WO2003104450A1 - Rapid dna selection method - Google Patents

Rapid dna selection method Download PDF

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Publication number
WO2003104450A1
WO2003104450A1 PCT/MX2002/000050 MX0200050W WO03104450A1 WO 2003104450 A1 WO2003104450 A1 WO 2003104450A1 MX 0200050 W MX0200050 W MX 0200050W WO 03104450 A1 WO03104450 A1 WO 03104450A1
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Prior art keywords
dna
solution
probe
vectors
selection method
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PCT/MX2002/000050
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Spanish (es)
French (fr)
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Federico Esteban Sanchez Rodriguez
Gabriel Guillen Solis
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Universidad Nacional Autonoma De Mexico
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Priority to AU2002314598A priority Critical patent/AU2002314598A1/en
Priority to PCT/MX2002/000050 priority patent/WO2003104450A1/en
Publication of WO2003104450A1 publication Critical patent/WO2003104450A1/en
Priority to MXPA04011642A priority patent/MXPA04011642A/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries

Definitions

  • the invention object of the present invention refers to a method of rapid DNA selection, which consists in the manipulation of certain properties of the DNA and the conditions necessary for its hybridization, which unlike some of the existing methods does not require the use of enzymes, nor of complex systems of immobilization of the probe of interest, so it turns out to be a simple, fast, reliable, low cost and efficient method for the recovery of positive clones.
  • cDNAs or genomic banks are obtained by a process of ligation of the fragments of interest in a specific vector.
  • One of the main problems in the banks is to obtain a good representation of all the genes of the genome of the organism or of all the genes that are being expressed in the cells that were chosen for the construction of the cDNA bank.
  • the representativeness of the bank is very important in order to achieve the isolation of cDNAs that have low expression or genes that are found in low copy numbers in the genome of the organism of interest.
  • genomic DNA preparation contains approximately 3 x 10 9 base pairs.
  • a fragment of interest of approximately 3000 bp comprises only one part per million of a genomic DNA preparation.
  • genomic DNA fragments or those of the cDNA are first prepared by insertion into the chosen vector.
  • the desired vector and DNA are ligated and introduced into E. cells. coli, either packaged in vitro in the phage capsid ⁇ or by direct transformation.
  • the strategies for the isolation of individual genomes or cDNA clones may be a bit different.
  • cDNA library selection processes involve positive identification of cDNA clones either with antibodies or by hybridization with nucleic acid probes.
  • a particular application of DNA selection methods is the cDNA subtracted libraries, which provide a method for the identification of mRNAs (by handling the respective cDNAs) that are differentially expressed in two different tissues.
  • the cDNAs of one of the tissues are synthesized and hybridized against the cDNAs of the other tissue, to obtain those cDNAs that are preferentially expressed in one of them.
  • the sequences that did not hybridize are cloned into a bacteriophage or a plasmid to obtain a subtracted library of cDNA. It is difficult to estimate the number of clones that must be screened, as this will vary with the type of cell and the gene to be identified.
  • Rigas et al. reported a method that is based on: 1) the ability of a protein to form stable complexes between linear single-stranded and double-stranded DNA molecules that share sequence identity; Y 2) a procedure that allows the isolation of biotinylated nucleic acids. That is, the protein coated with the single chain biotinylated DNA probe is hybridized with the plasmids to be selected, to form triple chain complexes with the plasmids having sequences homologous to the probe. Subsequently, these complexes are purified by streptavidin columns immobilized in agarose or iminodiacetic cupric acid. In this method, a recovery between 10 and 20% of the plasmids of interest is reported.
  • one of the disadvantages of this method is the low percentage of recovery of positive clones, which is due, among other things, to the purification steps that are necessary, another disadvantage is the high cost of the method, given that They use high quality specific reagents.
  • the ssDNA of interest are released from the biotinylated oligonucleotide.
  • an oligonucleotide (primer) is added to the cDNAs for in-chain synthesis of the complementary strand.
  • competent cells are transformed and plated. With this method, 20% to 100% of colonies are recovered with the plasmid containing the cDNA of interest.
  • this method has several disadvantages, first, the recovery percentage of clones of interest is very variable, so it turns out to be unreliable, secondly, to obtain a population of simple chains it is necessary to use enzymes that degrade DNA and thirdly, a special device is required to trap small magnetic spheres. For all the above, the procedure turns out to be laborious and relatively expensive.
  • recombinant DNA technology is widely used in the area of medicine, industry and agriculture. For example, it has been used in the generation of vaccines for different diseases, in obtaining recombinant bacterial strains that improve the quality and / or quantity of a certain product of industrial interest, the generation of transgenic plants that allow obtaining plants with a higher resistance to certain pathogens, etc.
  • This technology has made great progress in recent years thanks to the development of methodologies that have allowed us to isolate, analyze, manipulate and control the genes of interest.
  • one of the limiting steps in the generation of genetically modified organisms is the isolation of the genes of interest, which is achieved through the use of gene selection techniques from a genomic or expression bank.
  • FIG. 1 Illustrates the DNA Quick Selection Method of the present invention, in the case of an expression bank.
  • T Represents the different vectors with insert of a double-stranded cDNA Bank. (1) Obtaining the vectors with single chain insert concatenated by denaturation (2) Fragmentation of the sonication cloning vector; (3) prehybridization with the DNA of the sonicated vector with the concatenated single chain insert vector; (4) Immobilization of the DNA probe of interest in a Nylon membrane and blocking the free surface thereof; (5) hybridization of the vectors with single chain insert, particularly in the region of the insert, of the Bank with the immobilized DNA probe; for 8-10 h; (6) Washes with 2X SSC to eliminate the non-hybridized cDNA; (7) Heat treatment and membrane separation to recover the insert vectors that hybridized with the probe; (8) transformation of the vectors with insert recovered in the cells where the vector can be replicated (9) Pur
  • FIG. 2 Illustrates the cDNAs cloned by the method of the present invention.
  • the recovered DNAs (step 7 of Figure 1), were transformed, purified and digested with the restriction enzyme EcoR ⁇ (lanes 2-24 and 27-38). The digestion products were separated on the 1% agarose gel, transferred to a C-extra Hybond membrane and hybridized with the probe used in step 3 of Figure 1. (in this case the cDNA of clone pKG3 ) marked by random priming with 32 P. Lanes 1, 25 and 26 show the DNA of the probe.
  • Insert refers to a DNA fragment that was incorporated into the vector of choice to give rise to an vector with insert.
  • the inserts are of different sizes and of different sequence.
  • Probe refers to a DNA fragment that will be used to hybridize with a DNA bank to identify those clones that contain an insert of complementary sequence homologous and therefore be able to hybridize with it.
  • Choice vector refers to the molecular vector (Plasmid or Phagemid) in which either genome fragments or cDNAs obtained from a cell will be incorporated, to obtain genomic or cDNA banks, respectively.
  • Vector with insert refers to the vector of choice that already has a genome fragment or a cDNA incorporated and that together forms a genomic or cDNA bank, respectively.
  • Each vector molecule with a bank insert incorporates an insert of different sequence and size.
  • Sonic vector Refers to the same vector of choice after being subjected to a fragmentation process, preferably by means of an ultrasound pulse in such a way that the vector of choice is reduced to fragments of variable double-chain sizes. (Note: The sonicated vector must be the same as the vector of choice, to achieve adequate prehybridization with the vectors with single chain and concatenated inserts).
  • Positive clones host cells that contain an insert vector whose sequence turns out to be complementary and homologous to the nucleotide sequence of the probe.
  • Concatenated DNA Crosslinked DNA rings as the links in a chain. Result of the denaturation of double stranded circular DNA.
  • the methods must include at the end of the process some steps that allow regenerating the double-stranded circular DNA. This is necessary to be able to replicate the vectors with insert in the host cell, since the single-stranded DNAs when introduced into it, are degraded, in addition to the DNA polymerases require double-stranded DNA in order to replicate the DNA. In this regard, the use of DNA polymerases to synthesize the complementary chain is reported.
  • the strategies for the generation of single chain plasmids usually involve the use of specific enzymes, both nucleases to degrade one of the chains at the beginning, and DNA polymerases to regenerate the complementary chain at the end, which causes a high variability in the recovery efficiency of recovered positive clones (20 to 100%).
  • specific enzymes both nucleases to degrade one of the chains at the beginning
  • DNA polymerases to regenerate the complementary chain at the end
  • the inventors of the present method propose a solution to these limitations by making use of certain DNA properties and the conditions of their manipulation, which, unlike the methods described above, do not require enzymes such as nucleases (to generate single chains), nor polymerases (to regenerate the complementary chain), so it is a method, simple, fast, efficient, reliable, low cost and efficient recovery of positive clones and also does not require special equipment to recover the vectors with insert with sequence probe homologue.
  • This method comprises the following steps:
  • a) Obtaining the vectors with single chain insert and concatenated by denaturation and prehybridization with the DNA of the sonicated vector; b) on the other hand, the immobilization of the DNA probe of interest in a solid support and the blocking of the free surface thereof; c) hybridization of the vectors with single chain insert (particularly in the region of the insert) of the bank with the immobilized DNA probe; d) recovery of the insert vectors that hybridized with the probe by membrane separation; e) transformation of the vectors with insert recovered in the cells where it can be replicated for the subsequent analysis of the DNAs.
  • the rapid method of DNA selection of the present invention is schematized for the case of a cDNA bank.
  • Obtaining vectors with single chain insertion is done by denaturation and prehybridization of the vectors with double strand insert with the DNA of the sonicated vector, separating the chains from the vectors with insert, giving rise to two single strands of each vector with insert concatenated with each other and hybridized with the fragments of the vector of choice which It will keep them that way. In this way, the inserts are exposed as a single chain to allow hybridization with the specific probe.
  • the immobilization of the probe of interest is carried out, by means of its fixation and denaturation in a support and the blocking of the free surface of the support, in order to prevent vectors with single chain insert of the bank sticking unspecifically to the support.
  • hybridization of the immobilized probe with the vectors with DNA bank insert is carried out in order to select those with the insert of interest. Subsequently, the selected insert vectors are recovered and transformed into the cells where they can be replicated for later analysis.
