WO2012062202A1

WO2012062202A1 - Cot-1 dna, preparation method and use thereof

Info

Publication number: WO2012062202A1
Application number: PCT/CN2011/081954
Authority: WO
Inventors: 孔淑娟; 张俊青; 聂超; 程玲; 张秀清; 杨焕明
Original assignee: 深圳华大基因科技有限公司; 深圳华大基因研究院
Priority date: 2010-11-12
Filing date: 2011-11-08
Publication date: 2012-05-18
Also published as: CN102465178A; CN102465178B

Abstract

Provided are a method for preparing c _o t-1 DNA, a c _o t-1 DNA, a method for hybridizing nucleic acid, a method for capturing a nucleic acid sequence, a composition, a kit and a use of c _o t-1 DNA in preparing a reagent for nucleic acid hybridization or nucleic acid sequence capturing, wherein the method for preparing c _o t-1 DNA comprises the steps of: fragmenting the genomic DNA so as to obtain a first DNA fragment mixture, wherein the DNA fragment length in the first DNA fragment mixture is 100bp-1000bp; denaturing and re-naturing the first DNA fragment mixture successively so as to obtain a second DNA fragment mixture, wherein the second DNA fragment mixture contains single stranded DNA and double stranded DNA; removing the single stranded DNA from the second DNA fragment mixture so as to obtain a DNA fragment mixture with single stranded DNA removed; and purifying the DNA fragment mixture with single stranded DNA removed so as to obtain the c _o t-1 DNA.

Description

C ₀ t-1 DNA and its preparation method and use priority information

Priority is claimed on Japanese Patent Application No. 201010541872.0, the entire disclosure of which is hereby incorporated by reference.

Technical field

The present invention relates to the field of molecular biology, and in particular to DNA and methods for its preparation and use. More specifically, the present invention relates to a preparation c. DNA method, a c. DNA, a method of nucleic acid hybridization, a method of capturing a nucleic acid sequence, a composition, a kit, and the use of c ₀ tl DNA in the preparation of a reagent for performing nucleic acid hybridization or capturing a nucleic acid sequence.

Background technique

In molecular biology, a class of DNA with Cj = l is defined as Cj-1 DNA (sometimes also labeled as cj-l DNA). Among them, C. It is a unit in the renaturation kinetics of DNA, which represents the renaturation time (t, sec), C of a single DNA chain at a unit concentration (C., mol/L). Straight is actually the product of the single-chain starting concentration (C.;) and the reaction time (t) in the hybridization solution. Under the same conditions, the DNA repeats the C. The value is small, and the non-repetitive sequence has a larger C. straight.

C-l DNA is commonly used as a DNA competitive blocking agent to block repeats that may cause non-specific hybridization in genomic hybridization. For example, when using probes containing repeat sequences for Northern hybridization, Southern hybridization, FISH localization, and somatic in situ hybridization, the repeats present in the probe will produce a wide range of non-specific strong hybrid background signals. Thereby the hybridization signals of the low copy or single copy sequences to be detected are masked off. Therefore, in performing the above experimental procedures, these repeat sequences in the probe should be considered first to avoid non-specific hybridization signals, thereby capturing single-copy or low-copy sequences such as exons, and enhancing the sequences such as exons. The purpose of the detection signal. The most ideal method is to use an excess of genomic repeat DNA, such as cj-l DNA, to pre-block these probes to block the repetitive DNA present in the probe.

However, current methods for preparing cj-1 DNA are still not sufficient.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, in one aspect of the invention, a method of preparing cj-1 DNA is presented. According to an embodiment of the present invention, the method comprises the steps of: fragmenting genomic DNA to obtain a first DNA fragment mixture, wherein the length of the DNA fragment in the first DNA fragment mixture is from 100 bp to 1000 bp; The DNA fragment mixture is subjected to denaturation treatment and renaturation treatment in order to obtain a second DNA fragment mixture, the second DNA fragment mixture comprising single-stranded DNA and double-stranded DNA; and removing single-stranded DNA in the second DNA fragment mixture, To obtain a mixture of DNA fragments from which single-stranded DNA is removed; and to purify the DNA fragment mixture from which the single-stranded DNA is removed, to obtain the c. DNA. Based on this method, c can be prepared simply, efficiently, and with high throughput. -J DNA, and the resulting c. - J DNA works well for hybridization. Compared with the currently expensive commercial cj-1 DNA products, the preparation of cj-1 DNA by the method for preparing cj-1 DNA of the present invention is low in cost and easy to generalize. Further, the present invention also provides a CJ-1 DNA obtained by a method for producing c ₀ tl DNA according to an embodiment of the present invention. The c. DNA is inexpensive to manufacture and has a wide range of uses.

