WO2003104450A1 - Rapid dna selection method - Google Patents

Rapid dna selection method Download PDF

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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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dna
solution
probe
insert
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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    • 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

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

RAPID METHOD FOR SELECTING DNAs.

TECHNICAL FIELD OF THE INVENTION

The invention object of the present relates to a method of rapid screening of DNA, which involves the manipulation of certain properties of DNA and the necessary conditions for hybridization, unlike some existing methods do not require the use of enzymes, complex systems or immobilization of the probe of interest, so it turns out to be simple, fast, reliable, inexpensive method efficient for recovering positive clones.

BACKGROUND

The construction of cDNAs or genomic banks is obtained by a process of ligating the fragments of interest in a specific vector. One of the main problems in banks is able to obtain a good representation of all of the organism's genes or all genes that are being expressed in cells that were chosen for the construction of genome cDNA bank. The representativeness of the bank is very important to achieve isolation of cDNAs that have low expression or genes found in low copy number in the genome of the organism of interest.

The DNA of higher organisms is very complex: for example, a haploid mammalian genome contains approximately 3 x 10 9 - bp. As a fragment of approximately 3000 bp of interest comprises only one part per million of a preparation of genomic DNA. Clearly the main problem in generating a recombinant DNA library either genomic or cDNA is creating representative population of clones needed to ensure that the library contains at least one version of each sequence of interest. Solutions to this problem are basically similar to genomic and cDNA libraries. Generally, fragments of genomic DNA or cDNA, are prepared by first insertion into the chosen vector. The vector and DNA are desired ligated and introduced into E. coli cells, either in vitro packaging into phage λ capsid or by direct transformation. In some aspects, strategies for isolation of individual genomes or cDNA clones may be a little different.

Most selection processes involve cDNA libraries positive identification of cDNA clones of either antibody or hybridization with nucleic acid probes. A particular application with selection methods of DNA, are subtracted cDNA libraries, which provide a method for identifying mRNAs (by managing the respective cDNAs) that are differentially expressed in two different tissues. In this method cDNAs of the tissues they are synthesized and hybridized with cDNAs of other tissue, for those cDNAs preferentially expressed in one of them. Sequences not hybridized are cloned in a bacteriophage or a plasmid to obtain a cDNA library subtracted. It is difficult to estimate the number of clones that must be screened, as this will vary with the type of cell and gene to be identified.

The most common method for cloning cDNAs from a library is in situ hybridization of lytic plaques with a probe homologous or marked heterologous with 32 P. Usually this technique is performed in bacteriophages that were previously grown on agar plates and transferred a nitrocellulose membrane. However, to perform search of a cDNA of interest is necessary to conduct a survey of 200.000 to 400.000 plaque forming units (PFU) to have represented the total of approximately 30,000 transcripts per cell. On the other hand it is required at least three rounds of purification for individual lytic plaques containing one cDNA of interest.

In recent years there have been reported some methods that allow rapid cloning of cDNAs gene banks, then some of them are listed.

In 1986, Rigas et al. They reported a method based on: 1) the ability of a protein to form stable complexes between the DNA molecules of linear single-stranded and double-stranded circular share sequence identity; and 2) a process which allows the isolation of biotinylated nucleic acids. That is, the protein coated with biotinylated DNA probe single chain becomes plasmids hybridizing to select, for complexing triplex with plasmids having sequences homologous to the probe. These complexes are then purified by column of immobilized streptavidin agarose or cupric iminodiacetic acid. In this method a recovery between 10 and 20% of the plasmids of interest is reported. As shown, one of the disadvantages of this method is the low percentage recovery of positive clones, that is inter alia, purification steps are needed, another disadvantage is the high cost of the method, since it is They are using specific reagents high quality.

Furthermore, in 1992 Ito et al., Published a method of isolating DNA by forming triple-helix 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 coated magnetic beads with streptavidin and recovered in double stranded form by elution with mild alkaline buffer destabilizes the triple helix. The authors demonstrated the effectiveness of this procedure in an experimental model with an artificially and also reconstructed by isolating the polidinucleótidos (dT-dC) n '(dG -da) n of a library of the human genome library. Although not reported recoveries of clones, according to the authors, the advantage of this procedure is that the filtration steps which are required in other methodologies are minimized, however, it proves to be a relatively expensive method since it requires equipment or special system to trap small magnetic spheres and similarly specific reagents that increase their cost are used.