  • the molecules of the vector with insert that are selected are those that by having an insert with homologous sequence and complementary to the immobilized probe hybridize with it, while those molecules whose insert does not have a complementary homologous sequence remain in solution.
  • the recovery of the vectors with insert is performed by separating the membrane under certain temperature conditions in conventional equipment, which represents a comparative advantage, with respect to the reported methods.
  • the inventors propose to obtain the simple chain of the vectors with insert, by means of their denaturation and hybridization simultaneously with the small fragments of the sonicated vector, to keep the chains separated while remaining concatenated.
  • this previous hybridization it is avoided that the DNA of each vector with insert can hybridize with the complementary chain of the same, which would result in double-stranded DNA or with another molecule of the vector with insert which could give rise to a false positive clone , thus avoiding recovering multiple chains that could give rise to false positive clones, so that the method turns out to have a high efficiency, of easy implementation and, above all, of a relatively low cost.
  • one of the determining factors to obtain an efficient transformation in the cells of E colies the type of DNA to be transformed. For this, it is necessary to introduce DNA from double stranded vectors since single stranded DNA is degraded inside the cell. In this sense, an additional advantage of the method of the present invention is that in order to obtain DNA from double stranded vectors at the end, it is not necessary to regenerate one of the chains, because during the whole process, the two DNA chains remain attached concatenated.
  • the inventors propose the use of a solid support whose characteristics allow manipulating the reaction conditions, particularly the use of high temperatures that favor the immobilization of the DNA that is used as a probe and that at the end of the hybridization facilitate the separation of the vectors with single chain insert that hybridized with the probe of interest without requiring an intermediary, while retaining the probe.
  • the blocking of the free surface of the support (the one in which the probe was not glued) is also contemplated to prevent the bank's DNAs from nonspecifically sticking to the support, thereby achieving, substantially increasing efficiency and specificity of the method of the present invention.
  • the inventors propose using a support that meets the characteristics described above and especially that can be handled at high temperatures; in order to achieve greater specificity in hybridization; in this case, they suggest using nitrocellulose membrane preferably, obtaining better results, if the nitrocellulose membrane is positively charged.
  • the method of the present invention allows the use of probes of different size, that is, an oligonucleotide, a complete gene or a polynucleotide can be used as a probe.
  • single-stranded DNA is a macromolecule formed by a large number of deoxyribonucleotides that can bind to another DNA macromolecule with complementary sequence to form the double-stranded DNA or remain as a chain macromolecule.
  • double stranded DNA can be selected, without the need to use enzymes to obtain single strands.
  • DNA denaturation is the process by which the hydrogen bonds that bind the double strand of DNA can be broken and the two strands of DNA separated to obtain two single strands of DNA (that in the case of circular DNA, they will remain concatenated).
  • acids the addition of acids; the adition of alkalis such as sodium hydroxide or by heat treatment, using temperatures ranging from 72 to 105 ° C.
  • the method of the present invention contemplates the denaturation of the DNA of the vectors with insert, in the same step, with the prehybridization with the sonicated vector, which can be performed by any of the aforementioned denaturation methods, that is by means of the handling of pH or temperature;
  • this denaturation and prehybridization be carried out by means of a two-step heat treatment, which consists of heating the inserted vectors in the presence of the sonicated vector to the denaturation temperature of 72 to 105 ° C, during minus 5 minutes, subsequently a slight cooling or decrease of the temperature is carried out, to keep this mixture at the hybridization temperature of 42 to 65 ° C for a minimum of 5 minutes; and finally allowed to cool to room temperature.
  • the heat treatment has comparative advantages with respect to pH manipulation methods, since it turns out to be a simple, fast treatment and it is not necessary to use additional reagents that could interfere with the hybridizations that are carried out in the method of The present
  • Another advantage of the method of the present invention is that since the DNA handling properties and conditions are universal and not specific for certain inserts, probes or vectors of choice, the possibility of using the different vectors that are commonly used used in gene banks of prokaryotic cells, such as pCP13 and eukaryotic cells such as: ⁇ gt11 for genomic banks and ⁇ ZAP for cDNA banks.
  • Example No.1 The application of the method of the present invention in the selection of cDNA coding for the synthesis of a receptor-like protein kinase is illustrated.
  • This example shows the efficiency of the selection of DNA of interest from a 13-day bean nodule expression bank, by the rapid method of DNA selection of the present invention.
  • nucleotide sequence identity of the selected DNAs is shown with the probe used in the Rapid DNA Selection Method of the present invention.
  • example 2 The methodology of example 2 was followed. The 3 DNAs that hybridized in greater proportion to the probe were selected and sequenced with the "Big Dye TM Terminator Cycle sequencing Ready" Kit (PE Applied Biosystems Perkin Elmer) and the reactions were read in a ABI PRIMS Model 377 DNA sequence automatic sequencer.
  • Table I shows the percent identity of the nucleotide sequence of the cDNA of clone pKG3 used as a probe, with the sequence of 3 of the cDNAs that were selected by the Method of the present invention (pKG 1, pKG 8 and pKG 13).
  • Example 4 This example illustrates the industrial application of the Rapid DNA Selection Method, by integrating a case based on the method of the present invention. COMPONENTS
  • Solution I Prehybridization solution (1 ⁇ g / ⁇ l of sonic vector DNA).
  • Solution II DNA fixing solution (NaOH 0.4 N)
  • Solution III Washing solution.
  • Solution IV Hybridization solution.
  • Solution V Solution of circumvention.
  • Solution VI DNA precipitation solution. A set of positively charged Nylon membranes of 1cm 2 .
  • the hybridization temperature will depend on the identity of the probe's DNA sequence and the gene to be selected. A temperature of 65 ° C is recommended for a homologous probe. For a probe with low% identity the temperature can be lowered to 42 ° C. 7) Mix the prehybridized cDNA bank with 5 ml of the Hybridization Solution and incubate at the hybridization temperature for 1-8 hours.

Abstract

The invention relates to a rapid DNA selection method involving the manipulation of certain DNA properties and hybridisation conditions. Unlike some existing methods, the invention does not require the use of enzymes or complex probe immobilisation systems and, as a result, is a simple, quick, reliable, low-cost and efficient method of recovering positive clones. The inventive method provides a valuable tool for the selection and/or purification of DNA fragments from a gene or expression bank.

Description

MÉTODO RÁPIDO DE SELECCIÓN DE DNAs. QUICK METHOD OF DNA SELECTION.
CAMPO TÉCNICO DE LA INVENCIÓNTECHNICAL FIELD OF THE INVENTION
La invención objeto de la presente refiere un método de selección rápida de DNA, que consiste en la manipulación de ciertas propiedades del DNA y de las condiciones necesarias para su hibridación, que a diferencia de algunos de los métodos existentes no requiere del uso de enzimas, ni de sistemas complejos de inmovilización de la sonda de interés, por lo que resulta ser un método, sencillo, rápido, confiable, de bajo costo y eficiente para la recuperación de clonas positivas.The invention object of the present invention refers to a method of rapid DNA selection, which consists in the manipulation of certain properties of the DNA and the conditions necessary for its hybridization, which unlike some of the existing methods does not require the use of enzymes, nor of complex systems of immobilization of the probe of interest, so it turns out to be a simple, fast, reliable, low cost and efficient method for the recovery of positive clones.
ANTECEDENTESBACKGROUND
La construcción de bancos de cDNAs o genómicos se obtiene por un proceso de ligación de los fragmentos de interés en un vector específico. Uno de los principales problemas en los bancos es el lograr obtener una buena representatividad de todos los genes del genoma del organismo o de todos los genes que se están expresando en las células que se eligieron para la construcción del banco del cDNA. La representatividad del banco es muy importante para poder lograr el aislamiento de cDNAs que tienen baja expresión o de genes que se encuentran en bajo número de copias en el genoma del organismo de interés.The construction of cDNAs or genomic banks is obtained by a process of ligation of the fragments of interest in a specific vector. One of the main problems in the banks is to obtain a good representation of all the genes of the genome of the organism or of all the genes that are being expressed in the cells that were chosen for the construction of the cDNA bank. The representativeness of the bank is very important in order to achieve the isolation of cDNAs that have low expression or genes that are found in low copy numbers in the genome of the organism of interest.
El DNA de organismos superiores es muy complejo: por ejemplo, un genoma haploide de un mamífero contiene aproximadamente 3 x 109 pares de bases. Por lo que un fragmento de interés de aproximadamente 3000 bp comprende sólo una parte por millón de una preparación de DNA genómico. Claramente el principal problema en la generación de una biblioteca recombinante de DNA ya sea genómico o cDNA es la creación de población representativa de clonas necesaria para asegurar que la biblioteca contenga al menos una versión de cada secuencia de interés. Las soluciones a este problema son básicamente similares para bibliotecas genómicas y de cDNA. De manera general, los fragmentos del DNA genómico o los del cDNA, primero son preparados por inserción en el vector elegido. El vector y el DNA deseado son ligados e introducidos en células de E. coli, ya sea empaquetado in vitro en la cápside del fago λ o mediante transformación directa. En algunos aspectos, las estrategias para el aislamiento de genomas individuales o clonas de cDNA pueden ser un poco diferentes.The DNA of higher organisms is very complex: for example, a haploid genome of a mammal contains approximately 3 x 10 9 base pairs. Thus, a fragment of interest of approximately 3000 bp comprises only one part per million of a genomic DNA preparation. Clearly the main problem in generating a recombinant DNA library either genomic or cDNA is the creation of a representative population of clones necessary to ensure that the library contains at least one version of each sequence of interest. The solutions to this problem are basically similar for genomic and cDNA libraries. In general, the genomic DNA fragments or those of the cDNA are first prepared by insertion into the chosen vector. The desired vector and DNA are ligated and introduced into E. cells. coli, either packaged in vitro in the phage capsid λ or by direct transformation. In some aspects, the strategies for the isolation of individual genomes or cDNA clones may be a bit different.