According to still another aspect of the present invention, the present invention also provides a method of nucleic acid hybridization. According to some embodiments of the invention, the method comprises the step of blocking using c^ DNA according to an embodiment of the invention. Based on this method, an excess of c is used prior to nucleic acid hybridization. DNA, which can be conveniently and efficiently blocked to shield the repeat DNA, can effectively avoid the non-specific hybrid background signal generated by the repeat sequence in the subsequent nucleic acid hybridization process, and ultimately improve the efficiency of nucleic acid hybridization and enhance the hybridization effect. .

According to still another aspect of the present invention, the present invention also provides a method of capturing a nucleic acid sequence. According to an embodiment of the invention, the method comprises using c according to some embodiments of the invention. - J DNA to perform the blocking step. Based on the method, the probe can be conveniently and efficiently blocked, and the repeat sequence in the probe is shielded, thereby avoiding interference of non-specific hybridization signals during nucleic acid hybridization, thereby enhancing the effect of nucleic acid hybridization, and finally realizing the effect on the nucleic acid sample. Efficient capture of nucleic acid sequences.

According to yet another aspect of the invention, the invention also provides a composition. According to an embodiment of the invention, the composition comprises c according to an embodiment of the invention. DNA. Contains c according to an embodiment of the invention. This composition of DNA can be used to perform nucleic acid hybridization or to capture nucleic acid sequences. By carrying out nucleic acid hybridization and capture nucleic acid sequences using the composition comprising CJ-1 DNA, the effect of nucleic acid hybridization can be effectively enhanced, and the efficiency of capturing nucleic acid sequences can be improved.

According to another aspect of the present invention, the present invention also provides a kit. According to an embodiment of the invention, the kit comprises c according to some embodiments of the invention. DNA. With this kit, nucleic acid hybridization or nucleic acid sequences can be conveniently and efficiently performed.

According to still another aspect of the present invention, the present invention also provides the use of cj-1 DNA in the preparation of a reagent for performing nucleic acid hybridization or capturing a nucleic acid sequence according to an embodiment of the present invention. The reagent for nucleic acid hybridization or capture of a nucleic acid sequence prepared according to this can efficiently perform nucleic acid hybridization or capture of a nucleic acid sequence.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

Figure 1 : shows preparation c in accordance with one embodiment of the present invention. - Flow diagram of the method of _J ? DNA; Figure 2: shows preparation c according to one embodiment of the present invention. Schematic diagram of the thermal denaturation process and two cooling processes in the DNA method;

Figure 3: shows preparation c in accordance with one embodiment of the present invention. Electrophoretic detection results of a mixture of first DNA fragments obtained during the process of DNA;

Figure 4: shows a Mixer and chip for use in accordance with one embodiment of the present invention.

Detailed description of the invention

The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative only and not to limit the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or dark. Indicates relative importance or implicitly indicates the number of technical features indicated. Thus, features defining "first" and "second" may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, "multiple" means two or more unless otherwise stated.

According to one aspect of the invention, the invention provides an efficient preparation of c. The method of DNA. As shown in Fig. 1, a method of preparing cj-1 DNA according to an embodiment of the present invention includes the following steps:

S100: Fragmenting the genomic DNA to obtain a first DNA fragment mixture, wherein the DNA fragment in the first DNA fragment mixture has a length of 100 bp to 1000 bp.

