JP2004357701A - Method for extracting target gene and particle having probe dna bound thereto - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an extremely simple and easy method for extracting a target gene by which the target gene can be extracted and separated speedily, accurately and efficiently without being influenced by the configuration and size of the target gene and by which screening requiring about two weeks can be accomplished within about 24 hr. <P>SOLUTION: The method for extracting the target gene comprises mixing particles having a probe DNA bound to the surfaces thereof, a single-stranded bridge oligonucleotide having at its one side a sequence complementary for the probe DNA and at its other side a sequence complementary for at least a part of a base sequence of the target gene with a sample containing the target gene to be extracted and effecting hybridization of the probe DNA, single-stranded bridge oligonucleotide and target gene. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention provides a method for extracting a target gene having a specific base sequence using a single-stranded bridge oligonucleotide hybridized with both a target gene and probe DNA, and a probe DNA-binding particle used in the method. I do. The present invention is mainly suitable for uses such as gene cloning, extraction of a target gene from a cDNA library, separation and purification, and detection.

  Methods for separating and detecting a specific gene using hybridization between a probe DNA bound to a solid phase particle and a target gene have been devised for some time. In many cases, a probe DNA having a sequence complementary to the target gene is bound to the surface of the solid particle, and the target gene is immobilized by hybridization with the target gene, separated and detected. In this method, it is necessary to efficiently immobilize only the end of the single-stranded oligonucleotide having a specific sequence on the surface of the solid phase particle. Therefore, a method of introducing biotin to the end of an oligonucleotide serving as a probe, immobilizing avidin on the surface of solid phase particles by covalent bond, and immobilizing probe DNA using the reaction between avidin and biotin. Has been used (for example, see Patent Document 1). However, in this method, since the probe DNA bound to the surface of the solid particle and the target gene are directly hybridized, there is a disadvantage that the capture efficiency varies depending on the size of the solid particle. In particular, for the separation and purification of large genes, the size of the probe DNA bound to the solid phase particle surface becomes so small that it cannot be ignored compared to the size of the solid phase particle and the effective size of the target gene. I can't escape.

In order to improve the above-mentioned drawback of the method of directly hybridizing the probe DNA bound to the surface of the solid particle and the target gene, the hybridization between the biotinylated probe DNA and the target gene was first performed to prepare a hybrid. Then, a method is disclosed in which the hybrid body is bound to solid phase particles on which avidin is immobilized and extracted (for example, see Non-Patent Document 1). However, this method also uses the avidin-biotin reaction for immobilizing the probe DNA, so that when the size of the hybrid of the biotinylated probe DNA and the target gene is large, the extraction efficiency of the target gene is significantly reduced. Still not resolved. In particular, when the target gene is a double-stranded DNA or a circular DNA, extraction may not be performed.
JP-A-60-93355 "Biomagnetic Techniques in Molecular Biology", Dynal, 1995

When the probe DMA is immobilized using the avidin-biotin reaction described above, there is a problem that the size dependency of the biotin-labeled product cannot be excluded. Care must also be taken when handling solid particles in which avidin, which is a protein, is immobilized. Further, when the reaction conditions for avidin / biotin and the conditions for forming a hybrid are not always the same, problems such as a decrease in extraction efficiency and a collapse of the hybrid may occur. In particular, when the target gene is a double-stranded DNA or a circular DNA, extraction may not be performed. In addition, there is a problem in that the cost of biotinylation of an avidin protein or an oligonucleotide is high.

The present invention provides a probe DNA-binding particle having a probe DNA bound to its surface, one having a sequence complementary to the probe DNA-binding particle, and the other having a sequence complementary to at least a part of the base sequence of the target gene. Mixing a sample containing a single-stranded bridge oligonucleotide having a sequence with a target gene to be extracted, and allowing the probe DNA to hybridize with the single-stranded bridge oligonucleotide and the target gene; And a method for extracting the same.

  The present invention also provides a single-stranded oligonucleotide having a sequence of 5 to 80 nucleotides including a sequence in which at least one of deoxycytosic acid (dC) and deoxyguanic acid (dG) is continuous at least 5 nucleotides, and And a probe DNA-bound particle having a particle size of 0.1 to 15 μm.