In 1995, Li et al., Published the system called GeneTrapper. In this system it is part of a complex population of double-stranded plasmids containing the cDNA inserts. These double-stranded plasmids are converted to single-stranded DNA degradation of one strand of DNA via enzymatic reactions, then the plasmids containing the DNA of interest by solution hybridization with an oligonucleotide probe selected biotinylated complementary the sequence of interest. Hybrids are captured in small magnetic beads covered with streptavidin. With a magnet the magnetic fields of the solution are recovered, leaving it the single stranded DNA (ssDNA) unhybridized themselves to be discarded. Subsequently, the ssDNA of interest are released from the biotinylated oligonucleotide. After released, the cDNAs were added is an oligonucleotide (primer) in vltro for complementary strand synthesis. Subsequently, competent cells were transformed and plated. With this method they recover 20% to 100% of colonies with the plasmid containing the cDNA of interest. As shown, this method has several disadvantages, first, the recovery percentage of clones of interest is very variable, making it to be unreliable, secondly, to obtain a population of single strands it is necessary to use enzymes that degrade the DNA and thirdly, it requires a special device to catch small magnetic fields. Based on the foregoing, the procedure turns out to be laborious and relatively high cost.

At present recombinant DNA technology is widely used in the area of ​​medicine, industry and agriculture. For example, it has been used to generate vaccines for different diseases, obtaining bacterial strains recombinant improve the quality and / or quantity of a certain product of industrial interest, the generation of transgenic plants which can obtain plants with higher resistance to certain pathogens, etc. This technology has made great strides in recent years through the development of methodologies that have enabled isolate, analyze, manipulate and control the genes of interest. However, one of the limiting steps in the generation of genetically modified organisms today is the isolation of genes of interest, which is achieved with the use of techniques of gene selection of a genomic or expression.

DETAILED DESCRIPTION OF THE INVENTION

BRIEF descricion OF THE FIGURES Figure 1 Illustrates the rapid selection method DNAs of the present invention, in the case of an expression library. (T) represents the different vectors with insert a cDNA double stranded. (1) Preparation of vectors with single strand insert concatenated by denaturation (2) Fragmentation of the cloning vector by sonication; (3) prehybridization with vector DNA sonicated with the vector with insert concatenated single chain; (4) Immobilization of probe DNA of interest into a nylon membrane and blocking the free surface thereof; (5) hybridizing vectors with single strand insert, particularly in the region of the insert, the Bank with the DNA probe immobilized; for 8 to 10 h; (6) Washing with 2X SSC to remove the cDNA did not hybridize; (7) Heat treatment and membrane separation to recover the vectors with insert which hybridized with the probe; (8) transforming the vectors with insert recovered in cells where the vector can replicate (9) Purification of plasmids and hybridization with the probe used in the selection for further analysis of the DNAs.

Figure 2 illustrates the cDNA cloned by the method of the present invention. The recovered DNAs (step 7 of Figure 1), were transformed, purified and digested with restriction enzyme EcoR the \ (lanes 2-24 and 27-38). The digestion products were separated on agarose gel 1%, transferred to a membrane of Hybond C-extra and hybridized with the probe used in step 3 of Figure 1 (in this case the cDNA of the cloned pKG3 ) marked by random priming with 32 P. in lanes 1, 25 and 26 the probe DNA is shown.

For proper interpretation of the scope of the present invention, the inventors consider appropriate to provide the following definitions:

Inset: refers to a DNA fragment was incorporated into the vector of choice to give a vector with insert. At a bank, the inserts are of different sizes and different sequence.

Probe: refers to a DNA fragment which will be used to hybridize with DNA bank to identify those clones containing an insert of complementary homologous sequence and thus are capable of hybridizing to this.

Vector choice: the molecular vector (plasmid or phagemid) in which they are incorporated and the fragments of a genome or cDNAs obtained from a cell, to obtain genomic or cDNA refers respectively.

Vector with insert: vector of choice to which has already incorporated a fragment of genome or cDNA and forming together a genomic or cDNA, respectively refers bank. Each molecule of the vector with insert of a bench incorporates a different sequence and insert size.

sonicated vector: Refers to the same vector of choice after being subjected to a fragmentation process, preferably by an ultrasound pulse so that the vector of choice is reduced to fragments of varying sizes double stranded. (Note: The sonicate vector must be the same as the vector of choice for a suitable prehybridization with vectors with insert simple and concatenated string).

positive clones: host cells containing a vector with insert whose sequence is homologous to be complementary nucleotide sequence of the probe.