According to an embodiment of the present invention, the source of genomic DNA is not particularly limited. According to some embodiments of the invention, genomic DNA can be extracted directly from a biological sample. Further, according to an embodiment of the present invention, c is prepared. The method of DNA may further comprise the step of extracting genomic DNA from the biological sample. According to an embodiment of the present invention, the method of extracting genomic DNA from a biological sample is not particularly limited and may be carried out using any prior art in the art, for example, a commercially available genome extraction kit may be employed. Preferably, the following method can be used, for example, by using a nuclear lysate, proteinase K and SDS, and digesting the tissue at 37 ° C - 56 ° C, preferably 56 ° C for 12 - 16 hours to allow the tissue to be fully lysed. Since the digestive time is 12-16 hours, the released DNA has been fully digested, and it can be successfully separated by centrifugation for 5 _ 15 min at 3000 - 10000 rpm. Therefore, it is not necessary to deliberately arrange a high-speed centrifuge, for example, by centrifugation at 4,700 rpm for 10 minutes to separate impurities such as DNA and protein, thereby eliminating the step of purifying the DNA, and not only saving a part of the purification of the DNA. Reagents also avoid the loss of proposed DNA during purification. Thereby, the genomic DNA of the biological sample can be obtained efficiently and conveniently, so as to carry out subsequent operations, and the efficiency of preparing cj-1 DNA is improved.

According to an embodiment of the present invention, a biological sample for extracting genomic DNA is not particularly limited. It will be understood by those skilled in the art that, preferably, the biological sample for extracting genomic DNA to prepare cj-1 DNA is derived from the same species as the sample to be hybridized or to be blocked, whereby the subsequent blocking, nucleic acid can be accurately and efficiently performed. Hybridization and other experiments. According to some specific examples of the invention, the biological sample that can be used is a tissue or cell selected from a human. Further, the tissue site or cell type for extracting genomic DNA is not particularly limited. Although the abundance of the genome of different tissues is different, an appropriate amount of genomic DNA can be obtained by adjusting the amount of tissue or cells.

According to an embodiment of the present invention, a method of fragmenting genomic DNA is not particularly limited. According to some specific examples of the invention, genomic DNA can be fragmented using techniques known in the art, such as ultrasonic, nitrogen, mechanical disruption, and the like. Preferably, the genomic DNA is fragmented by ultrasound. Thus, the length of the obtained genomic DNA fragment can be easily controlled by adjusting the parameters of the ultrasonic treatment. Specifically, the following steps can be used: 釆 Using the ultrasonic cell pulverizer, the extracted genomic DNA is interrupted into a DNA fragment of 100 bp - 1000 b by setting parameters according to the instructions. In addition, the ultrasonic cell pulverizer can interrupt 40 ml of DNA at a time, and can quickly obtain large quantities of interrupted DNA compared to existing interrupting equipment. The inventors have found that the instrument itself emits a large amount of heat during the fragmentation of genomic DNA by ultrasonic waves, and the interruption time is long, so that DNA denaturation is easily caused, and it can be cooled by ultrasonic treatment, for example, it will contain The container of DNA is placed in crushed water to effectively solve the above problems.

After fragmenting the genomic DNA, a first DNA fragment mixture is obtained, the first DNA fragment Fragments in the mixture are mainly composed of double-stranded DNA, and these DNA fragments are 100-1000 bp in length. The inventors have found that if the length of the DNA fragment in the first DNA fragment mixture is greater than 1000 bp, the diffusion rate is low, and in the subsequent renaturation process, the chance of the linear strands of the DNA fragments finding complementary strands is reduced; If the length is less than 100 bp, the diffusion speed is too fast, and it is difficult to grasp the time when the renaturation is renatured.

According to one embodiment of the present invention, genomic DNA can be interrupted into a DNA fragment of 200 bp - 800 bp. According to an embodiment of the present invention, genomic DNA can be interrupted into a DNA fragment of 200 bp - 600 bp. According to one embodiment of the present invention, genomic DNA can be interrupted into a DNA fragment of 250 bp to 600 bp. According to one embodiment of the present invention, genomic DNA can be interrupted into a DNA fragment of 300 bp - 500 bp, for example: 300, 350, 400, 450, or 500 bp. According to some embodiments of the invention, the length of the DNA fragment in the first DNA fragment mixture is 300 bp. The inventors have surprisingly found that when the length of the DNA fragment in the first DNA fragment mixture is 300 bp, subsequent steps of denaturation and renaturation treatment can be smoothly performed, thereby significantly improving the efficiency of preparing cj-1 DNA, and The obtained cj-1 DNA works very well in applications such as blocking probes, nucleic acid hybridization, and capture nucleic acid sequences.

