JP2007195441A - Dna library selectively enriched in dna repeat sequence and method for preparing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA library selectively enriched in a DNA repeat base sequence, and to provide a method for preparing the same. <P>SOLUTION: This DNA library selectively enriched in repeat sequence is characterized by comprising a step for separating genome DNA from the tissue of a mammal, a step for subjecting an ultrasonic disintegration to the genome DNA to cleave the DNA in a constant size, and a step for thermally denaturing the product, rehybridizing the denatured product, and then removing non-reacted single chain portions with a single chain-selective nuclease. And the method for producing the same. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a DNA library in which DNA repeat sequences are selectively concentrated and a method for producing the same.

Comparative genomic hybridization (CGH) is a method in which the gain and loss of DNA replication can be confirmed by a single gene hybridization reaction.
Examples of diverse DNA hybridization studies include comparative genomic hybridization (CGH), chromosome painting, cDNA-microarray, cDNA-microarray, microarray-CGH, or nucleic acid probe (probe). Examples include fluorescent in situ hybridization for detecting specific mRNA and DNA on a chromosome.
Currently, an array-CGH improved in usefulness by combining microarray technology and comparative genomic hybridization (CGH) has been developed. The array-CGH method is commonly used to study DNA replication number abnormalities in various diseases.

  DNA libraries with selective enrichment of repetitive sequences are used in various DNA hybridization studies to block nonspecific reactions such as DNA probes, eg, cosmids, YACs and chromosomal coloring probes. .

  Since repetitive nucleotide sequences are distributed around the DNA of mammals, non-specific reactions between probes and repetitive nucleotide sequences can be used to determine the position or expression level of a gene. Therefore, it is necessary to perform a process of previously blocking the probe from being bound to the repetitive base sequence of the sample through the process of adding an excessive repetitive base sequence to the probe mixture. The DNA library in which the repetitive nucleotide sequences used at this time are selectively concentrated cannot be artificially created because the sequences are so diverse that they are extracted from the genomic DNA of animals and used.

  Examples of such a repetitive nucleotide sequence gene include Alu, Mir, LINE, and MER repetitive sequences in the case of a mouse. For humans, there are LINE and Alu repeat sequences. These repetitive sequences cannot be artificially synthesized and used, but must be selectively extracted from animal genome genes and used.

  Therefore, there is a need for a method for efficiently concentrating repetitive base sequences and a DNA library selectively enriched with repetitive base sequences having excellent operational performance.

A technical problem to be solved by the present invention is to provide a DNA library having excellent operation performance and selectively enriched DNA repeat sequences.
Another technical problem to be solved by the present invention is to provide a method for producing a DNA library that efficiently and selectively concentrates DNA repetitive sequences.

The present invention provides a method for producing a DNA library that efficiently selects and concentrates DNA repetitive sequences. More specifically, the present invention includes a step of extracting genomic DNA from animal cells, a step of cleaving DNA to a certain size using ultrasonic waves, a step of re-crossing after modifying the cleaved DNA strand, DNA live comprising a selective enrichment of repetitive nucleotide sequences comprising the step of selectively degrading a single strand from the recrossed DNA muscle using a nuclease that is selective for one strand A method for manufacturing a rally is provided.
In addition, the present invention provides a DNA library excellent in operating performance, which is produced by the above method and is selectively enriched with repetitive base sequences.

  The DNA library according to the present invention which selectively concentrates the DNA repeat sequences efficiently blocks non-specific reactions in the DNA hybridization research method, and the DNA library production method according to the present invention efficiently selects and concentrates repeat base sequences. Thus, a repetitive base sequence having excellent operating performance is provided.

  The present invention will be described in detail below.

  All mammalian cells can be used as animal cells for extracting genomic DNA. For humans, placental tissue is preferred over other tissues. Genomic DNA can also be extracted from blood, but it is difficult to obtain a sufficient biomass.

  DNA extraction is performed by a known method. Conventional methods for extracting DNA in large quantities include a method using phenol (US Pat. No. 3,838,148) and a method using an anionic surfactant and a high concentration of sodium chloride (Korea Patent No. 35972). ) Etc. are presented.