La mayoría de los procesos de selección de las bibliotecas de cDNA involucran la identificación positiva de las clonas de cDNA ya sea con anticuerpos o por la hibridación con sondas de ácidos nucleicos. Una aplicación particular que tienen los métodos de selección de DNA, son las bibliotecas sustraídas de cDNA, las cuales proporcionan un método para la identificación de mRNAs (mediante el manejo de los cDNAs respectivos) que son expresados diferencialmente en dos tejidos distintos. En este método los cDNAs de uno de los tejidos son sintetizados e hibridados contra los cDNAs del otro tejido, para obtener aquellos cDNAs que se expresan preferencialmente en uno de ellos. Las secuencias que no hibridaron son clonadas en un bacteriófago o un plásmido obteniéndose una biblioteca sustraída de cDNA. Es difícil la estimación del número de clonas que deben ser tamizadas, ya que esto variará con el tipo de célula y el gene a ser identificado.Most cDNA library selection processes involve positive identification of cDNA clones either with antibodies or by hybridization with nucleic acid probes. A particular application of DNA selection methods is the cDNA subtracted libraries, which provide a method for the identification of mRNAs (by handling the respective cDNAs) that are differentially expressed in two different tissues. In this method the cDNAs of one of the tissues are synthesized and hybridized against the cDNAs of the other tissue, to obtain those cDNAs that are preferentially expressed in one of them. The sequences that did not hybridize are cloned into a bacteriophage or a plasmid to obtain a subtracted library of cDNA. It is difficult to estimate the number of clones that must be screened, as this will vary with the type of cell and the gene to be identified.
El método más comúnmente utilizado para la clonación de cDNAs de una biblioteca es la hibridación in situ de placas líticas con una sonda homologa o heteróloga marcada con 32P. Normalmente esta técnica es realizada en bacteriófagos que previamente fueron crecidos en placas de agar y transferidos en una membrana de nitrocelulosa. Sin embargo, para llevar a cabo la búsqueda de un cDNA de interés es necesario hacer un sondeo de 200,000 - 400,000 unidades formadoras de placa (UFP) para tener representados el total de transcritos de aproximadamente 30,000 por célula. Por otro lado es necesario realizar por lo menos tres rondas de purificación para obtener placas líticas individuales que contengan un solo cDNA de interés.The most commonly used method for cloning cDNAs from a library is in situ hybridization of lytic plates with a homologous or heterologous probe labeled with 32 P. Normally this technique is performed on bacteriophages that were previously grown on agar plates and transferred into agar plates. a nitrocellulose membrane. However, in order to carry out the search for a cDNA of interest, it is necessary to conduct a survey of 200,000-400,000 plate-forming units (UFPs) to have the total number of transcripts of approximately 30,000 per cell represented. On the other hand, it is necessary to carry out at least three rounds of purification to obtain individual lithic plates containing a single cDNA of interest.
En los últimos años se han reportado algunos métodos que permiten la clonación rápida de genes de bancos de cDNAs, a continuación se detallan algunos de ellos.In recent years, some methods that allow rapid cloning of cDNA bank genes have been reported, some of them are detailed below.
En 1986, Rigas et al. reportaron un método que se basa en: 1) la habilidad de una proteína para formar complejos estables entre las moléculas de DNA de cadena sencilla lineal y de doble cadena circular que comparten identidad de secuencia; y 2) un procedimiento que permite el aislamiento de ácidos nucleicos biotinilados. Esto es, la proteína cubierta con la sonda de DNA biotinilada de cadena sencilla se hace hibridar con los plásmidos a seleccionar, para formar complejos de cadena triple con los plásmidos que tienen secuencias homologas a la sonda. Posteriormente estos complejos son purificados por columnas de estreptavidina inmovilizada en agarosa o bien de ácido cúprico iminodiacético. En este método se reporta una recuperación entre el 10 y el 20% de los plásmidos de interés. Como se puede apreciar, una de las desventajas de este método es el bajo porcentaje de recuperación de clonas positivas, que se debe entre otras cosas, a los pasos de purificación que son necesarios, otra desventaja es el alto costo del método, dado que se utilizan reactivos específicos de alta la calidad.In 1986, Rigas et al. reported a method that is based on: 1) the ability of a protein to form stable complexes between linear single-stranded and double-stranded DNA molecules that share sequence identity; Y 2) a procedure that allows the isolation of biotinylated nucleic acids. That is, the protein coated with the single chain biotinylated DNA probe is hybridized with the plasmids to be selected, to form triple chain complexes with the plasmids having sequences homologous to the probe. Subsequently, these complexes are purified by streptavidin columns immobilized in agarose or iminodiacetic cupric acid. In this method, a recovery between 10 and 20% of the plasmids of interest is reported. As can be seen, one of the disadvantages of this method is the low percentage of recovery of positive clones, which is due, among other things, to the purification steps that are necessary, another disadvantage is the high cost of the method, given that They use high quality specific reagents.
Por otro lado, en 1992 Ito et al., publicaron un procedimiento de aislamiento de DNA mediante la formación de triple-hélice y separación magnética. En este procedimiento, el DNA de interés es capturado mediante un oligonucleótido biotinilado vía formación triple intermolecular, posteriormente el "complejo" es seleccionado mediante su unión a pequeñas esferas magnéticas cubiertas con estreptavidina y recuperado en forma de doble cadena por elución con buffer alcalino suave que desestabiliza la triple hélice. Los autores demostraron la efectividad de este procedimiento mediante un modelo experimental con una biblioteca reconstruida artificialmente y, también, por el aislamiento de los polidinucleótidos (dT-dC)n »(dG -dA)n de una biblioteca del genoma humano. Si bien, no reportan porcentajes de recuperación de clonas, según los autores, la ventaja de este procedimiento es que se minimizan las etapas de filtración que son requeridas en otras metodologías, no obstante, resulta ser un método relativamente costoso, ya que se requiere de un equipo o sistema especial para atrapar las pequeñas esferas magnéticas y de igual manera, se utilizan reactivos específicos que incrementan su costo.On the other hand, in 1992 Ito et al., Published a method of DNA isolation by triple-helix formation and magnetic separation. In this procedure, the DNA of interest is captured by a biotinylated oligonucleotide via triple intermolecular formation, then the "complex" is selected by binding to small magnetic spheres covered with streptavidin and recovered as a double strand by elution with a soft alkaline buffer that destabilizes the triple helix. The authors demonstrated the effectiveness of this procedure by means of an experimental model with an artificially reconstructed library and, also, by the isolation of polydinucleotides (dT-dC) n » (dG-dA) n from a library of the human genome. Although they do not report recovery percentages of clones, according to the authors, the advantage of this procedure is that the filtration stages that are required in other methodologies are minimized, however, it turns out to be a relatively expensive method, since it requires a special equipment or system to trap small magnetic spheres and similarly, specific reagents are used that increase their cost.
En 1995, Li et al., publicaron el sistema denominado GeneTrapper. En este sistema se parte de una población compleja de plásmidos de doble cadena que contienen los insertos de cDNA. Estos plásmidos de doble cadena se convierten a DNA de cadena sencilla por la degradación de una de las cadenas de DNA a través de reacciones enzimáticas, posteriormente se seleccionan los plásmidos que contienen el DNA de interés por hibridación en solución con una sonda de oligonucleótido biotinilado complementario a la secuencia de interés. Los híbridos son capturados en pequeñas esferas magnéticas cubiertas con estreptavidina. Con un imán se recuperan las esferas magnéticas de la solución, dejando en la misma los DNA de cadena sencilla (ssDNA) no hibridados mismos que serán descartados. Posteriormente, los ssDNA de interés son liberados del oligonucleótido biotinilado. Después de liberados, a los cDNAs se les adiciona un oligonucleótido (primer) para la síntesis in vltro de la cadena complementaria. Posteriormente, se transforman células competentes y se plaquean. Con este método se recuperan del 20% al 100% de colonias con el plásmido que contiene el cDNA de interés. Como se puede apreciar, este método presenta varias desventajas, en primer lugar, el porcentaje de recuperación de clonas de interés es muy variable, por lo que resulta ser poco confiable, en segundo lugar, para obtener una población de cadenas sencillas es necesario utilizar enzimas que degraden el DNA y en tercer lugar, se requiere de un aparato especial para atrapar las pequeñas esferas magnéticas. Por todo lo anterior, el procedimiento resulta ser laborioso y de un costo relativamente alto.In 1995, Li et al., Published the system called GeneTrapper. This system is based on a complex population of double chain plasmids that contain cDNA inserts. These double stranded plasmids are converted to single stranded DNA by degradation of one of the strands of DNA through enzymatic reactions, then the plasmids are selected. containing the DNA of interest by hybridization in solution with a biotinylated oligonucleotide probe complementary to the sequence of interest. The hybrids are captured in small magnetic spheres covered with streptavidin. With a magnet, the magnetic spheres of the solution are recovered, leaving in it the single stranded DNA (ssDNA) that will not be discarded. Subsequently, the ssDNA of interest are released from the biotinylated oligonucleotide. After release, an oligonucleotide (primer) is added to the cDNAs for in-chain synthesis of the complementary strand. Subsequently, competent cells are transformed and plated. With this method, 20% to 100% of colonies are recovered with the plasmid containing the cDNA of interest. As can be seen, this method has several disadvantages, first, the recovery percentage of clones of interest is very variable, so it turns out to be unreliable, secondly, to obtain a population of simple chains it is necessary to use enzymes that degrade DNA and thirdly, a special device is required to trap small magnetic spheres. For all the above, the procedure turns out to be laborious and relatively expensive.