According to some embodiments of the present invention, after obtaining the first DNA fragment mixture, the step of purifying the obtained first DNA fragment mixture may be further included before performing the denaturation treatment. Purification of the first DNA fragment mixture according to an embodiment of the present invention may be carried out by any prior art in the art, for example, by using a commercially available DNA purification kit or by purification with phenol chloroform (see, for example, Herrmann). BG, Frischauf AM: Isolation of genomic DNA. Methods Enzymol 1987, 152: 180-183, which is incorporated herein in its entirety by reference. Specifically, according to an embodiment of the present invention, the following method can be employed: Purify with phenol chloroform and precipitate DNA with an equal volume of isopropanol. The inventors have found that the purification of the first DNA fragment mixture by this method is not only convenient, rapid, but also suitable for large-scale operations, and can also significantly reduce reagent consumption, while the effect of equal volume of isopropanol precipitation is compared with conventional Compared to ethanol precipitation, the purification effect is very good, and the thus obtained purified first DNA fragment mixture can meet the needs of subsequent experiments.

S200: The first DNA fragment mixture is subjected to denaturation treatment and renaturation treatment in order to obtain a second DNA fragment mixture comprising single-stranded DNA and double-stranded DNA.

According to an embodiment of the present invention, the first DNA fragment mixture can be denatured and renatured by any technique known in the art, such as denaturation and renaturation of the DNA fragments by heating and cooling, respectively. Specifically, according to some specific examples of the present invention, the following steps can be employed: 变性 The first DNA fragment mixture is denatured by a boiling water bath, and then the DNA is rapidly cooled to achieve the renaturation temperature of the DNA to ensure Follow the formula c. =l = mol / L x T s The accuracy of the predetermined time T for which the DNA is subjected to renaturation treatment is continued, and then the DNA is renatured at the renaturation temperature for a predetermined time T. According to an embodiment of the invention, the denaturation treatment is carried out at about 100 degrees Celsius. According to some embodiments of the invention, the renaturation treatment is performed at about 65 degrees Celsius. Specifically, according to an embodiment of the present invention, when the DNA is renatured, the denatured DNA is placed in a 65 ° C water bath, and the temperature is lowered to 65 ° C before the DNA is renatured. After treatment, the physicochemical properties of the DNA after renaturation treatment can be restored. The inventors found that if the DNA is subjected to thermal denaturation treatment and rapidly cooled, the DNA may not be renatured, as shown in FIG. According to an embodiment of the invention, NaCl may be added to the first DNA fragment mixture during the renaturation treatment. The inventors of the present invention have surprisingly found that the efficiency of renaturation can be remarkably improved by the addition of NaCl. The expression used herein "in the process of renaturation, the addition of NaCl to the first DNA fragment mixture" should be understood in a broad sense, either by adding NaCl to the first DNA fragment mixture before renaturation, or After the first DNA fragment mixture is placed under renaturation conditions, NaCl is added to the DNA fragment mixture. According to an embodiment of the present invention, after the addition of NaCl, the final concentration of NaCl in the renaturation reaction system may be from 0.1 to 0.2 M, preferably from 0.12 to 0.13 M. It will be understood by those skilled in the art that the final concentration can be achieved by any known method, for example, by using a concentration of 3 M NaCl, according to stoichiometric calculation, an appropriate amount of NaCl is added to the DNA fragment mixture to reach the predetermined final concentration. NaCl. According to a specific example of the present invention, the NaCl solution can be preheated to the denaturation temperature before the addition of NaCl to the DNA fragment mixture, whereby the temperature change due to the addition of NaCl can be reduced, thereby improving the renaturation efficiency of the DNA fragment.