  The method using phenol is a process of extracting DNA using phenol / chloroform (phenol / chloroform) or phenol / chloroform / isoamyl alcohol (phenol / chloroform / isoamyl alcohol), and recovering DNA by ethanol (or isopropanol) precipitation. And the process of separating the source of contamination from DNA.

The ethanol (or isopropanol) precipitation extracts DNA from a liquid solution of DNA extracted with phenol / chloroform and removes residual phenol and chloroform from the DNA.
Also, short oligonucleotides consisting of nucleoside triphosphates and 30 bases or less can be removed partially through the ethanol precipitation process.

However, the phenol / chloroform extraction process is very dangerous because it requires many steps and time to extract DNA and uses phenol and chloroform harmful to human body.
Phenol is so dangerous that it burns when in contact with the skin, and chloroform is also very volatile, toxic and flammable.
In order to use such dangerous phenol and chloroform, there is a disadvantage that it must be experimented in a hood.
Also, the phenol / chloroform extraction process greatly damages the DNA due to the oxidation of phenol, so only redistilled phenol must be used.

  Accordingly, various methods have been devised that are more effective and simple than the traditional DNA separation process through the phenol / chloroform extraction process and the ethanol precipitation process, and try to minimize the DNA separation time.

  Examples of such methods include methods using chaotropic salts and silica resins, methods using affinity resins, ion exchange resin chromatography methods and magnetic properties in addition to conventional phenol / chloroform treatment and salting out methods. There are methods using beads (US Pat. No. 5,057,426, US Pat. No. 4,923,978, EP 0512767A1, EP 0515484B, WO 97/10331, WO 96/18731).

  Recently, column-based kits (kits) tend to be the basis of genomic DNA extraction in order to overcome the inconvenience of phenol / chloroform extraction and ethanol precipitation methods.

The principle of extracting genomic DNA through such a column is the structural interaction between water molecules and DNA by using a glass fiber or silica membrane that specifically binds to the DNA. the principle of interaction).
Such a glass fiber or silica membrane does not bind to salts, proteins, and other intracellular substances, so that only DNA can be separated purely.

In order to create a DNA library in which repetitive sequences having excellent operation performance are selectively concentrated, genomic DNA extracted in advance so as to have an average size of 200 to 400 base pairs is disrupted.
When the average molecular weight of DNA is less than 200, the molecular size is too small, so that the blocking effect of nonspecific reaction is reduced. When the average molecular weight is 400 or more, non-repetitive sequences are also present. In some cases, the blocking effect of non-specific reactions is also reduced.

The genomic DNA fragmentation method includes enzymatic treatment and physical treatment, but ultrasonic treatment is preferred.
Ultrasonic waves change electrical energy into longitudinal mechanical vibrations with a converter, and when immersed in a solution, mechanical vibrations are transmitted by pressure waves to cause cavitation development.
Bubbles are formed by a local pressure drop when the pressure wave cycle is negative (-), and the formed bubbles are squeezed and distorted when the pressure wave is positive (+).
While the formation and destruction of the bubbles are repeated, a severe impact is generated in the solution.
In the case of enzymatic treatment, there is a disadvantage that the DNA size cannot be adjusted to a constant value. However, in the case of ultrasonic treatment, it is easy to adjust the average size to a constant value.

Genomic DNA processed to a certain size by sonication is heated and denatured.
It is sufficient if the heating temperature is such that the DNA can be denatured into a single strand, but heating at 95-100 ° C. can save reaction time.

The denatured genomic DNA is re-crossed by heating and cooling.
Preferably, the reaction is carried out at 55 to 65 ° C. for 5 to 6 hours, and among the DNA transformed into a single strand in this process, a repetitive sequence having a large number of copies is recrossed and double strand. However, single copy genes are not recrossed and remain single stranded.
At this stage, it is necessary to adjust the reaction temperature and reaction time so that the single replication gene is not hybridized to the duplex.
If the reaction time is extended, the single replication gene may become double-stranded, so that the reaction time must not be extended.