En la actualidad la tecnología del DNA recombinante es ampliamente usada en el área de la medicina, la industria y la agricultura. Por ejemplo, se ha utilizado en la generación de vacunas de diferentes enfermedades, en la obtención de cepas bacterianas recombinantes que mejoran la calidad y/o la cantidad de cierto producto de interés industrial, la generación de plantas transgénicas que permiten obtener plantas con una mayor resistencia a ciertos patógenos, etc. Esta tecnología ha tenido grandes avances en los últimos años gracias al desarrollo de metodologías que han permitido aislar, analizar, manipular y controlar los genes de interés. No obstante en la actualidad, uno de los pasos limitantes en la generación de organismos modificados genéticamente es el aislamiento de los genes de interés, el cual se logra con la utilización de técnicas de selección de genes de un banco genómico o de expresión.Today, recombinant DNA technology is widely used in the area of medicine, industry and agriculture. For example, it has been used in the generation of vaccines for different diseases, in obtaining recombinant bacterial strains that improve the quality and / or quantity of a certain product of industrial interest, the generation of transgenic plants that allow obtaining plants with a higher resistance to certain pathogens, etc. This technology has made great progress in recent years thanks to the development of methodologies that have allowed us to isolate, analyze, manipulate and control the genes of interest. However, at present, one of the limiting steps in the generation of genetically modified organisms is the isolation of the genes of interest, which is achieved through the use of gene selection techniques from a genomic or expression bank.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
BREVE DESCRICIÓN DE LAS FIGURAS Figura 1.- Ilustra el Método de selección rápida de DNAs de la presente invención, para el caso de un banco de expresión. (T ) Representa los diferentes vectores con inserto de un Banco de cDNA de doble cadena. (1) Obtención de los vectores con inserto de cadena sencilla concatenados mediante la desnaturalización (2) Fragmentación del vector de clonación por sonicación; (3) prehibridación con el DNA del vector sonicado con el vector con inserto de cadena sencilla concatenado; (4) Inmovilización de la sonda de DNA de interés en una membrana de Nylon y el bloqueo de la superficie libre del mismo; (5) hibridación de los vectores con inserto de cadena sencilla, particularmente en la región del inserto, del Banco con la sonda de DNA inmovilizada; durante 8 - 10 h; (6) Lavados con SSC 2X para eliminar el cDNA que no híbridaron; (7) Tratamiento térmico y separación de la membrana para recuperar los vectores con inserto que hibridaron con la sonda; (8) transformación de los vectores con inserto recuperados en las células donde puede replicarse el vector (9) Purificación de los plásmidos e hibridación con la sonda utilizada en la selección para el posterior análisis de los DNAs.BRIEF DESCRIPTION OF THE FIGURES Figure 1.- Illustrates the DNA Quick Selection Method of the present invention, in the case of an expression bank. (T) Represents the different vectors with insert of a double-stranded cDNA Bank. (1) Obtaining the vectors with single chain insert concatenated by denaturation (2) Fragmentation of the sonication cloning vector; (3) prehybridization with the DNA of the sonicated vector with the concatenated single chain insert vector; (4) Immobilization of the DNA probe of interest in a Nylon membrane and blocking the free surface thereof; (5) hybridization of the vectors with single chain insert, particularly in the region of the insert, of the Bank with the immobilized DNA probe; for 8-10 h; (6) Washes with 2X SSC to eliminate the non-hybridized cDNA; (7) Heat treatment and membrane separation to recover the insert vectors that hybridized with the probe; (8) transformation of the vectors with insert recovered in the cells where the vector can be replicated (9) Purification of the plasmids and hybridization with the probe used in the selection for the subsequent analysis of the DNAs.
Figura 2.- Ilustra los cDNA clonados por el método de la presente invención. Los DNAs recuperados (paso 7 de la Figura 1), fueron transformados, purificados y digeridos con la enzima de restricción EcoR\ (carriles 2 - 24 y 27 - 38). Los productos de la digestión fueron separados en el gel de agarosa al 1 %, transferidos a una membrana de Hybond C-extra e hibridados con la sonda utilizada en el paso 3 de la Figura 1. (en este caso el cDNA de la clona pKG3) marcada por random priming con 32P. En los carriles 1 , 25 y 26 se muestra el DNA de la sonda.Figure 2.- Illustrates the cDNAs cloned by the method of the present invention. The recovered DNAs (step 7 of Figure 1), were transformed, purified and digested with the restriction enzyme EcoR \ (lanes 2-24 and 27-38). The digestion products were separated on the 1% agarose gel, transferred to a C-extra Hybond membrane and hybridized with the probe used in step 3 of Figure 1. (in this case the cDNA of clone pKG3 ) marked by random priming with 32 P. Lanes 1, 25 and 26 show the DNA of the probe.
Para la adecuada interpretación del alcance de la presente invención, los inventores consideran conveniente establecer las siguientes definiciones:For the proper interpretation of the scope of the present invention, the inventors consider it convenient to establish the following definitions:
Inserto: Se refiere a un fragmento de DNA que fue incorporado en el vector de elección para dar lugar a un vector con inserto. En un banco, los insertos son de diferentes tamaños y de secuencia distinta.Insert: Refers to a DNA fragment that was incorporated into the vector of choice to give rise to an vector with insert. In a bank, the inserts are of different sizes and of different sequence.
Sonda: Se refiere a un fragmento de DNA que será utilizado para hibridar con un banco de DNA para identificar aquellas clonas que contengan un inserto de secuencia complementaria homologa y que por tanto sean capaces de hibridar con esta.Probe: Refers to a DNA fragment that will be used to hybridize with a DNA bank to identify those clones that contain an insert of complementary sequence homologous and therefore be able to hybridize with it.
Vector de elección: Se refiere al vector molecular (Plásmido o Fagémido) en el cual se incorporarán ya sean los fragmentos de un genoma o los cDNAs obtenidos de una célula, para obtener bancos genómicos o de cDNA, respectivamente.Choice vector: Refers to the molecular vector (Plasmid or Phagemid) in which either genome fragments or cDNAs obtained from a cell will be incorporated, to obtain genomic or cDNA banks, respectively.
Vector con inserto: Se refiere al vector de elección que tiene ya incorporado un fragmento de genoma o un cDNA y que forma en conjunto un banco genómico o de cDNA, respectivamente. Cada molécula del vector con inserto de un banco incorpora un inserto de diferente secuencia y tamaño.Vector with insert: Refers to the vector of choice that already has a genome fragment or a cDNA incorporated and that together forms a genomic or cDNA bank, respectively. Each vector molecule with a bank insert incorporates an insert of different sequence and size.
Vector sonicado: Se refiere al mismo vector de elección luego de ser sometido a un proceso de fragmentación, preferentemente mediante un pulso de ultrasonido de tal manera, que el vector de elección se reduce a fragmentos de tamaños variables de doble cadena. (Nota: El vector sonicado debe ser el mismo que el vector de elección, para lograr una adecuada prehibridación con los vectores con inserto de cadena sencilla y concatenados).Sonic vector: Refers to the same vector of choice after being subjected to a fragmentation process, preferably by means of an ultrasound pulse in such a way that the vector of choice is reduced to fragments of variable double-chain sizes. (Note: The sonicated vector must be the same as the vector of choice, to achieve adequate prehybridization with the vectors with single chain and concatenated inserts).
Clonas positivas: células hospederas que contienen un vector con inserto cuya secuencia resulta ser complementaria y homologa a la secuencia de nucleótidos de la sonda.Positive clones: host cells that contain an insert vector whose sequence turns out to be complementary and homologous to the nucleotide sequence of the probe.
DNA concatenado: Anillos de DNA entrecruzados como los eslabones de una cadena. Resultado de la desnaturalización de DNA circular de doble cadena.Concatenated DNA: Crosslinked DNA rings as the links in a chain. Result of the denaturation of double stranded circular DNA.
Los métodos reportados para la selección y/o purificación de fragmentos de DNA a partir de un banco de genes o de expresión son ampliamente utilizados para la generación de organismos modificados genéticamente y en el desarrollo de tecnologías de DNA recombinante. Estos métodos tienen dos aspectos importantes que determinan la eficiencia y especificidad de la hibridación con la sonda de interés: por un lado, la generación de vectores con inserto de cadena sencilla y por otro, la adecuada inmovilización de la sonda utilizada. Con relación a la obtención de vectores con inserto de cadena sencilla, en los métodos antes mencionados, Pruitt 1988, se describen algunas estrategias tales como: la utilización de enzimas específicas (nucleasas) para que degraden una sola cadena del DNA de doble cadena. En otros casos, se utiliza la propiedad del DNA para formar una triple hélice bajo ciertas condiciones de reacción. En cualquiera de estas estrategias, los métodos deben incluir al final del proceso algunos pasos que permitan regenerar el DNA circular de doble cadena. Esto es necesario para poder replicar los vectores con inserto en la célula hospedera, ya que los DNAs de cadena sencilla al ser introducidas en ésta, son degradados, además de que las DNA polimerasas requieren DNA de doble cadena para poder replicar el DNA. Al respecto, se reporta la utilización de DNA polimerasas para sintetizar la cadena complementaria.Reported methods for the selection and / or purification of DNA fragments from a gene or expression bank are widely used for the generation of genetically modified organisms and in the development of recombinant DNA technologies. These methods have two important aspects that determine the efficiency and specificity of hybridization with the probe of interest: on the one hand, the generation of vectors with single chain insert and on the other, the adequate immobilization of the probe used. With regard to obtaining vectors with a single chain insert, in the aforementioned methods, Pruitt 1988, some strategies are described such as: the use of specific enzymes (nucleases) to degrade a single double strand DNA chain. In other cases, the property of DNA is used to form a triple helix under certain reaction conditions. In any of these strategies, the methods must include at the end of the process some steps that allow regenerating the double-stranded circular DNA. This is necessary to be able to replicate the vectors with insert in the host cell, since the single-stranded DNAs when introduced into it, are degraded, in addition to the DNA polymerases require double-stranded DNA in order to replicate the DNA. In this regard, the use of DNA polymerases to synthesize the complementary chain is reported.