  Hereinafter, the present invention will be described in detail.

  The present invention relates to a method for extracting a target gene from the specificity of a gene sequence, and a probe DNA-immobilized particle used in the method. In the present invention, the target gene can be separated, purified, and rapidly, accurately, and efficiently without being restricted by its origin, shape, size, sequence, and the like. The effect of the present invention is particularly remarkable when the target gene is a double-stranded DNA or a circular DNA.

  In order to strengthen the bond between the probe DNA immobilized on the surface of the solid particle and the single-stranded bridge oligonucleotide, the immobilized probe DNA-binding particles used in the present invention must contain at least deoxycytosic acid (dC). It is preferable that a single-stranded oligonucleotide having a 5- to 80-base sequence containing a sequence in which at least one of deoxyguanic acid (dG) and at least 5 bases are continuous is bonded to the surface. Here, it is more preferable that the single-stranded oligonucleotide includes a sequence in which at least one of dC and dG is continuous for 10 bases or more. It is not preferable that the sequence in which at least one of dC and dG is continuous is less than 5, since a strong bond is hardly formed between the probe DNA and the single-stranded bridge oligonucleotide. Further, the entire base sequence of the probe DNA is preferably composed of 5 to 80 bases, more preferably 10 to 50 bases. If the total nucleotide sequence of the probe DNA exceeds 80 bases, the efficiency of synthesizing the probe DNA decreases, and the ratio of functional portions decreases, which is not preferable.

  In the present invention, in order to efficiently separate a target gene, a melting point (Tm1) when a double strand is formed between a probe DNA and a single-stranded bridge oligonucleotide which are bound to a solid phase particle by a covalent bond. It is preferable that the relationship of Tm1 ≧ Tm2 be established between the single-stranded bridge oligonucleotide and the melting point (Tm2) when a double strand is formed between the target gene and the target strand. When Tm1 is lower than Tm2, there are restrictions on hybrid conditions between the target gene and the single-stranded bridge oligonucleotide, and the target gene may be forced to have a low recognition level in some cases. In this case, a sequence other than the target gene may be picked up, which is not preferable.

  Specifically, the base sequence of the probe DNA to be immobilized in the present invention is, for example, a sequence having 5 or more, preferably 10 continuous dC or dG. In order to specifically fix the terminal of these base sequences to the particle surface, a functional group necessary for the terminal to be fixed may be modified. Further, depending on the type of the immobilization reaction, the functional group activity in the base may be blocked as necessary.

The probe DNA used for the probe DNA-binding particles of the present invention can be prepared using a conventional DNA synthesizer. If necessary, a functional group for covalent bonding can be introduced into the terminal to be fixed. For example, when a carboxyl group is introduced on the surface of the solid particle, the probe D
An amino group may be introduced into the terminal of NA. The reverse case is also possible. Moreover, you may introduce | transduce SH group etc. as needed.

The probe DNA-binding particle of the present invention is obtained by covalently binding a single-stranded oligonucleotide to a solid phase particle, and preferably has a particle size of 0.1 to 15 μm, more preferably 0.5 to 10 μm. More preferred. When the particle diameter is less than 0.1 μm, it takes a long time for solid-liquid separation, which is not preferable. On the other hand, when the particle size exceeds 15 μm, the surface area of the solid phase particles when a certain amount of the solid phase particles is used decreases, and it is necessary to increase the density of the probe in order to introduce the same amount of the probe. Is not preferred because it may cause steric hindrance derived from the high-density probe. As a result, the Brownian motion of the particles is reduced, and the natural sedimentation due to the particle's own weight is accelerated, which is not preferable.