According to an embodiment of the invention, the renaturation process lasts for a predetermined time, which is calculated according to T=330 / c X 1000, where c is the concentration of DNA in ng/μ ΐ» specifically, according to In an embodiment of the present invention, the renaturation treatment time Τ (in seconds) of the DNA fragment in the first DNA fragment mixture of each tube is according to the formula T=330 / c χ 1000 (DNA average molecular weight M=330, initial concentration C. = ng / μ 1 = g / L 10- 3, the C. = mol / L = c / M 10- 3 = c / 330 x 10- 3; defined cj-1 DNA can be obtained from C. χ Ts = l = mol / L s; then Ts = l / C. = 330 / c χ 1000 ) Calculated, where c is the concentration of DNA [refers to the concentration of DNA (including single-stranded and double-stranded), the unit is ng / μ ΐ] , for example, if the DNA concentration of the Nth tube is 550 ng/μ 1, the renaturation time required for the tube is 330 _Ν 330 330 = 330 / 550 χ 1000 = 600 seconds. According to the embodiment of the present invention, the inventors have found that the doubling effect is optimal when the tempering process continues for a predetermined period of time. Among them, the higher the repeatability of the sequence, the faster the renaturation speed, so when the renaturation treatment continues for the predetermined time ,, the double-stranded repeat sequence obtained by the renaturation can only be highly repetitive or a small amount. Repeating the sequence, the resulting second DNA fragment mixture can meet the requirement for preparing cj-1 DNA. Here, the term "highly repeating sequence" refers to a DNA sequence having hundreds or even millions of copies in one genome. The average length of the repeating unit is 300 bp, and the renaturation time is in seconds; the term "moderate repeat sequence" refers to a DNA sequence of 10 to several hundred copies, and the renaturation time is divided into minutes. Among them, the moderate repeat sequence is usually a non-coding sequence, and the average length of the repeating unit is 300 bp, which often constitutes a sequence family, which is arranged by a single sequence, dispersed in the genome, and may play a role in gene regulation. In the obtained second DNA fragment mixture, in addition to the double-stranded DNA fragment, it also contains single-stranded DNA, which may be generated during the process of interrupting the genomic DNA, or may be denaturing. After renaturation and treatment, no renaturation of single-stranded DNA occurred. For these single-stranded DNA, these single-stranded DNAs can be removed in a subsequent step, such as by S1 nuclease.

S300: The single-stranded DNA in the second DNA fragment mixture is removed to obtain a DNA fragment mixture in which single-stranded DNA is removed.

According to an embodiment of the present invention, single-stranded DNA and unreplicated DNA molecules in the second DNA fragment mixture can be removed using any technique known in the art. According to some specific examples of the present invention, S1 nuclease can be used to remove single-stranded DNA and unreplicated DNA molecules in the second DNA fragment mixture.

According to the practice of the present invention, the amount of S1 nuclease used is not particularly limited. Preferably, the formula S1 can be adopted The amount of nuclease = cx 20 / 320 μ ΐ Calculate the amount of SI nuclease used, where c is the concentration of DNA (refers to the concentration of DNA [including single-stranded and double-stranded] in ng/μ ΐ λ. The inventors found By using the amount of the S1 enzyme calculated by the formula, the single-stranded DNA and the unreplicated DNA molecule in the second DNA fragment mixture can be effectively removed without affecting the double-stranded DNA therein, thereby enabling The preparation efficiency of c ₀ tl DNA and the quality of the obtained cj-1 DNA are guaranteed.

S400: Purification of a DNA fragment mixture for removing single-stranded DNA to obtain cj-1 DNA.