En conclusión, las estrategias para la generación de plásmidos de cadena sencilla por lo general involucran el uso de enzimas específicas, tanto nucleasas para degradar una de las cadenas al inicio, como polimerasas de DNA para regenerar la cadena complementaria al final, lo que origina una alta variabilidad en la eficiencia de recuperación de clonas positivas recuperadas (20 al 100%). Por otro lado, es evidente que conforme aumenta la complejidad de los métodos debido al incremento en el número de pasos o etapas, se disminuye la confiabilidad de ios mismos. Por último y no menos importante, es el alto costo asociado a los reactivos y enzimas específicas que se emplean en estos métodos.In conclusion, the strategies for the generation of single chain plasmids usually involve the use of specific enzymes, both nucleases to degrade one of the chains at the beginning, and DNA polymerases to regenerate the complementary chain at the end, which causes a high variability in the recovery efficiency of recovered positive clones (20 to 100%). On the other hand, it is evident that as the complexity of the methods increases due to the increase in the number of steps or stages, the reliability of the same is reduced. Last but not least, it is the high cost associated with the specific reagents and enzymes used in these methods.
En cuanto al otro de los aspectos importantes, la adecuada inmovilización de la sonda utilizada, en los métodos reportados se identifican algunas estrategias asociadas particularmente a sistemas complejos de inmovilización; al respecto, podemos mencionar el uso de pequeñas esferas magnéticas acopladas con estraptividina con el fin de atrapar aquellos vectores con inserto que hibridan con la sonda, que por lo general se encuentra marcada con biotina, Hawkins 1999, Li et al, 1995. Este sistema presenta varias desventajas, entre las que podemos mencionar: 1) no se puede trabajar con temperaturas elevadas que permitan una mayor especificidad en la hibridación (debido a que la temperatura afecta la interacción biotina-estraptividina), dando como resultado una selección de un alto número de DNAs con secuencias no relacionadas con la sonda, es decir de falsas clonas positivas; 2) es necesario contar con el equipo especializado para la recuperación de las esferas magnéticas. Como se puede apreciar, en estos métodos se obtienen bajas eficiencias de selección de DNAs y además resultan ser laboriosos y costosos.As for the other important aspects, the adequate immobilization of the probe used, in the reported methods some strategies are identified, particularly associated with complex immobilization systems; In this regard, we can mention the use of small magnetic spheres coupled with straptividin in order to trap those insert vectors that hybridize with the probe, which is usually marked with biotin, Hawkins 1999, Li et al, 1995. This system It has several disadvantages, among which we can mention: 1) it is not possible to work with elevated temperatures that allow a greater specificity in hybridization (because the temperature affects the biotin-straptividin interaction), resulting in a selection of a high number of DNAs with sequences not related to the probe, ie false positive clones; 2) it is necessary to have specialized equipment for the recovery of magnetic spheres. As can be seen, in these methods low efficiencies of DNA selection are obtained and also prove to be laborious and expensive.
Así pues, es clara la existencia de varios problemas técnicos o limitaciones en los métodos de selección de DNA reportados y en el "Kit" comercial basado en el método de "Gene Trapper", USP 5,898,071 , que de cierta manera impiden o limitan el desarrollo de la tecnología de DNA recombinante.Thus, it is clear that there are several technical problems or limitations in the methods of DNA selection reported and in the commercial "Kit" based on the method of "Gene Trapper", USP 5,898,071, which somehow prevent or limit the development of recombinant DNA technology.
Los inventores del presente método proponen una solución a estas limitaciones haciendo uso de ciertas propiedades del DNA y de las condiciones de su manipulación, que a diferencia de los métodos anteriormente descritos, no requieren de enzimas como las nucleasas (para generar cadenas sencillas), ni polimerasas (para regenerar la cadena complementaria), por lo que resulta un método, sencillo, rápido, eficiente, confiable, de bajo costo y con eficiente recuperación de clonas positivas y que además no requiere de equipos especiales para recuperar los vectores con inserto con secuencia homologa a la sonda. Este método comprende los siguientes pasos:The inventors of the present method propose a solution to these limitations by making use of certain DNA properties and the conditions of their manipulation, which, unlike the methods described above, do not require enzymes such as nucleases (to generate single chains), nor polymerases (to regenerate the complementary chain), so it is a method, simple, fast, efficient, reliable, low cost and efficient recovery of positive clones and also does not require special equipment to recover the vectors with insert with sequence probe homologue. This method comprises the following steps:
a) Obtención de los vectores con inserto de cadena sencilla y concatenados mediante su desnaturalización y prehibridación con el DNA del vector sonicado; b) por otro lado, la inmovilización de la sonda de DNA de interés en un soporte sólido y el bloqueo de la superficie libre del mismo; c) hibridación de los vectores con inserto de cadena sencilla (particularmente en la región del inserto) del banco con la sonda de DNA inmovilizada; d) recuperación de los vectores con inserto que hibridaron con la sonda por separación de la membrana; e) transformación de los vectores con inserto recuperados en las células donde puede replicarse para el posterior análisis de los DNAs.a) Obtaining the vectors with single chain insert and concatenated by denaturation and prehybridization with the DNA of the sonicated vector; b) on the other hand, the immobilization of the DNA probe of interest in a solid support and the blocking of the free surface thereof; c) hybridization of the vectors with single chain insert (particularly in the region of the insert) of the bank with the immobilized DNA probe; d) recovery of the insert vectors that hybridized with the probe by membrane separation; e) transformation of the vectors with insert recovered in the cells where it can be replicated for the subsequent analysis of the DNAs.
En la Figura No. 1 , se esquematiza el método rápido de selección de DNAs de la presente invención para el caso de un banco de cDNA. La obtención de los vectores con inserto de cadena sencilla se realiza mediante la desnaturalización y prehibridación de los vectores con inserto de doble cadena con el DNA del vector sonicado, separando las cadenas de los vectores con inserto, dando lugar a dos cadenas sencillas de cada vector con inserto concatenadas entre sí e hibridadas con los fragmentos del vector de elección lo que los mantendrá en esa forma. De esta manera, los insertos quedan expuestos como cadena sencilla para permitir la hibridación con la sonda específica.In Figure No. 1, the rapid method of DNA selection of the present invention is schematized for the case of a cDNA bank. Obtaining vectors with single chain insertion is done by denaturation and prehybridization of the vectors with double strand insert with the DNA of the sonicated vector, separating the chains from the vectors with insert, giving rise to two single strands of each vector with insert concatenated with each other and hybridized with the fragments of the vector of choice which It will keep them that way. In this way, the inserts are exposed as a single chain to allow hybridization with the specific probe.
Por otro lado y de manera independiente, se lleva a cabo la inmovilización de la sonda de interés, mediante su fijación y desnaturalización en un soporte y el bloqueo de la superficie libre del soporte, a fin de evitar que los vectores con inserto de cadena sencilla del banco se peguen de manera inespecífica al soporte.On the other hand and independently, the immobilization of the probe of interest is carried out, by means of its fixation and denaturation in a support and the blocking of the free surface of the support, in order to prevent vectors with single chain insert of the bank sticking unspecifically to the support.
Con los elementos antes referidos; se lleva a cabo la hibridación de la sonda inmovilizada con los vectores con inserto del banco de DNA a fin de seleccionar aquellos con el inserto de interés. Posteriormente los vectores con inserto seleccionados son recuperados y transformados en las células donde pueden replicarse para su posterior análisis.With the aforementioned elements; hybridization of the immobilized probe with the vectors with DNA bank insert is carried out in order to select those with the insert of interest. Subsequently, the selected insert vectors are recovered and transformed into the cells where they can be replicated for later analysis.
Esto es, las moléculas del vector con inserto que se seleccionan son aquellas que por tener un inserto con secuencia homologa y complementaria a la sonda inmovilizada hibridan con esta, mientras que aquellas moléculas cuyo inserto no tiene una secuencia homologa complementaria permanecen en solución. En el método de la presente invención, la recuperación de los vectores con inserto se realiza mediante la separación de la membrana en determinadas condiciones de temperatura en equipo convencional, lo cual representa una ventaja comparativa, con respecto a los métodos reportados.That is, the molecules of the vector with insert that are selected are those that by having an insert with homologous sequence and complementary to the immobilized probe hybridize with it, while those molecules whose insert does not have a complementary homologous sequence remain in solution. In the method of the present invention, the recovery of the vectors with insert is performed by separating the membrane under certain temperature conditions in conventional equipment, which represents a comparative advantage, with respect to the reported methods.
Con el método de la presente invención quedan resueltos los problemas técnicos o limitaciones que se presentan en los métodos existentes, como se indica a ° continuación:With the method of the present invention, the technical problems or limitations presented in the existing methods are resolved, as indicated below:
A) En el estado de la técnica, un factor limitante y determinante en los métodos para la purificación de DNAs, es la obtención de vectores con inserto de cadena sencilla, reportándose algunas alternativas, siendo la más común el utilizar enzimas específicas que degraden una sola cadena de DNA, no obstante, esto repercute directamente en un alto costo de los métodos que siguen esta estrategia.A) In the state of the art, a limiting and determining factor in the methods for the purification of DNAs is the obtaining of vectors with a single chain insert, some alternatives being reported, the most common being the using specific enzymes that degrade a single DNA chain, however, this directly impacts the high cost of the methods that follow this strategy.
Particularmente sobre esta limitación, los inventores proponen obtener la cadena sencilla de los vectores con inserto, mediante su desnaturalización e hibridación simultánea con los pequeños fragmentos del vector sonicado, para mantener separadas las cadenas a la vez que permanecen concatenadas. Con esta previa hibridación se evita que el DNA de cada vector con inserto pueda hibridar con la cadena complementaria del mismo, lo que resultaría en DNA de doble cadena o bien con otra molécula del vector con inserto lo que podría dar origen a una falsa clona positiva, evitando de esta manera recuperar cadenas múltiples que pudieran dar origen a clonas positivas falsas, por lo que el método resulta tener una alta eficiencia, de fácil implementación y sobre todo, de un costo relativamente bajo.Particularly on this limitation, the inventors propose to obtain the simple chain of the vectors with insert, by means of their denaturation and hybridization simultaneously with the small fragments of the sonicated vector, to keep the chains separated while remaining concatenated. With this previous hybridization it is avoided that the DNA of each vector with insert can hybridize with the complementary chain of the same, which would result in double-stranded DNA or with another molecule of the vector with insert which could give rise to a false positive clone , thus avoiding recovering multiple chains that could give rise to false positive clones, so that the method turns out to have a high efficiency, of easy implementation and, above all, of a relatively low cost.