  It is preferable that at least a part of the surface of the solid particles used in the present invention is coated with a synthetic polymer. This is particularly necessary for increasing the selectivity for chemical bonding of the probe DNA, and is suitable for preventing non-specific adsorption of DNA other than the target gene. Depending on the type of the probe DNA immobilization reaction, it can be introduced at the time of polymerizing the necessary functional group on the surface of the solid phase particle or by post-modification. Further, if necessary, at least one or more magnetically responsive magnetic substances, fluorescent substances, dyes, pigments and the like may be contained in the solid phase particles. When a magnetic material is introduced, magnetic separation is possible, and the work process can be replaced with an automatic machine, which is particularly suitable for a large amount of work. Also, if the fluorescent substance can be introduced into the solid phase particles, the solid phase particles themselves become the label, which is suitable for detection. Further, if the solid phase particles are colored, the solid phase particles can be colored according to the type of the probe DNA.

  The organic polymer particles constituting the solid phase particles (particles for immunoagglutination reaction) of the present invention are polymers of one kind of monomer selected from the group consisting of aromatic vinyl compounds, polyacrylates, and polymethacrylates. A carboxylic acid group can be introduced to the surface of the organic polymer particles by copolymerizing an α, β-unsaturated carboxylic acid monomer as required.

  Although the base sequence size of the single-stranded bridge oligonucleotide used in the present invention is not particularly limited, it is usually preferably 10 to 500 bases. If the number of bases is less than 10 bases, it is difficult to form a hybrid, which is not preferable. On the other hand, when the number of bases exceeds 500, the length of the single-stranded bridge oligonucleotide becomes longer than necessary to satisfy the original function, and thus steric hindrance may be caused in the hybrid reaction. This base sequence may be prepared by chemical synthesis, or may be prepared artificially using an enzymatic reaction. Alternatively, the nucleic acid may be artificially modified using at least a part of a natural nucleic acid. It is obvious that avoiding formation of a complementary base sequence inside the single-stranded bridge oligonucleotide as much as possible is effective for high-efficiency extraction.

  The single-stranded bridge oligonucleotide used in the present invention has a sequence complementary to the probe bound to the solid phase particle on one side, and a sequence complementary to at least a part of the base sequence of the target gene on the other side. Have. These complementary sequences need not necessarily be located at the termini, but are preferably located near the termini to minimize steric hindrance during hybrid formation. The length of each hybridization base sequence is naturally determined by the base sequence of the probe DNA and the target site of the target gene. Specifically, when the solid-phase probe DNA contains a portion where 5 or more dGs are continuously arranged at the 5 ′ end, dC is continuously arranged at 5 or more at the 3 ′ end of the single-stranded bridge oligonucleotide. It is clear that. The single-stranded bridge oligonucleotide of the present invention can be prepared by ordinary chemical synthesis. The complementary strand of the nucleic acid sequence to be hybridized can be introduced at an appropriate position.

In the present invention, the target gene to be extracted is not particularly limited with respect to its shape, size, presence status and the like. The target gene to be extracted may be a single-stranded DNA, a double-stranded DNA such as a plasmid, a circular DNA, or a supercoiled DNA.

  Specific examples of the single-stranded DNA include those obtained by subjecting genomic DNA obtained from animal cells or cultured cells thereof, or fragments thereof to a single treatment, samples containing cDNA and cDNA libraries, mRNA, and the like. Can be Further, specific examples of the double-stranded DNA include a plasmid, a circular DNA, a supercoiled DNA, particularly a plasmid DNA into which a cDNA library is incorporated.

  Samples containing these target DNAs can be prepared by a conventional nucleic acid extraction method, for example, a phenol chloroform extraction method. Usually, when the target gene to be extracted is double-stranded DNA, it is necessary to hybridize with a single-stranded bridge oligonucleotide at a higher temperature than single-stranded DNA. For example, when the target gene to be extracted is a double-stranded plasmid, it can be extracted in the form of a double-stranded plasmid according to the extraction method of the present invention. Therefore, the DNA recovered by the extraction method of the present invention can be used as it is for applications such as an enzymatic reaction used, and infection into Escherichia coli and the like.