Concatenated DNA: DNA rings crosslinked as links in a chain. Result denaturing double-stranded circular DNA.

Reported methods for selection and / or purification of DNA fragments from a gene bank or expression are widely used for the generation of genetically modified organisms and the development of recombinant DNA technologies. These methods have two important aspects determining the efficiency and specificity of hybridization with the probe of interest: first, the generation of vectors to insert single chain and the other, proper immobilization of the probe used. the use of specific enzymes (nucleases) to degrade single strand dsDNA: Regarding obtaining vectors with single strand insert, in the above methods, Pruitt 1988, some strategies such as described. In other cases, the property of DNA is used to form a triple helix under certain reaction conditions. In any of these strategies, methods must include the end of the process steps which will regenerate some double-stranded circular DNA. This is necessary to replicate vectors with insert in the host cell as simple DNAs to be introduced in this chain are, in addition to DNA polymerases require double stranded DNA to replicate the DNA degraded. In this regard, the use of DNA polymerases reported to synthesize the complementary strand.

In conclusion, the strategies for generating plasmids single stranded usually involve the use of specific enzymes, both nuclease to degrade one strand at the beginning, as DNA polymerases to regenerate the complementary strand at the end, which results in a high variability in the recovery efficiency of positive clones recovered (20 to 100%). Furthermore, it is obvious that as the complexity of the methods due to the increase in the number of steps or stages, ios reliability thereof is lowered. Last but not least, it is the high cost associated with specific enzymes and reagents used in these methods.

Concerning the other important aspects, proper immobilization of the probe used in the methods reported some strategies particularly associated with complex systems of immobilization are identified; in this respect, we can mention the use of small magnetic beads coupled with streptavidin in order to catch those vectors with insert hybridizing to the probe, which usually is labeled with biotin, Hawkins 1999, Li et al, 1995. This system It has several disadvantages, among which we can mention: 1) can not work with high temperatures which allow greater specificity in hybridization (because temperature affects the biotin-streptavidin interaction), resulting in a selection of a high number DNAs with unrelated probe sequences, ie false positive clones; 2) there is a need for specialized recovery equipment magnetic fields. As can be seen, in these methods of selecting low efficiencies are obtained and also DNAs are found to be laborious and costly.

Thus it is clear the existence of a number of technical problems or limitations in the methods of selection of DNA reported and the commercial "Kit" based on the method of "Gene Trapper" USP 5,898,071, that in some way prevent or limit development of recombinant DNA technology.

The inventors of the present method proposes a solution to these limitations by using certain properties of DNA and handling conditions, unlike the methods described above, do not require enzymes like nucleases (to generate single strands), or polymerases (to regenerate the complementary strand), so it is a method, simple, fast, efficient, reliable, inexpensive and efficient recovery of positive clones and further does not require special equipment to recover the vectors with insert with sequence homologous to the probe. This method comprises the following steps:

a) Preparation of vectors with insert simple and concatenated string by denaturation and prehybridization with sonicated DNA vector; b) on the other hand, immobilization of the probe DNA of interest to a solid support and blocking the free surface thereof; c) hybridizing vectors with single strand insert (particularly in the region of the insert) bank with the DNA probe immobilized; d) recovery of vectors with insert hybridized with the probe by membrane separation; e) transformation of the vectors with insert recovered into cells where it can replicate for further analysis of the DNAs.

In Figure No. 1, the quick selection of DNAs of the present invention for the case of a cDNA diagrammed. Obtaining vectors to insert single chain it is performed by denaturing and prehybridization of vectors with insert double stranded DNA sonicated vector, separating chains vectors with insert, resulting in two single strands of each vector with insert concatenated together and hybridized with the fragments of the vector of choice which will keep them in that form. Thus, the inserts are exposed as single-chain to allow hybridization with the specific probe.

On the other hand, and independently, it is performed immobilizing the probe of interest, by fixing and denaturation in a support and blocking the free surface of the support in order to prevent vectors with insert single chain bank stick nonspecifically to the support means.

With the aforementioned elements; It is carried out hybridization of the immobilized probe vectors with insert DNA bank to select those with the insert of interest. Subsequently vectors with selected insert are recovered and transformed into cells where it can replicate for further analysis.