Por otro lado, uno de los factores determinantes para obtener una transformación eficiente en las células de E colies el tipo de DNA que se va a transformar. Para esto, es necesario introducir DNA de vectores de doble cadena ya que el DNA de cadena sencilla es degradado en el interior de la célula. En este sentido una ventaja adicional del método de la presente invención, es que para obtener al final DNA de vectores de doble cadena, no es necesario regenerar una de las cadenas, debido a que durante todo el proceso, las dos cadenas de DNA permanecen unidas de manera concatenada.On the other hand, one of the determining factors to obtain an efficient transformation in the cells of E colies the type of DNA to be transformed. For this, it is necessary to introduce DNA from double stranded vectors since single stranded DNA is degraded inside the cell. In this sense, an additional advantage of the method of the present invention is that in order to obtain DNA from double stranded vectors at the end, it is not necessary to regenerate one of the chains, because during the whole process, the two DNA chains remain attached concatenated.
Con relación al otro aspecto limitante en los métodos reportados en la selección de DNAs, referente a la manera de inmovilización de la sonda, los inventores proponen la utilización de un soporte sólido cuyas características permitan manipular las condiciones de reacción, particularmente el uso de altas temperaturas que favorezcan la inmovilización del DNA que es utilizado como sonda y que al final de la hibridación facilite la separación de los vectores con inserto de cadena sencilla que hibridaron con la sonda de interés sin que sea requerido un intermediario, mientras que retiene la sonda. Por otro lado, también se contempla el bloqueo de la superficie libre del soporte (aquella en la que no se pegó la sonda) para evitar que los DNAs del banco se peguen de manera inespecífica al soporte, logrando con ello, incrementar sustancialmente la eficiencia y especificidad del método de la presente invención.Regarding the other limiting aspect in the methods reported in the selection of DNAs, referring to the way of immobilization of the probe, the inventors propose the use of a solid support whose characteristics allow manipulating the reaction conditions, particularly the use of high temperatures that favor the immobilization of the DNA that is used as a probe and that at the end of the hybridization facilitate the separation of the vectors with single chain insert that hybridized with the probe of interest without requiring an intermediary, while retaining the probe. On the other hand, the blocking of the free surface of the support (the one in which the probe was not glued) is also contemplated to prevent the bank's DNAs from nonspecifically sticking to the support, thereby achieving, substantially increasing efficiency and specificity of the method of the present invention.
Si bien existen diferentes tipos de soportes, los inventores proponen utilizar un soporte que cumpla con las características antes descritas y sobre todo que pueda manipularse a altas temperaturas; a fin de lograr una mayor especificidad en la hibridación; en este caso, sugieren utilizar preferentemente membrana de nitrocelulosa, obteniéndose mejores resultados, si la membrana de nitrocelulosa se encuentra cargada positivamente.Although there are different types of supports, the inventors propose using a support that meets the characteristics described above and especially that can be handled at high temperatures; in order to achieve greater specificity in hybridization; in this case, they suggest using nitrocellulose membrane preferably, obtaining better results, if the nitrocellulose membrane is positively charged.
Adicionalmente, con relación a la sonda de DNA de interés que se inmoviliza en el soporte, el método de la presente invención permite el uso de sondas de diferente tamaño, es decir, puede utilizarse como sonda un oligonucleótido, un gen completo o un polinucleótido.Additionally, in relation to the DNA probe of interest that is immobilized in the support, the method of the present invention allows the use of probes of different size, that is, an oligonucleotide, a complete gene or a polynucleotide can be used as a probe.
Por otro lado, como es sabido, el DNA de cadena sencilla, es una macromolécula formada por un gran número de deoxirribonucleótidos que pueden unirse a otra macromolécula de DNA con secuencia complementaria para formar el DNA de doble cadena o bien permanecer como una macromolécula de cadena sencilla. En este sentido, una de las ventajas de la presente invención es que mediante el presente método puede ser seleccionado DNA de doble cadena, sin que sea necesario utilizar enzimas para obtener cadenas sencillas.On the other hand, as is known, single-stranded DNA is a macromolecule formed by a large number of deoxyribonucleotides that can bind to another DNA macromolecule with complementary sequence to form the double-stranded DNA or remain as a chain macromolecule. simple. In this sense, one of the advantages of the present invention is that by means of the present method, double stranded DNA can be selected, without the need to use enzymes to obtain single strands.
En el estado de la técnica, es conocido que la desnaturalización del DNA es el proceso por el cual se pueden romper los puentes de hidrógeno que unen a la doble cadena de DNA y separar las dos cadenas de DNA para obtener dos cadenas sencillas de DNA (que en el caso de DNA circular, permanecerán concatenadas). Entre los métodos más comunes para llevar a cabo la desnaturalización del DNA, podemos mencionar la adición de ácidos; la adición de álcalis como el hidróxido de sodio o bien mediante un tratamiento térmico, utilizando temperaturas que varían de 72 a 105 °C.In the state of the art, it is known that DNA denaturation is the process by which the hydrogen bonds that bind the double strand of DNA can be broken and the two strands of DNA separated to obtain two single strands of DNA ( that in the case of circular DNA, they will remain concatenated). Among the most common methods for carrying out DNA denaturation, we can mention the addition of acids; the adition of alkalis such as sodium hydroxide or by heat treatment, using temperatures ranging from 72 to 105 ° C.
Asimismo, también es conocido en el estado de la técnica que para llevar a cabo una hibridación, es necesario primero desnaturalizar o separar las cadenas de DNA para favorecer la exposición de los posibles sitios de hibridación; y posteriormente revertir las condiciones desnaturalizantes en presencia de la sonda de interés para propiciar su unión a una de las cadenas con secuencia complementaria y homologa a la secuencia de la sonda utilizada.Likewise, it is also known in the state of the art that in order to carry out a hybridization, it is first necessary to denature or separate the DNA chains to favor the exposure of the possible hybridization sites; and subsequently reverse the denaturing conditions in the presence of the probe of interest to promote its binding to one of the chains with complementary sequence and homologous to the sequence of the probe used.
El método de la presente invención contempla la desnaturalización del DNA de los vectores con inserto, en un mismo paso, con la prehibridación con el vector sonicado, que puede ser realizada por cualquiera de los métodos de desnaturalización antes mencionados, es decir mediante el manejo de pH o de temperatura; particularmente los inventores sugieren que esta desnaturalización y prehibridación se lleve a cabo mediante un tratamiento térmico de dos pasos, que consiste en el calentamiento de los vectores con inserto en presencia del vector sonicado hasta la temperatura de desnaturalización de 72 a 105 °C, durante al menos 5 minutos, posteriormente se realiza un ligero enfriamiento o disminución de la temperatura, para mantener esta mezcla a la temperatura de hibridación de 42 a 65 °C por un mínimo de 5 minutos; y finalmente se deja enfriar a temperatura ambiente. Cabe señalar que el tratamiento térmico tiene ventajas comparativas con respecto a los métodos de manipulación de pH, ya que resulta ser un tratamiento sencillo, rápido y no es necesario utilizar reactivos adicionales que pudieran interferir con las hibridaciones que se llevan a cabo en el método de la presente invención.The method of the present invention contemplates the denaturation of the DNA of the vectors with insert, in the same step, with the prehybridization with the sonicated vector, which can be performed by any of the aforementioned denaturation methods, that is by means of the handling of pH or temperature; In particular, the inventors suggest that this denaturation and prehybridization be carried out by means of a two-step heat treatment, which consists of heating the inserted vectors in the presence of the sonicated vector to the denaturation temperature of 72 to 105 ° C, during minus 5 minutes, subsequently a slight cooling or decrease of the temperature is carried out, to keep this mixture at the hybridization temperature of 42 to 65 ° C for a minimum of 5 minutes; and finally allowed to cool to room temperature. It should be noted that the heat treatment has comparative advantages with respect to pH manipulation methods, since it turns out to be a simple, fast treatment and it is not necessary to use additional reagents that could interfere with the hybridizations that are carried out in the method of The present invention.
Otra de las ventajas del método de la presente invención, es que dado que las propiedades y condiciones de manipulación del DNA son universales y no específicos para determinados insertos, sondas o vectores de elección, se contempla la posibilidad de utilizar los diferentes vectores que comúnmente son usados en los bancos de genes de células procarióticas, como por ejemplo pCP13 y de células eucarióticas tales como: λgt11 para bancos genómicos y λZAP para bancos de cDNA. E J E M P L O SAnother advantage of the method of the present invention is that since the DNA handling properties and conditions are universal and not specific for certain inserts, probes or vectors of choice, the possibility of using the different vectors that are commonly used used in gene banks of prokaryotic cells, such as pCP13 and eukaryotic cells such as: λgt11 for genomic banks and λZAP for cDNA banks. EXAMPLES
A fin de ilustrar la aplicación del método de la presente invención para la rápida selección de DNAs, a continuación se describen algunos ejemplos, mismos que no deberán considerarse como limitantes de su alcance.In order to illustrate the application of the method of the present invention for the rapid selection of DNAs, some examples are described below, which should not be considered as limiting their scope.
Ejemplo No.1 Se ilustra la aplicación del método de la presente invención en la selección de cDNA que codifica para la síntesis de una proteína cinasa tipo receptor.Example No.1 The application of the method of the present invention in the selection of cDNA coding for the synthesis of a receptor-like protein kinase is illustrated.
Para aislar el cDNA de interés se partió de un banco de expresión de doble cadena clonados en el vector λ-ZAP de nodulos de frijol de 13 días y se le aplicó el método de la presente invención de la siguiente manera:To isolate the cDNA of interest, a double-stranded expression bank cloned into the 13-day bean λ-ZAP vector was started and the method of the present invention was applied as follows:
1.- Desnaturalización y prehibridación del banco de cDNA. Calentamiento a 95°C durante 5 min de 500 μl del banco de cDNA de doble cadena (banco de expresión de nodulos de frijol de 13 días) en presencia de 10 μ I («10 μg de vector) de DNA del vector previamente sonicado ( mediante cinco ciclos de 20 segundos a 10 watts), permitiendo enfriar a la temperatura de hibridación (50 °C).1.- Denaturation and prehybridization of the cDNA bank. Heating at 95 ° C for 5 min of 500 μl of the double-stranded cDNA bank (13-day bean nodule expression bank) in the presence of 10 μ I (“10 μg vector) of DNA from the previously sonicated vector ( by five cycles of 20 seconds at 10 watts), allowing to cool to the hybridization temperature (50 ° C).