  In the present invention, the order of the hybrid reaction of the probe DNA, the single-stranded bridge oligonucleotide, and the target gene to be extracted is not particularly limited, and may be adjusted according to the target gene. That is, a hybrid of the single-stranded bridge oligonucleotide and the target gene may be formed first, and then a hybrid complex of the solid-phase probe DNA and the hybrid may be formed. A hybrid with the strand bridge oligonucleotide may be formed first, and then a hybrid complex of the target gene and the hybrid may be formed. Alternatively, the three may be mixed simultaneously to form a hybrid complex. The specific working order may be selected according to the selected single-stranded bridge oligonucleotide and the target gene to be extracted.

  In the present invention, by forming a hybrid complex (composite hybrid), the target gene is immobilized on solid phase particles (probe DNA binding), solid-liquid separated from a liquid sample, and extracted. By a simple heating and quenching treatment, the extracted target gene can be released again intact into the liquid phase.

  There are a plurality of methods for confirming the extracted target gene, for example, the target gene is complementary to a position other than hybridizing with a single-stranded oligonucleotide, and is hybridized with a detection probe labeled with fluorescence or the like, There is a method of measuring the fluorescence intensity after washing by solid-phase separation. As a more sensitive and reliable method, for example, the plasmid containing the extracted target gene can be reinfected into Escherichia coli, the Escherichia coli is cultured, and then the type and size of the target gene can be verified by colony PCR. By using the extraction method of the present invention, such a series of operations conventionally taking several days to several weeks can be completed in only about 24 hours.

  Hereinafter, specific examples of the present invention will be described, but the present invention is not limited thereto.

1. Example 1
1.1. Solid Phase Particles All solid phase particles used in the present invention were JSR products. The particle diameter was measured using a photodynamic scattering device PhotalLPA3100 (Otsuka Electronics Co., Ltd.) or an optical diffraction device SOLD2000 (Shimadzu Corporation). The amount of the carboxyl group (functional group) introduced on the surface was measured by conductivity measurement by acid-alkali titration (Potentiograph E536 Metrohm, Herisan). In Table 1,
The physical properties of the used solid particles (particles 1 to 4) are shown.

1.2. Preparation of Probe DNA Next, a sequence in which 30 dCs were continued was prepared by an ordinary amidide synthesis method. A c7 column was used so that an amino group could be introduced at the 3 'end. The synthesis was performed according to the manufacturer's manual (PERKIN ElUMER). The synthesized oligonucleotide was purified with an OPC column. The oligonucleotide synthesized in this manner was designated as dC30-N.

(DC30-N)
5'-CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-NH ₂ -3 '
In the same manner, the nucleotide sequence of dG30 in which an amino group was not introduced into the 3 ′ end and the nucleotide sequence of dG30 in which a fluorescent dye FAM was introduced into the 5 ′ end were synthesized. These were dG30 and dG30-F, respectively.

Subsequently, the concentrations of dC30-N and dG30 were adjusted to 10 nmol / 100 μl, respectively,
Take out 00 μl, place in a tube containing 0.8 ml TE / 1.5 M NaCl solution, heat to 100 ° C., then slowly cool to room temperature, and obtain 10 nmol / dG / dC double-stranded DNA having an amino group introduced at the 3 ′ end of dG. ml was prepared. This was designated as dC30-P. From the measurement of the absorbance at 260 nm, it was confirmed that a double strand was formed.

1.3. Immobilization of probe DNA 1 ml of a 1% by weight dispersion of each carboxyl group-introduced particle prepared in Example 1 was centrifuged, and a 5 mM MES buffer solution (pH = 5.5) was added to the solid substance. The operation of centrifugation was repeated three times to wash the particles. Thereafter, 0.1 ml of a 5 mM MES solution obtained by dissolving 5 mg of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (manufactured by Dojindo Co.) was added to the particle dispersion, and the mixture was rotated and stirred at 40 ° C. for 2 hours. 100 μl of a −P solution (10 nmol / ml) was added, and the mixture was rotated and stirred at room temperature for 10 hours. Subsequently, the particle dispersion is washed three times with distilled water, and then 1 ml of the particle dispersion is heated to 100 ° C., then cooled with ice water at 0 ° C., and immediately centrifuged to perform solid-liquid separation, whereby heat denaturation is performed. The released dG sequence was removed. This operation was repeated three times, and it was confirmed from the absorbance measurement of the centrifuged supernatant that dG had been removed. Thus, a 1% by weight dispersion of probe DNA-immobilized particles having 30 bases of dC immobilized thereon was obtained.