According to an embodiment of the present invention, the method of purifying the DNA fragment mixture for removing single-stranded DNA is not particularly limited, and may be the same as or different from the method of purifying the first DNA fragment mixture. Specifically, the DNA fragment mixture from which single-stranded DNA is removed can be purified by any technique known in the art, for example, using a commercially available DNA purification kit, or purified by phenol chloroform (for example, Herrmann can be referred to). BG, Frischauf AM: Isolation of genomic DNA. Methods Enzymol 1987, 152: 180-183). According to some specific examples of the present invention, purification of a DNA fragment mixture from which single-stranded DNA is removed is carried out by a phenol chloroform method in which DNA is precipitated using an equal volume of isopropanol. The inventors have surprisingly found that when phenol chloroform is used to purify a mixture of DNA fragments of single-stranded DNA, not only can large-scale DNA be purified conveniently and quickly, but also reagents can be saved, costs can be reduced, and the purification effect is very good. Prepared c. DNA can well meet the needs of experimentation and production. Further, according to still another aspect of the present invention, the present invention provides a c. DNA, which is prepared by a method for producing cj-1 DNA according to an embodiment of the present invention. According to still another aspect of the present invention, the present invention also provides a method of nucleic acid hybridization. According to some embodiments of the invention, the method comprises the step of blocking using cj-1 DNA according to an embodiment of the invention. According to an embodiment of the present invention, the expression herein "blocking using cj-1 DNA according to an embodiment of the present invention" should be understood broadly, and in the process of nucleic acid hybridization, a cj-1 DNA pair according to an embodiment of the present invention may be used. The nucleic acid probe or the repeat sequence in the nucleic acid sequence of the sample to be detected is subjected to blocking hybridization. According to an embodiment of the invention, it is preferred to use c. The DNA is blocked by a nucleic acid probe. The inventors have found c according to an embodiment of the invention. When the DNA is subjected to blocking hybridization to the nucleic acid probe, the repeat sequence existing in the probe is shielded, thereby preventing the non-specific strong hybridization background signal generated by the repeated sequence in the nucleic acid hybridization process from being low-copy or single-copy in the sample to be detected. The hybridization signal of the sequence is masked off, thereby enabling the nucleic acid sequence of the sample to be detected to be successfully subjected to nucleic acid hybridization, and finally capable of efficiently capturing a single copy or a low copy sequence, for example, an exon sequence having a relatively small content in the nucleic acid sample. When c according to an embodiment of the invention is used. When the nucleic acid sequence of the DNA to be tested is subjected to blocking hybridization, the repeat sequence in the nucleic acid sequence of the sample to be detected can also be effectively removed, thereby avoiding non-specific hybridization signal interference of the repeated sequence in the subsequent nucleic acid hybridization to ensure nucleic acid hybridization. s efficiency. Further, according to an embodiment of the present invention, c according to an embodiment of the present invention is used. -J DNA is blocked when c. DNA is used in excess. Specifically, according to some specific examples of the present invention, regardless of the kind of nucleic acid hybridization, the amount of cj-1 DNA used is a specific calculation of the amount of nucleic acid of the sample to be detected. According to an embodiment of the invention, c. The amount of DNA used is about 20 to 30 times the amount of nucleic acid of the sample to be detected. In addition, according to an embodiment of the present invention, the nucleic acid hybridization thereof is Northern hybridization, Southern Hybridization, or in situ hybridization, more specifically, the in situ hybridization is tissue in situ hybridization, somatic in situ hybridization, or fluorescence in situ hybridization. For in situ hybridization, a sample refers to tissue; for nucleic acid hybridization, a sample refers to a nucleic acid. Based on the method of nucleic acid hybridization according to an embodiment of the present invention, an excess of DNA can be used in a convenient and efficient manner to block the repeat DNA before the nucleic acid hybridization, thereby effectively avoiding the repeat sequence in the subsequent nucleic acid hybridization process. The interference generated by the non-specific strong hybrid background signal enhances the detection signal of the single copy or low copy sequence exon, which can ultimately improve the efficiency of nucleic acid hybridization and enhance the hybridization effect.

According to still another aspect of the present invention, the present invention also provides a method of capturing a nucleic acid sequence. According to an embodiment of the invention, the method comprises the step of blocking using cj-1 DNA according to an embodiment of the invention. According to an embodiment of the present invention, the expression "closed using cj-1 DNA according to an embodiment of the present invention" is to be understood broadly, and has been explained in detail above, and will not be described again. Based on the method, the repeat sequence in the nucleic acid sequence of the probe or the sample to be detected can be conveniently and effectively blocked, and the repeat sequence is shielded, thereby avoiding interference of non-specific hybridization signals during nucleic acid hybridization, so as to enhance the effect of nucleic acid hybridization. Eventually an efficient capture of the nucleic acid sequence in the nucleic acid sample to be detected is achieved.

According to yet another aspect of the invention, the invention also provides a composition. According to an embodiment of the invention, the composition comprises c according to an embodiment of the invention. -J DNA. According to some specific examples of the present invention, cj-1 DNA according to an embodiment of the present invention can be applied to nucleic acid hybridization and capture nucleic acid sequences, and in particular, cj-1 DNA according to an embodiment of the present invention can be effectively used for probes or The repeat sequence in the nucleic acid sequence of the sample to be detected is blocked to reduce the interference of the non-specific strong hybridization signal, thereby improving the efficiency of nucleic acid hybridization and nucleic acid capture. Thus, those skilled in the art will appreciate that compositions comprising cj-1 DNA in accordance with embodiments of the present invention may contain other conventional components depending on their particular use, and are not listed here. The composition according to an embodiment of the present invention can be applied to carry out nucleic acid hybridization or capture nucleic acid sequences. By performing nucleic acid hybridization and capturing nucleic acid sequences using the composition comprising cj-1 DNA, the effect of nucleic acid hybridization can be effectively enhanced, and the efficiency of capturing nucleic acid sequences can be improved.

According to still another aspect of the present invention, the present invention also provides a kit. According to an embodiment of the invention, the kit comprises c according to an embodiment of the invention. DNA. With this kit, nucleic acid hybridization or nucleic acid sequence can be carried out simply and efficiently, and the cost is low.