That is, the molecules of the vector with insert which are selected are those which have an insert with homologous and complementary to the immobilized probe hybridize with this sequence, whereas those molecules which insert has a complementary sequence homologous remain in solution. In the method of the present invention, the recovery of vectors with insert is performed by membrane separation under certain temperature conditions in conventional equipment, which represents a comparative advantage over the reported methods.

With the method of the present invention are solved the technical problems or limitations that arise in existing methods, as indicated below °:

A) In the prior art, a limiting and determining methods for purifying DNAs factor is obtaining vectors with insert single chain, reporting some alternatives, the most common use specific enzymes that degrade one DNA chain, however, this has a direct impact on high cost methods that follow this strategy.

Particularly this limitation, the inventors propose obtain single-stranded vector with insert, by denaturation and simultaneous hybridization with small sonicated fragments vector, to maintain separate chains while remaining concatenated. With this prehybridization prevents the DNA of each vector with insert can hybridize with the complementary strand thereof, resulting in double-stranded DNA or with another molecule of the vector with insert which could lead to a false positive clones , thereby preventing retrieve multiple chains that could give rise to false positive clones, so the method is to have a high efficiency, easy implementation and above all, a relatively low cost.

Furthermore, one determinant for efficient transformation in cells of E coli is the type of DNA to be transformed factors. For this it is necessary to introduce DNA double-stranded vector and the single-stranded DNA is degraded within the cell. In this connection an additional advantage of the method of the present invention is that for the end DNA vector double-stranded, it is not necessary to regenerate one of the chains, because throughout the process, the two DNA strands remain bound concatenated manner.

Relative to the other limiting aspect methods reported in selecting DNAs, regarding the manner of immobilizing the probe, the inventors propose the use of a solid support that will permit manipulate the reaction conditions, particularly the use of high temperatures favoring the immobilization of DNA it is used as a probe and the end of the hybridization facilitate separation of vectors with inserted single chain hybridized with the probe of interest without required intermediary while retaining the probe. Furthermore, blocking the free surface of the support (one in which no probe stuck) to prevent DNAs bank stick so unspecific to the support, thereby achieving substantially increased efficiency is also contemplated and specificity of the method of the present invention.

While different types of media, the inventors propose to use a support that meets the characteristics described above and particularly which can be operated at high temperatures; to achieve greater specificity in the hybridization; in this case, preferably suggest using nitrocellulose membrane, better results are obtained if the nitrocellulose membrane is positively charged.

Additionally, relative to the DNA probe of interest is immobilized on the support, the method of the present invention allows the use of probes of different size, ie can be used as a probe an oligonucleotide, an entire gene or a polynucleotide.

Furthermore, as is known, the single stranded DNA is a macromolecule formed by a large number of deoxyribonucleotides that can bind other macromolecule DNA with sequence complementary to form double-stranded DNA or remain as a macromolecule chain simple. In this regard, one advantage of the present invention is that by the present method can be selected DNA double strand, without it being necessary to use enzymes to obtain single strands.

In the prior art, it is known that the DNA denaturation is the process which can break the hydrogen bonds that bind double-stranded DNA and to separate the two DNA strands to produce two single strands of DNA ( in the case of circular DNA remain concatenated). The most common to carry out denaturation of DNA methods, we can mention the addition of acid; adding alkalis such as sodium hydroxide or by heat treatment, using temperatures ranging from 72 to 105 ° C.

Moreover, also known in the prior art that to carry out hybridization, it is necessary to first denature or separate the strands of DNA to promote exposure potential hybridization sites; and subsequently reversing the denaturing conditions in the presence of the probe of interest to promote its binding to one strand with complementary sequence and the sequence homologous to the probe used.

The method of the present invention contemplates the denaturation of DNA vectors with insert, in one step, with prehybridization with sonicated vector, which can be performed by any of the methods mentioned above denaturation, i.e. by handling pH or temperature; particularly the inventors suggest that this denaturation and prehybridization is carried out by heat treatment of two steps, consisting in heating vectors with insert in the presence of sonicated vector to the denaturation temperature of 72 to 105 ° C for at least 5 minutes, then slight cooling or temperature decrease is performed to maintain this mixture to the hybridization temperature of 42 to 65 ° C for at least 5 minutes; and finally allowed to cool to room temperature. It notes that the heat treatment has advantages in comparison to methods of manipulating pH, since it be a simple, rapid processing and is not necessary to use additional reagents that could interfere with the hybridizations which take place in the method the present invention.