2.- Inmovilización de la sonda en la membrana y su bloqueo. a) Colocar 1-2 μl de sonda de interés («1-2 μg), en este caso un fragmento del cDNA de una proteína cinasa del tipo receptor, en 1 c 2 de membrana de Nylon cargada positivamente (Hybon N+; Amershan Pharmacia Biotech UK Ltd. b) Fijar y desnaturalizar la sonda en la membrana con 500 μl de 400 mM de NaOH. c) Adicionar 5 mi de una solución de fosfatos (300 mM NaH2 PO 4, 7%2.- Immobilization of the probe in the membrane and its blockage. a) Place 1-2 μl of the probe of interest («1-2 μg), in this case a cDNA fragment of a receptor-like protein kinase, in 1 c 2 of positively charged Nylon membrane (Hybon N + ; Amershan Pharmacia Biotech UK Ltd. b) Fix and denature the probe in the membrane with 500 μl of 400 mM NaOH. c) Add 5 ml of a phosphate solution (300 mM NaH 2 PO 4 , 7%
SDS, 1 mM EDTA) para bloquear la superficie libre del soporte mediante calentamiento a una temperatura de 50 °C por un tiempo de 5 min; 3.- Hibridación con la sonda inmovilizada. a) Incubar una solución con los vectores con inserto en presencia de la sonda inmovilizada a una temperatura de 50 °C por 8 h, dependiendo del porcentaje de identidad de las secuencias de DNA con la sonda de interés (NOTA: A mayor porcentaje de identidad mayor temperatura de hibridación); b) Incubar la membrana con 5 mi de la solución de lavado (600mMSDS, 1 mM EDTA) to block the free surface of the support by heating at a temperature of 50 ° C for a time of 5 min; 3.- Hybridization with the immobilized probe. a) Incubate a solution with the inserted vectors in the presence of the immobilized probe at a temperature of 50 ° C for 8 h, depending on the percentage of identity of the DNA sequences with the probe of interest (NOTE: A higher percentage of identity higher hybridization temperature); b) Incubate the membrane with 5 ml of the wash solution (600mM
NaCI, 60 mM citrato de sodio; 1% SDS) durante 10 min a la temperatura de hibridación, repetir este paso tres veces. 4.- Recuperación de los vectores con el inserto de interés. a) Incubar la membrana con 500 μl de H20 a 95 °C durante 5 min y retirar la membrana a esta temperatura. b) Recuperar el sobrenadante y precipitar con 300 mM de acetato de sodio y dos volúmenes de etanol.NaCI, 60 mM sodium citrate; 1% SDS) for 10 min at the hybridization temperature, repeat this step three times. 4.- Recovery of the vectors with the insert of interest. a) Incubate the membrane with 500 μl of H 2 0 at 95 ° C for 5 min and remove the membrane at this temperature. b) Recover the supernatant and precipitate with 300 mM sodium acetate and two volumes of ethanol.
10.- Secar y resuspender en 20 μl de H20. 5.- Tansformación con los 20 μl de los vectores con inserto recuperados, en las células electrocomponentes de E. coli XL1 Blue (preparadas como recomienda el proveedor del electroporador, Bio Rad Laboratories), para la amplificación de los vectores con inserto y el posterior análisis.10.- Dry and resuspend in 20 μl of H 2 0. 5.- Transformation with the 20 μl of the vectors with insert recovered, in the electrocomponent cells of E. coli XL1 Blue (prepared as recommended by the electroporator supplier, Bio Rad Laboratories), for the amplification of the vectors with insert and the subsequent analysis.
Ejemplo No. 2Example No. 2
En este ejemplo se muestra la eficiencia de la selección de DNA de interés de un banco de expresión de nodulos de frijol de 13 días, mediante el método rápido de selección de DNAs de la presente invención.This example shows the efficiency of the selection of DNA of interest from a 13-day bean nodule expression bank, by the rapid method of DNA selection of the present invention.
Después de utilizar la metodología descrita en el ejemplo 1 , se obtuvieron 100 clonas positivas, de las cuales se seleccionaron 35 al azar. A estas clonas se les extrajo el DNA, posteriormente fueron separadas en un gel de agarosa y transferidos a una membrana y se hibridaron con la misma sonda utilizada para la selección pero en solución (marcada con P32). Se expusieron en una película de rayos X. Los resultados obtenidos se ilustran en la figura 2. Como se puede observar, con el método de la presente invención se obtiene que 28 de las 35 clonas (80%) hibridan con la sonda utilizada en la selección.After using the methodology described in example 1, 100 positive clones were obtained, of which 35 were randomly selected. These clones were extracted with DNA, subsequently separated on an agarose gel and transferred to a membrane and hybridized with the same probe used for selection but in solution (labeled with P 32 ). They were exposed in an X-ray film. The results obtained are illustrated in Figure 2. As can be seen, with the method of the present invention it is obtained that 28 of the 35 clones (80%) hybridize with the probe used in the selection.
Ejemplo 3Example 3
En este ejemplo se muestra la identidad a nivel de secuencia de nucleótidos de los DNAs seleccionados con la sonda utilizada en el Método Rápido de Selección de DNA de la presente invención.In this example, the nucleotide sequence identity of the selected DNAs is shown with the probe used in the Rapid DNA Selection Method of the present invention.
Se siguió la metodología del ejemplo 2. Se seleccionaron los 3 DNA que hibridaron en mayor proporción con la sonda y se secuenciaron con el "Kit" Big Dye™ Terminator Cycle sequencing Ready (PE Applied Biosystems Perkin Elmer) y las reacciones se leyeron en un secuenciador automático ABI PRIMS Modelo 377 DNA sequence.The methodology of example 2 was followed. The 3 DNAs that hybridized in greater proportion to the probe were selected and sequenced with the "Big Dye ™ Terminator Cycle sequencing Ready" Kit (PE Applied Biosystems Perkin Elmer) and the reactions were read in a ABI PRIMS Model 377 DNA sequence automatic sequencer.
Tabla ITable I
Figure imgf000016_0001
Figure imgf000016_0001
En la tabla I se muestra el porcentaje de identidad de la secuencia de nucleótidos del cDNA de la clona pKG3 utilizada como sonda, con las secuencia de 3 de los cDNAs que se seleccionaron por el Método de la presente invención ( pKG 1 , pKG 8 y pKG 13).Table I shows the percent identity of the nucleotide sequence of the cDNA of clone pKG3 used as a probe, with the sequence of 3 of the cDNAs that were selected by the Method of the present invention (pKG 1, pKG 8 and pKG 13).
Ejemplo 4 En este ejemplo se ilustra la aplicación industrial del Método Rápido de Selección de DNA, mediante la integración de un estuche basado en el método de la presente invención. COMPONENTES.Example 4 This example illustrates the industrial application of the Rapid DNA Selection Method, by integrating a case based on the method of the present invention. COMPONENTS
Solución I: Solución de prehibridación (1 μg/μl de DNA del vector sonicado). Solución II: Solución fijadora de DNA (NaOH 0.4 N) Solución III: Solución de lavado. Solución IV: Solución de hibridación. Solución V: Solución de elusión. Solución VI: Solución de precipitación de DNA. Un juego de membranas de Nylon cargada positivamente de 1cm2.Solution I: Prehybridization solution (1 μg / μl of sonic vector DNA). Solution II: DNA fixing solution (NaOH 0.4 N) Solution III: Washing solution. Solution IV: Hybridization solution. Solution V: Solution of circumvention. Solution VI: DNA precipitation solution. A set of positively charged Nylon membranes of 1cm 2 .
MATERIAL Y EQUIPO ADICIONALMATERIAL AND ADDITIONAL EQUIPMENT
Baño de 95 °C.95 ° C bath.
Horno de hibridación.Hybridization oven.
Células electrocompetentes.Electrocompetent cells
Banco de DNA en vectores de doble cadena. (NOTA: El vector debe ser el mismo que el vector sonicado de la solución I)DNA bank in double stranded vectors. (NOTE: The vector must be the same as the sonic vector of solution I)
Sonda de interés.Probe of interest
PROTOCOLOPROTOCOL
1 ) Mezclar 500 μl del banco de DNA con 50 μl de la Solución I. 2) Calentar la mezcla a 95 °C durante 5 minutos.1) Mix 500 μl of the DNA bank with 50 μl of Solution I. 2) Heat the mixture at 95 ° C for 5 minutes.
3) Dejar enfriar a temperatura de hibridación ( 42 a 65 °C) durante 10 minutos.3) Allow to cool to hybridization temperature (42 to 65 ° C) for 10 minutes.
4) Colocar los μl equivalentes a 1-2μg de la sonda de DNA de interés en la membrana de Nylon, previamente hidratada con la Solución II.4) Place the μl equivalent to 1-2μg of the DNA probe of interest on the Nylon membrane, previously hydrated with Solution II.
5) Lavar durante 10 minutos con 5 mi de la Solución III. 6) Incubar la membrana con 1 ml de la Solución IV durante 10 minutos a la temperatura de hibridación requerida.5) Wash for 10 minutes with 5 ml of Solution III. 6) Incubate the membrane with 1 ml of Solution IV for 10 minutes at the required hybridization temperature.
NOTA: La temperatura de hibridación dependerá de la identidad de la secuencia de DNA de la sonda y el gen que se desea seleccionar. Para una sonda homologa se recomienda una temperatura de 65 °C. Para una sonda con bajo % identidad se puede bajar la temperatura hasta 42 °C. 7) Mezclar el banco de cDNA prehibridado con 5 ml de la Solución de hibridación e incubar a la temperatura de hibridación durante 1-8 horas.NOTE: The hybridization temperature will depend on the identity of the probe's DNA sequence and the gene to be selected. A temperature of 65 ° C is recommended for a homologous probe. For a probe with low% identity the temperature can be lowered to 42 ° C. 7) Mix the prehybridized cDNA bank with 5 ml of the Hybridization Solution and incubate at the hybridization temperature for 1-8 hours.