Since the single-replicated gene portion exists as a single strand in the recrossed DNA, the single-stranded DNA sequence is selectively degraded using the S1 nuclease (S1 nuclease).
The reaction temperature and reaction time are preferably determined according to the instructions recommended by the nucleolytic enzyme manufacturer.

  The method may additionally include performing a protein removal and purification process by a known method. At this time, the removal and purification process of the added protein may use a method such as chromatography, precipitation and filtration.

  Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

  <Production of a DNA library in which DNA repetitive sequences are selectively concentrated from the mouse genome>

  The DNA library according to the present invention can be extracted from a mouse tissue, and the process is as follows.

  1. Mouse genomic DNA isolation

1) 200 ml of cell lysate is added to 5 g of mouse tissue, and the tissue is cut finely to increase the surface area (cell lysate: 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% SDS, Proteolytic enzyme (proteinase K) 1 mg / ml).
2) React for 12 hours at 55 ° C. on a mixing shaker.
3) After confirming that the tissue is completely lysed, 200 ml of isopropanol is added thereto to primarily separate genomic DNA.
4) The primary separated DNA is washed with alcohol and dried, and then dissolved in 50 ml of TE buffer solution (pH 8.0).
5) Add the same amount of phenol / chloroform / isoamyl alcohol mixed solution to the dissolved DNA solution, and centrifuge at 3,000 rpm for 15 minutes to primarily remove proteins.
6) Add the same amount of chloroform to the supernatant again and centrifuge at 3,000 rpm for 15 minutes to additionally remove phenol and protein.
7) Add the same amount of isopropanol to the supernatant and separate the purified genomic DNA again.

  2. Repeated DNA library extraction and purification

1) The genomic DNA obtained in the first step 1-7 is dissolved in a TE solution. After dissolution, the final DNA concentration should be 1.3 μg / μl or more. The total absolute amount of DNA entering sonication is calculated in advance at this stage.
2) The DNA solution is crushed using an ultrasonic sonicator. The average size of the disrupted genomic DNA is set to 300 base pairs (see FIG. 2).
3) Put NaCl into the ultrasonically disrupted genomic DNA to a final concentration of 1 M, and heat at 90-100 ° C. for 7-12 minutes.
4) Immediately after heating, react at 55-65 ° C. for 5-6 hours.
5) After adding the same amount of isopropanol here, it is made to react at -20 degreeC for 1 hour or more.
6) Centrifuge to precipitate (14,000 rpm, 10 minutes).
7) Wash with 70% alcohol.
8) Dissolve the dried precipitate with distilled water.
9) Add (10X) S1 nucleolytic enzyme buffer solution to 1/10 of the final volume.
10) Calculate and add 1 unit of S1 nucleolytic enzyme per 1 μg of DNA (relative to the total amount calculated in step 2-1).
11) React at 30 ° C. (room temperature) for 30 minutes.
12) Perform a DNA concentration test on the reaction mixture.
At this stage, if about 30 to 35% of the initially calculated amount remains, a DNA library in which appropriate DNA repetitive sequences are selectively enriched can be obtained.
If more DNA remains, this is evidence that the non-repetitive sequence remaining as a single strand has not been completely removed by S1 nuclease.
13) Immediately after ultrasonic disruption, after 2-4 steps, before S1 nucleolytic enzyme treatment, after 2-4 steps, after S1 nucleolytic enzyme treatment, each sample was electrophoresed to determine whether the recrossing process was performed successfully. It was verified whether the degradation enzyme treatment was performed successfully, and the result is shown in FIG.
In lane 1 of FIG. 2, it is possible to confirm that about 300 base pairs of DNA are collected as sonicated DNA before 2-4 steps of DNA extraction and purification. It can be confirmed that the DNA having increased in size by re-crossing seems to be drawn upward in the state before the treatment with S1 nuclease after 4 steps.
In lane 3, DNA of about 300 bp is recovered in the state where the 2-4 process and the S1 nuclease treatment are all completed, but all single strands that are not repetitive sequences have been removed by S1 nuclease. It can be confirmed that the amount of DNA was reduced to about one third of the initial value.
14) S1 nucleolytic enzyme and protein were finally removed by treatment with the same amounts of phenol-chloroform and chloroform, respectively.
15) 3 mol NaOAc was added here so that it might become 1/10 of the final volume, and after adding the same amount of isopropanol, it was made to incubate at -20 degreeC for 1 hour or more.
16) It was centrifuged and precipitated (14,000 rpm, 10 minutes).
17) Washed with 70% alcohol.
18) Dissolve in a sterile TE solution (TE + sodium azide), measure the amount of DNA, and adjust the concentration at 1 μg / μl.
19) After treatment at 100 ° C. for 10 minutes, immediately cool and store frozen.
20) In the DNA hybridization research method, a DNA library in which DNA repetitive sequences produced are selectively concentrated is applied to the array comparison gene re-crossing reaction that can be quantitatively analyzed, and finally the possibility of blocking non-specific reaction is confirmed. (See FIG. 3).
FIG. 3 shows a result of mouse array-CGH using the DNA library prepared according to the present invention. As a result, when male and female genes are crossed, male X It is precisely seen that the chromosome is one replica less than the female.