Another advantage of the method of the present invention is that since the properties and conditions of manipulation of DNA are universal and not specific to particular inserts, probes or vectors of choice, the possibility of using different vectors are contemplated to be commonly used in gene banks prokaryotic cell, such as pCP13 ​​and eukaryotic cells such as for gt11 genomic libraries and cDNA libraries for λZAP. EXAMPLES

To illustrate the application of the method of the present invention for the rapid selection of DNAs, a few examples, which should not be same as limiting its scope are described.

Example No.1 applying the method of the present invention in the selection of cDNA coding for the synthesis of a protein kinase receptor type is illustrated.

To isolate the cDNA of interest was started from an expression library Double stranded cloned into the λ-ZAP bean nodule 13 days vector and applied the method of the present invention as follows:

1. Denaturation and prehybridization of the cDNA library. Heating at 95 ° C for 5 min 500 .mu.l of cDNA library Double stranded (expression library nodules bean 13 days) in the presence of 10 μ I ( "10 .mu.g of vector) vector DNA previously sonicated ( by five cycles of 20 seconds at 10 watts), allowing cooling to the annealing temperature (50 ° C).

2. Immobilizing the probe on the membrane and blocking. a) Place 1-2 ul probe of interest ( "02.01 g), in this case a cDNA fragment of a protein kinase receptor type 1 c 2 positively charged nylon membrane (HYBON N +; Amersham Pharmacia Biotech UK Ltd. b) setting and denature the probe on the membrane with 500 .mu.l of 400 mM NaOH. c) Add 5 ml of a phosphate solution (300 mM NaH 2 PO 4, 7%

SDS, 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) incubating a solution vectors with insert in the presence of the immobilized probe at a temperature of 50 ° C for 8 h, depending on the percent identity of the DNA sequences with the probe of interest (NOTE: A higher percentage of identity higher annealing temperature); b) Incubate the membrane with 5 ml of washing solution (600mm

NaCI, 60 mM sodium citrate; 1% SDS) for 10 min at the hybridization temperature, repeat this step three times. 4. Recovery of vectors with the insert of interest. a) Incubate the membrane with 500 ul of H 2 0 at 95 ° C for 5 min and remove the membrane at this temperature. b) recovering the supernatant and precipitated with 300 mM sodium acetate and two volumes of ethanol.

10. Drying and resuspend in 20 ul of H 2 0. 5. Tansformación with 20 .mu.l of vectors with insert recovered in Electrocomponentes E. coli cells XL1 Blue (prepared as recommended by the supplier electroporator, Bio Rad Laboratories), for amplification of the vector with insert and subsequent analysis.

Example No. 2

In this example the efficiency of selection of DNA of interest in an expression library bean nodule 13 days is shown by the quick selection of DNAs of the present invention.

After using the methodology described in Example 1, 100 positive clones, of which 35 randomly selected were obtained. These clones were DNA extracted subsequently were separated on an agarose gel and transferred to a membrane and hybridized with the same probe used for selection but solution (labeled with P 32). They were exposed in a X-ray film The results are illustrated in Figure 2. As can be seen, with the method of the present invention is obtained that 28 of the 35 clones (80%) hybridizing with probe used in selection.

example 3

In this example the identity level nucleotide sequence of the DNAs selected with the probe used in the quick selection of DNA of the present invention.

The methodology of Example 2. followed 3 DNA hybridizing to a greater extent with the probe were selected and sequenced with the "Kit" Big Dye ™ Terminator Cycle Sequencing Ready (PE Applied Biosystems Perkin Elmer) and reactions were read on a PRIMS ABI automated sequencer Model 377 DNA sequence.

table I

Figure imgf000016_0001

Table I shows the percent identity of the nucleotide sequence of cDNA pKG3 clone used as a probe is shown, with the sequence of three of the cDNAs selected by the method of the present invention (PKG 1 pkg 8 PKG 13).

Example 4 In this example the industrial application of the quick selection of DNA is illustrated, by integrating a system based on the method of the present invention case. COMPONENTS.

Solution I: prehybridization solution (1 ug / ul sonicated DNA vector). Solution II: DNA fixing solution (0.4 N NaOH) solution III: Wash Solution. Solution IV: Solution hybridization. Solution V: elution solution. Solution VI: Solution DNA precipitation. A set of positively charged Nylon membranes 1cm 2.

MATERIAL AND ADDITIONAL EQUIPMENT

95 ° C bath.