100 μl of a 1% by weight dispersion of the above probe DNA-immobilized particles and 100 μl of a 200 pmold G30-F solution were mixed, 200 μl of a 2 M NaCl solution was added, and the mixture was incubated at 50 ° C. for 15 minutes, and then the supernatant was collected by centrifugation. The fluorescence intensity of the supernatant was measured, and the ratio of the fluorescence intensity of the 200 pmoldG30-F solution before being added to the particle dispersion to the dG component hybridized to the probe particles,
That is, an effectively fixed dC component was obtained.

In the same manner, particles having dG30 immobilized thereon were prepared. Table 2 shows the probe binding amount (pmol / mg) of each probe solid phase particle.

2. Example 2 Extraction of actin gene 2.1. Preparation of Single-Stranded Bridge Oligonucleotide In the same manner as for the probe DNA, the following sequence for single-stranded bridge oligonucleotide (Actin-polyC) was prepared.

(Actin-polyC: corresponding to SEQ ID NO: 1 in the sequence listing)
5'-GGGGCCACACGCAGCTCATTGTAGAAGGTGTGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGG-3 '

2.2. Extraction of Actin Gene 10 pmol / 10ul of the single-stranded bridge oligonucleotide solution prepared in Example 2 above and 1,20 μl of the probe DNA binding particles prepared in Example 1 were heated at 70 ° C., and then cooled on ice. 10 μl of 5 MnaCl was added and incubated at 45 ° C. for 1 hour. The mixture was centrifuged at 12000 rpm for 3 minutes and washed twice with 0.1 M NaCl. Subsequently, a total of 15 μg / 20 μl of a plasmid (Takara Bio Inc.) incorporating a cDNA library previously heated at 95 ° C. for 10 minutes and ice-cooled was added. The mixture was incubated at 55 ° C. for 1 hour and washed three times with 0.1 mM NaCl. The pellet was added with 25 μl of distilled water, heated at 70 ° C. for 10 minutes, and cooled with ice. The supernatant was collected by centrifugation.

  Similar experiments were performed using probe DNA-bound particles 1 to 4 and particles to which dG30 and dC30 were respectively fixed.

2.3. Verification of the extracted actin gene 10 μl of the above separated and purified actin gene was added to 100 μl of an E. coli solution having an absorbance of 0.05 at 600 nm, cooled on ice for 20 minutes, heated at 42 ° C. for 50 seconds, and further ice-cooled for 2 minutes. . The Escherichia coli was cultured in an SOC medium at 37 ° C. for 2 hours, and then cultured in an Arp (+) agar medium for 16 hours. Subsequently, colony PCR was performed. PCR was performed at 94 ° C for 3 minutes, 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 30 seconds for 30 cycles. Table 3 shows the actin gene extraction results. The required time was 24 hours.

(Actin primer 1: corresponding to SEQ ID NO: 2 in the sequence listing)
5'-TAGGAGCGACAGCAGTAATC-3 '
(Actin primer 2: corresponding to SEQ ID NO: 3 in the sequence listing)
5'-GCTGTGCTATGTTGCTCTAG-3 '

From the cDNA plasmid library, in the system (A) to which actin-poly C (Actin-PolyC) bridge oligonucleotide was added or in the system (B) to which actin-PolyC was not added, actin cDNA was added using particles 1 (dG30 immobilized). The plasmid was extracted. FIG. 1 shows the results of PCR using the same actin primers directly as above without introducing the extracted DNA into E. coli. Similar results were obtained from the other particles 2 to 4.

  In FIG. 1, sample A (the band on the left side in FIG. 1) was extracted in the presence of an actin-poly C (Actin-poly C) bridge oligonucleotide, and sample B was an actin-poly C (Actin-poly C) bridge oligo. This is the result of extraction in the absence of nucleotides.