<Manufacture of a DNA library in which DNA repetitive sequences are selectively concentrated from the human genome>

  1. Human genomic DNA isolation

1) 200 ml of cell lysis solution is added to 5 g of placental tissue, and the tissue is cut finely to increase the surface area (cell lysis solution: 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% SDS, proteolysis) Enzyme K 1 mg / ml).
2) React for 12 hours at 55 ° C. on a mixing shaker.
3) After confirming that the tissue is completely lysed, 200 ml of isopropanol is added thereto to primarily separate genomic DNA.
4) The primary separated DNA is washed with alcohol and dried, and then dissolved in 50 ml of TE buffer solution (pH 8.0).
5) Add the same amount of phenol / chloroform / isoamyl alcohol mixed solution to the dissolved DNA solution, and centrifuge at 3000 rpm for 15 minutes to remove proteins primarily.
6) Add the same amount of chloroform to the supernatant again and centrifuge at 3000 rpm for 15 minutes to additionally remove phenol and protein.
7) Add the same amount of isopropanol to the supernatant and separate the purified genomic DNA again.

  2. Repeated DNA library extraction and purification

1) The genomic DNA obtained in the first step 1-7 is dissolved in a TE solution. After dissolution, the final DNA concentration should be 1.3 ug / ul or more. The total absolute amount of DNA entering sonication is calculated in advance at this stage.
2) The DNA solution is crushed using an ultrasonic sonicator. The average size of the disrupted genomic DNA is set to 300 base pairs (see FIG. 4).
3) NaCl is added to the sonicated genomic DNA to a final concentration of 1 mol, and heated at 90-100 ° C. for 7-12 minutes.
4) Immediately after heating, react at 55-65 ° C. for 5-6 hours.
5) After adding the same amount of isopropanol here, it is made to react at -20 degreeC for 1 hour or more.
6) Centrifuge to precipitate (14,000 rpm, 10 minutes).
7) Wash with 70% alcohol.
8) Dissolve the dried precipitate with distilled water.
9) Add (10X) S1 nucleolytic enzyme buffer solution to 1/10 of the final volume.
10) Calculate and add 1 unit of S1 nucleolytic enzyme per 1 μg of DNA (relative to the total amount calculated in step 2-1).
11) React at 30 ° C. (room temperature) for 30 minutes.
12) Measure the DNA concentration of the reaction mixture. At this stage, if about 30-35% of the initially calculated amount remains, a DNA library in which DNA repetitive sequences are selectively enriched can be obtained.
If more DNA remains, this is evidence that the non-repetitive sequence remaining as a single strand has not been completely removed by S1 nuclease.
13) Immediately after ultrasonic disruption, after 2-4 steps, before S1 nucleolytic enzyme treatment, after 2-4 steps, after S1 nucleolytic enzyme treatment, each sample was electrophoresed to determine whether the recrossing process was performed successfully. It was verified whether the degrading enzyme treatment was performed successfully, and the results are shown in FIG.
In lane 1 of FIG. 5, it can be confirmed that about 300 base pairs of DNA are collected as sonicated DNA before 2-4 steps of DNA extraction and purification process. It can be confirmed that the DNA having increased in size by re-crossing is drawn upward in the state before the treatment with S1 nuclease after 4 steps.
In lane 3, DNA of about 300 bp is recovered in the state where the 2-4 process and the S1 nuclease treatment are all completed, but all single strands that are not repetitive sequences have been removed by S1 nuclease. It can be confirmed that the amount of DNA was reduced to about one third of the initial value.
14) S1 nucleolytic enzyme and protein were finally removed by treatment with the same amounts of phenol-chloroform and chloroform, respectively.
15) 3 mol NaOAc was added here so that it might become 1/10 of the final volume, and after adding the same amount of isopropanol, it was made to incubate at -20 degreeC for 1 hour or more.
16) It was centrifuged and precipitated (14,000 rpm, 10 minutes).
17) Washed with 70% alcohol.
18) Dissolve in a sterile TE solution (TE + sodium azide), measure the amount of DNA, and adjust the concentration to 1 μg / μl.
19) After treatment at 100 ° C. for 10 minutes, immediately cool and store frozen.
20) In the DNA hybridization research method, a DNA library in which DNA repetitive sequences produced are selectively concentrated is applied to the array comparison gene re-crossing reaction that can be quantitatively analyzed, and finally the possibility of blocking non-specific reaction is confirmed. (See FIG. 6).
FIG. 6 shows the result of array-CGH using the DNA library produced according to the present invention. Looking at FIG. 6, if normal male and female genes are crossed, It is precisely seen that in females, the X chromosome has one more copy than males.
It is also clearly shown that the Y chromosome is missing.