8) Lavar la membrana con 5 ml de la Solución III durante 15 minutos a la misma temperatura de hibridación. 9) Repetir este paso dos veces más.8) Wash the membrane with 5 ml of Solution III for 15 minutes at the same hybridization temperature. 9) Repeat this step two more times.
10) Retirar la membrana y colocarla en un tubo de microcentrífuga de 1.5 ml y adicionarle 500 μl de la Solución V.10) Remove the membrane and place it in a 1.5 ml microcentrifuge tube and add 500 μl of Solution V.
11) Calentar el tubo a 95 °C durante 5 minutos e inmediatamente retirar la membrana del tubo de microcentrífuga. PRECAUCIÓN: No quitar la membrana cuando la temperatura este por debajo de los 95 °C.11) Heat the tube at 95 ° C for 5 minutes and immediately remove the membrane from the microcentrifuge tube. CAUTION: Do not remove the membrane when the temperature is below 95 ° C.
12) Adicionar 1 ml de la Solución VI al sobrenadante.12) Add 1 ml of Solution VI to the supernatant.
13) Centrifugar durante 10 minutos a 13,000 rpm y resuspender en 10 ml de la Solución Vil.13) Centrifuge for 10 minutes at 13,000 rpm and resuspend in 10 ml of the Vil Solution.
14) Transformar en células preparadas para electroporación. 15) Plaquear en cajas de LB con el antibiótico de selección del vector utilizado para hacer la librería.14) Transform into cells prepared for electroporation. 15) Plate in LB boxes with the vector selection antibiotic used to make the library.
R E F E R E N C I A SREFERENCES
Ito, T., Smith, C.L. & Cantor, Ch. R. (1992) Sequence-specific DNA purification by triplex affinity capture. Proc. Nati. Acad. Sci. 89, 495 - 498.Ito, T., Smith, C.L. & Cantor, Ch. R. (1992) Sequence-specific DNA purification by triplex affinity capture. Proc. Nati Acad. Sci. 89, 495-498.
Pruitt, S.C. (1988) Expression vectors permitting cDNA cloning and enrichment for specific sequences by hybridization/selection. Gene 66, 121 - 134.Pruitt, S.C. (1988) Expression vectors permitting cDNA cloning and enrichment for specific sequences by hybridization / selection. Gene 66, 121-134.
Hawkins, T. (1999) US Pat. No. 5,898.071 DNA purificación and isolation using magnetic particles.Hawkins, T. (1999) US Pat. No. 5,898,071 DNA purification and isolation using magnetic particles.
Rigas, B., Welcher A.A., Ward, D.C. & Weissman, S.M. (1986) Rapid plasmid library screening using RecA-coated biotinylated probes. Proc. Nati. Acad. Sci. 83: 9591-9595.Rigas, B., Welcher A.A., Ward, D.C. & Weissman, S.M. (1986) Rapid plasmid library screening using RecA-coated biotinylated probes. Proc. Nati Acad. Sci. 83: 9591-9595.
The Rapid Isolation of Specific Genes From CDNA Libraries with the GeneTrapper cDNA Positive Selection System The Rapid Isolation of Specific Genes From CDNA Libraries with the GeneTrapper cDNA Positive Selection System

Claims

R E I V I N D I C A C I O N E SHabiendo descrito la invención se reclama lo contenido en las siguientes reivindicaciones: CLAIMING Having described the invention, the content of the following claims is claimed:
1.- Un método rápido de selección de DNA que comprende los siguientes pasos:1.- A quick method of DNA selection that includes the following steps:
a) Obtención de los vectores con inserto de cadena sencilla y concatenados mediante su desnaturalización y prehibridación con el DNA del vector sonicado; b) por otro lado, la inmovilización de la sonda de DNA de interés en un soporte sólido que permita manipular las condiciones de reacción y el bloqueo de la superficie libre del mismo; c) hibridación de los vectores con inserto de cadena sencilla del banco con la sonda de DNA inmovilizada; d) recuperación de los vectores con inserto que hibridaron con la sonda por separación de la membrana; e) transformación de los vectores con inserto recuperados, en las células donde puede replicarse para el posterior análisis de los DNAs.a) Obtaining the vectors with single chain insert and concatenated by denaturation and prehybridization with the DNA of the sonicated vector; b) on the other hand, the immobilization of the DNA probe of interest in a solid support that allows manipulating the reaction conditions and blocking the free surface thereof; c) hybridization of the vectors with single chain insert of the bank with the immobilized DNA probe; d) recovery of the insert vectors that hybridized with the probe by membrane separation; e) transformation of the vectors with insert recovered, in the cells where it can be replicated for the subsequent analysis of the DNAs.
2.- Método rápido de selección de conformidad con la reivindicación 1 , caracterizado porque el DNA puede ser de doble cadena.2. Fast selection method according to claim 1, characterized in that the DNA can be double stranded.
3.- Método rápido de selección de conformidad con la reivindicación 1 , caracterizado porque la desnaturalización y prehibridación con el DNA del vector sonicado para la obtención de los vectores con inserto de cadena sencilla concatenados se lleva a cabo mediante cualquiera de los siguientes tratamientos: térmico, adición de soluciones alcalinas o de soluciones acidas.3. Rapid selection method according to claim 1, characterized in that denaturation and prehybridization with the DNA of the sonicated vector for obtaining the vectors with concatenated single chain insert is carried out by any of the following treatments: thermal , addition of alkaline solutions or acid solutions.
4.- Método rápido de selección de conformidad con la reivindicación 3, caracterizado porque la desnaturalización y prehibridación del DNA con el vector sonicado se llevan a cabo mediante tratamientos térmicos. 4. Quick selection method according to claim 3, characterized in that the denaturation and prehybridization of the DNA with the sonic vector are carried out by thermal treatments.
5.- Método rápido de selección de conformidad con la reivindicación 4, caracterizado porque el tratamiento térmico para la desnaturalización del DNA se realiza en un rango de temperatura de 62 a 105 °C por un tiempo de 3 a 15 min.5. Quick selection method according to claim 4, characterized in that the heat treatment for DNA denaturation is carried out in a temperature range of 62 to 105 ° C for a time of 3 to 15 min.
6.- Método rápido de selección de conformidad con la reivindicación 1 , caracterizado porque los vectores utilizados pueden ser plásmidos o fagos de doble cadena.6. Fast selection method according to claim 1, characterized in that the vectors used can be plasmids or double stranded phage.
7.- Método rápido de selección de conformidad con la reivindicación 1 , caracterizado porque la sonda de DNA de interés que se inmoviliza en el soporte puede ser un oligo, un gen o un polinucleótido.7. Quick selection method according to claim 1, characterized in that the DNA probe of interest that is immobilized on the support can be an oligo, a gene or a polynucleotide.
8.- Método rápido de selección de conformidad con la reivindicación 1 , caracterizado porque el soporte en el que se inmoviliza la sonda permite el uso de altas temperaturas; favorece la inmovilización de la sonda y al final de la hibridación, facilita el desprendimiento de los insertos que hibridaron con la sonda.8.- Quick selection method according to claim 1, characterized in that the support in which the probe is immobilized allows the use of high temperatures; it favors the immobilization of the probe and at the end of the hybridization, it facilitates the detachment of the inserts that hybridized with the probe.
9.- Método rápido de selección de conformidad con la reivindicación 8, caracterizado porque el soporte es una membrana de nitrocelulosa.9. Quick selection method according to claim 8, characterized in that the support is a nitrocellulose membrane.
10.- Método rápido de selección de conformidad con la reivindicación 8, caracterizado porque el soporte es una membrana de nitrocelulosa cargada positivamente.10. Quick selection method according to claim 8, characterized in that the support is a positively charged nitrocellulose membrane.
11.- Método rápido de selección de conformidad con la reivindicación 1 , caracterizado porque los insertos recuperados son aquellos que contienen un DNA con un alto porcentaje de identidad con la secuencia de la sonda.11.- Quick selection method according to claim 1, characterized in that the recovered inserts are those that contain a DNA with a high percentage of identity with the probe sequence.
12.- Estuche para la selección rápida de DNA basado en método de la reivindicación 1.12. Case for rapid DNA selection based on the method of claim 1.
13.- Estuche para la selección rápida de DNA de conformidad con la reivindicación 12, que comprende los siguientes elementos:13. Case for the rapid selection of DNA according to claim 12, comprising the following elements:
" Solución I: Solución de prehibridación (1 μg/μl de DNA del vector sonicado). Solución II: Solución fijadora de DNA (NaOH 0.4 N)"Solution I: Prehybridization solution (1 μg / μl of sonic vector DNA). Solution II: DNA fixation solution (0.4 N NaOH)
Solución III: Solución de lavado.Solution III: Wash solution.
Solución IV: Solución de hibridación.Solution IV: Hybridization solution.
Solución V: Solución de elusión.Solution V: Solution of circumvention.
Solución VI: Solución de precipitación de DNA.Solution VI: DNA precipitation solution.
Un juego de membranas de Nylon cargada positivamente de 1 cm2. A set of positively charged Nylon membranes of 1 cm 2 .
PCT/MX2002/000050 2002-06-05 2002-06-05 Rapid dna selection method WO2003104450A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5473060A (en) * 1993-07-02 1995-12-05 Lynx Therapeutics, Inc. Oligonucleotide clamps having diagnostic applications

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5473060A (en) * 1993-07-02 1995-12-05 Lynx Therapeutics, Inc. Oligonucleotide clamps having diagnostic applications

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ROCKMAN ET AL., AUSTRALIAN JOURNAL OF MEDICAL SCIENCE, vol. 15, May 1994 (1994-05-01), pages 56 - 57, XP002960012 *
SHAHJAHAN A. ET AL., PLANT MOLECULAR BIOLOGY REPORTER, vol. 18, 2000, pages 123 - 132, XP002960013 *

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