Hybridization oven.

electrocompetent cells.

DNA bank in double stranded vectors. (NOTE: The vector must be the same as the vector sonicated solution I)

Probe interest.

PROTOCOL

1) Mix 500 ul of DNA bank with 50 .mu.l of solution I. 2) Heat the mixture to 95 ° C for 5 minutes.

3) Allow to cool to hybridization temperature (42-65 ° C) for 10 minutes.

4) Place equivalents 1-2μg DNA probe of interest in the nylon membrane, previously hydrated with Solution II ul.

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 hybridization temperature required.

NOTE: The hybridization temperature depends on the identity of the DNA sequence of the probe and the gene to be selected. For a homologous probe is recommended a temperature of 65 ° C. For a probe with low% identity may lower the temperature to 42 ° C. 7) Mix the cDNA library prehybridized with 5 ml of hybridization solution and incubate at the annealing temperature for 1-8 hours.

8) Wash the membrane with 5 ml of Solution III for 15 minutes at the same annealing temperature. 9) Repeat this step twice.

10) Remove the membrane and place in a microcentrifuge tube and adding thereto 1.5 ml 500 ul of Solution V.

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) Add 1 ml of the supernatant solution VI.

13) Centrifuge for 10 minutes at 13,000 rpm and resuspended in 10 ml of Solution Vil.

14) Change in cells prepared for electroporation. 15) plating boxes LB with antibiotic selection of the vector used for the library.

REFERENCES

Ito, T., Smith, CL & Cantor, Ch. R. (1992) Sequence-specific DNA purification by triplex affinity capture. Proc. Nati. Acad. Sci 89, 495 -. 498.

Pruitt, SC (1988) Expression vectors cDNA cloning and permitting enrichment for specific sequences by hybridization / selection. Gene 66, 121-134.

Hawkins, T. (1999) US Pat. No. 5,898.071 DNA purification and isolation using magnetic particles.

Rigas, B., Welcher AA, Ward, DC & Weissman, SM (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

Claims

REIVINDICACIONE SHabiendo described the invention what is claimed content in the following claims:
1. A quick selection of DNA comprising the following steps:
a) Preparation of vectors with insert simple and concatenated string by denaturation and prehybridization with sonicated DNA vector; b) on the other hand, immobilization of the probe DNA of interest to a solid support that allows manipulating the reaction conditions and blocking the free surface thereof; c) hybridizing vectors with single strand insert bank with the DNA probe immobilized; d) recovery of vectors with insert hybridized with the probe by membrane separation; e) transformation of the vectors with insert recovered into cells where it can replicate for further analysis of the DNAs.
2. fast selection method according to claim 1, wherein the DNA can be double-stranded.
3. fast selection method according to claim 1, wherein the denaturation and prehybridization with vector DNA sonicated for obtaining vectors with insert concatenated single chain is performed by any of the following treatments: heat , addition of alkaline solutions or acid solutions.
4.- fast selection method according to claim 3, wherein the denaturation and prehybridization with sonicated DNA vector are carried out by thermal treatments.
5. fast selection method according to claim 4, wherein the heat treatment for the DNA denaturation is performed in a temperature range of 62 to 105 ° C for a time of 3-15 min.
6. fast selection method according to claim 1, wherein the vectors used can be plasmids or phages double stranded.
7. fast selection method according to claim 1, wherein the DNA probe is immobilized interest in the carrier may be an oligo-, a gene or polynucleotide.
8. rapid selection method according to claim 1, wherein the support on which the probe is immobilized allows the use of high temperatures; promotes the immobilization of the probe and the end of hybridization, it facilitates removal of the inserts hybridized with the probe.
9. rapid selection method according to claim 8, wherein the support is a nitrocellulose membrane.
10.- fast selection method according to claim 8, wherein the support is a positively charged nitrocellulose membrane.
11.- fast selection method according to claim 1, wherein the recovered insert are those containing a DNA with a high percentage of sequence identity probe.
12. Kit for rapid screening of DNA-based method of claim 1.
13. Kit for rapid screening of DNA according to claim 12, comprising the following elements:
"Solution I: prehybridization solution (1 ug / ul sonicated DNA vector) Solution II. DNA fixing solution (0.4 N NaOH)
Solution III: wash solution.
Solution IV: Solution hybridization.
Solution V: elution solution.
Solution VI: Solution DNA precipitation.
A set of positively charged Nylon membranes 1 cm2.
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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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