It is a photograph prepared by sonicating the mouse genomic DNA into slices having an average size of about 200 to 300 base pairs. Lane 1 shows the sonicated DNA 2-4 before the DNA extraction and purification process, where about 300 base pairs of DNA are gathered. Lane 2 shows the state after the 2-4 process and before the treatment with the S1 nucleolytic enzyme, and shows that the DNA size is increased by recrossing, and the lane 3 shows the 2-4 process and the S1. Although all nucleolytic enzyme treatments have been completed and about 300 base pairs of DNA have been recovered, single strands that are not repetitive sequences have all been removed by S1 nucleolytic enzyme, so the amount of DNA is Is reduced to about one third of The result of mouse array CGH (comparative genomic hybridization) using the DNA library which selectively enriched the repetitive sequence produced by this invention is shown. It is a photograph made by sonicating human genomic DNA into sections having an average size of about 300 base pairs. Lane 1 shows the state after 2-4 of the DNA extraction and purification process and before the S1 nuclease treatment, with the DNA size increased due to recrossing. Indicates. Lane 2 shows sonicated DNA from 2-4 steps before, about 300 base pairs in size, and Lane 3 shows all 2-4 steps and S1 nuclease treatment. In the finished state, about 300 bp of DNA was recovered, but all single strands that were not repetitive sequences were removed by S1 nuclease, so the amount of DNA was reduced to about 1/3 of the first. Shows what The result of the mouse array CGH using the DNA library which selectively enriched the repetitive sequence produced by this invention is shown.

Claims (5)

Extracting genomic DNA from animal cells;
Cutting the DNA into an average size of 200-400 bp using ultrasound;
Modifying the cleaved DNA strand and then recrossing;
Using a nucleolytic enzyme to selectively degrade single strands from the recrossed DNA muscle,
A method for producing a DNA library in which repetitive nucleotide sequences are selectively concentrated. The denaturation of the cleaved DNA strand is heated at 95 to 100 ° C for 7 to 10 minutes, and the recross reaction is cooled at 55 to 65 ° C for 5 to 6 hours. A method for producing a DNA library in which repetitive nucleotide sequences are selectively concentrated. 2. The method for producing a DNA library selectively enriched for repetitive nucleotide sequences according to claim 1, wherein the animal cells are mice cells. 2. The method for producing a DNA library selectively enriched with repetitive base sequences according to claim 1, wherein the animal cells are human cells. A DNA library in which repetitive base sequences produced by the production method according to any one of claims 1 to 4 are selectively concentrated.