells is integrated into the vector DNA by using the cell's endogenous homologous recombination system. Thus, efficient cloning requires no in vitro added enzyme.
(FR) La présente invention concerne une nouvelle procédure de clonage D'ADN moléculaire, au cours de laquelle un fragment d'ADN donné (stretch d'ADN soumis à un clonage, également appelé " insert ") destiné à être multiplié est introduit dans un vecteur d'ADN ("véhicule") sans utilisation d'enzymes ajoutées in vitro telles que des endonucléases de restriction, des ligases, des ADN polymérases et des 5' exonuclérases. Ce procédé est appelé auto-clonage d'ADN car il ne nécessite aucune réaction enzymatique in vitro pour introduire un fragment d'ADN (insert) dans un vecteur de clonage pour générer un ADN de synthèse. Plus spécifiquement, l'invention concerne un nouveau procédé de clonage d'ADN moléculaire, selon lequel un fragment d'ADN donné (insert) qui contient deux bras homologues spécifiques d'une séquence d'ADN cible est introduit dans un ADN vecteur (le plus fréquemment un ADN plasmidique d'origine bactérienne) portant deux copies de la séquence d'ADN cible et, en outre, quatre sites de reconnaissance de Cas9. L'ADN vectoriel se trouve dans une cellule bactérienne compétente (le plus fréquemment Escherichia coli) exprimant la nucléase de Cas9 (enzyme) et l'ARN guide spécifique au site de reconnaissance de Cas9 (gARN). Ainsi, le procédé de clonage ne nécessite que l'ADN insert amplifié précédemment par réaction en chaîne à la polymérase (PCR) et des bactéries compétentes portant l'ADN vectoriel et exprimant Cas9 et gARN, mais pas de réaction enzymatique in vitro . L'ADN insert transformé en cellules compétentes est intégré dans l'ADN vectoriel au moyen du système de recombinaison homologue endogène de la cellule. Ainsi, ce clonage efficace ne nécessite aucune enzyme ajoutée in vitro .
Latest bibliographic data on file with the International Bureau