3. Example 3 Extraction of HPRT gene In the same manner as in the actin gene extraction, HPRT-polyC (HPRT-polyC) was used as a bridge oligonucleotide instead of actin-polyC. The probe particles 1, immobilized dG30, the amount of the bridge oligonucleotide used, and the method of use were the same as in the actin gene extraction. The same method as in Example 2 was used except that the following primers were used for PCR detection. Table 4 shows the results of HPRT gene extraction. The required time was 24 hours.
Single-stranded bridge oligonucleotide:
(HPRT-polyC (HPRT-polyC): corresponding to SEQ ID NO: 4 in the sequence listing)
5'-GGGGTAGGCTGCCCTATAGGCTCATAGTGCACCCCCCCC
CCCCCCCCCCCCCCCCCCCCCC-3 '
(HPRT primer 1 (HPRT primer 1): corresponding to SEQ ID NO: 5 in the sequence listing)
5'-GAGAGCTTCAGACTCGTCTA-3 '

4. Comparative Example 1
Instead of this test method, actin and HPRT genes were respectively extracted by the following method using the colony hybridization method.
1) A cDNA library plasmid (library plasimid) was replaced with a competent E. coli.
Cells (E. coli) were transfected.
2) The transfected E. coli cells (Escherichia coli) were spread on an agar medium plate containing ampicillin at a rate of 2,000 to 50,000 cells per plate. About 10 to 20 plates were prepared in the same manner.
3) These plates were used for hybridization after confirming that the bacteria had grown sufficiently after 16 to 24 hours.
4) In the hybridization, first, two or three filter membranes made of nitrocellulose cut into a plate shape were prepared per plate. Therefore, a total of 20 to 60 filter membranes were produced.
5) A filter membrane made of nitrocellulose was aligned with the bacterial surface of the plate, and the bacteria were transferred to the filter membrane made of nitrocellulose, thereby creating two to three replicas for each plate.
6) Thereafter, the bacteria on the replica membrane were lysed, DNA denaturation treatment and the like were performed, and used for colony hybridization.
7) Next, hybridization was carried out for all the plates for 24 hours using a DNA fragment of the target gene labeled with a radioisotope as a probe.
8) Thereafter, the membrane was thoroughly washed to remove non-specific binding, dried, and then exposed to an X-ray film for about 2 to 5 days.
9) Bacteria on the replica plate corresponding to the portion of the light-sensitive signal were collected, and hybridization was repeated again by the procedures from 1) to 8) to obtain a single positive colony. Table 5 shows the results. The required time was 14 days.

5. Effects According to the method for extracting a target gene of the present invention using the probe DNA-immobilized particles and the single-stranded bridge oligonucleotide, the probe DNA-bound particles having the probe DNA bound to the surface, and one of the probe DNA-bound particles complementary to the probe DNA Having a sequence, a single-stranded bridge oligonucleotide having a sequence complementary to at least a part of the base sequence of the target gene, and a sample containing the target gene to be extracted,
Hybridizing the probe DNA, the single-stranded bridge oligonucleotide and the target gene, without affecting the shape and size of the target gene, quickly, accurately, and efficiently convert the target gene. It can be extracted and separated. Specifically, conventional screening for about two weeks can be completed in about 24 hours, so that it can be implemented as an extremely simple method.

It is a photograph of the band which shows the PCR amplification reaction result of an actin gene. The results are shown.

Claims (2)

A probe DNA-binding particle having probe DNA bound to its surface, and a single strand having a sequence complementary to the probe DNA on one side and a sequence complementary to at least a part of the base sequence of the target gene on the other side Mixing a sample containing a bridge oligonucleotide and a target gene to be extracted,
A method for extracting a target gene, comprising: hybridizing the probe DNA binding particle, the single-stranded bridge oligonucleotide, and the target gene. A single-stranded oligonucleotide consisting of a sequence of 5 to 80 bases including a sequence in which at least one of deoxycytosic acid (dC) and deoxyguanic acid (dG) is continuous at least 5 bases is bound to the surface, and has a particle diameter of 0. Probe DNA-bound particles that are 1-15 μm.