According to still another aspect of the present invention, the present invention also provides the use of cj-1 DNA in the preparation of a reagent for nucleic acid hybridization or capture of a nucleic acid sequence according to an embodiment of the present invention, which will not be described herein. According to an embodiment of the present invention, an agent for nucleic acid hybridization or capture of a nucleic acid sequence prepared by using the same can efficiently perform nucleic acid hybridization or capture of a nucleic acid sequence, and the hybridization result is good.

It should be noted that the method for preparing Cot-1 DNA, the method for nucleic acid hybridization, and the method for capturing sequences according to an embodiment of the present invention are all completed by the inventor of the present application through arduous creative labor and optimization work. Embodiments of the present invention will be described in detail below with reference to the embodiments. Those skilled in the art will understand that the following examples are merely illustrative of the invention and should not be construed as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. Reagent used Or if the instrument does not indicate the manufacturer, it is a regular product that can be obtained through the market. Example 1: Preparation of Cot-1 DNA Sample 1

Ultrasonic interruption

1) Dispense human genomic DNA into 50 ml centrifuge tubes, each tube not exceeding 40 ml.

2) Interrupted with a cleaned ultrasonic cell pulverizer (model: scientz08-II, produced by Zhejiang Ningbo Xinzhi Biotech Co., Ltd.).

Set the parameters (40 ml system) as shown in Table 3 below:

Table 1: Interrupt condition parameter settings

Since the instrument itself emits a lot of heat during the interruption with ultrasonic waves, and the interruption is long, the DNA may be denatured without adding water. Therefore, a water tank is placed in the processing space of the ultrasonic cell pulverizer, filled with crushed water, the DNA is placed in a centrifuge tube, and the centrifuge tube is placed in the crushed water. Be careful not to allow the broken water to accumulate in the nozzle of the centrifuge tube to avoid contamination.

3) The interrupted DNA is subjected to electrophoresis detection. The ideal fragment size is 100 bp - 1000 bp, and the main band is preferably located at 300 bp. If the interrupted fragment fails to meet the requirements, the interruption parameter should be re-adjusted according to the interruption to perform a second interruption.

The results of the electrophoresis test are shown in Figure 3. In Figure 3, the left lane is a 100 bp ladder marker, the right lane is a DL2000 marker, and the 1 - 3 in the figure represents three samples, which are parallel samples. In the electrophoresis test, the loading of three parallel samples was 1 microliter, and the loading of both Markers was 6 microliters. The results show that the fragment range is 100 - 1000 bp and the main band is concentrated at 200 - 600 bp, which meets the requirements for interruption.

2. Purification of DNA after interruption

The DNA was purified by phenol chloroform method as follows:

1) Add an equal volume of phenol chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1), mix well, place for 3 min to 5 min, and centrifuge at 4700 rpm for 5 min.

2) Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform, mix well, and place for 3 min to 5 min.

Centrifuge at 4700 rpm for 5 min.

3) Transfer the supernatant to a new centrifuge tube, add 1/10 volume of 3M sodium acetate, mix well, add 0.8 times volume pre-cooled at -20 °C to an equal volume of isopropanol, -20 °C The precipitate was placed for more than 1 hour.

4) Remove at -20 °C for 10 min at room temperature and centrifuge at 10700 rpm for 10 min.

5) Remove the supernatant, add 40 ml of 70% ethanol, and centrifuge at 4700 rpm for 5 min.

6) Remove the supernatant, wash it with 20 ml of absolute ethanol, and centrifuge at 5700 rpm for 5 min. 7) Remove the supernatant and dry the DNA in a constant temperature incubator at 50 °C.

8) Dissolved in 20 ml of mim-Q water.

Nanodro measures DNA concentration a.

In the purification of DNA, a commercially available DNA purification kit such as a product of PALL, QIAGEN, or the like can also be used.

3. Denaturation and renaturation of DNA after interruption

Preparation before performing renaturation DNA:

Open the water bath and adjust the water volume. Set the three temperatures to 100 °C, 65 °C and 37 °C respectively. Calculate the required reagent amount: Set a total of N tubes, then the required 3M NaCl is 2.22 χ N The ml, 10 x SI buffer was 2.47 χ N ml, and the amount of SI nuclease required per tube was c X 20 / 320 μl. Prepare a water box.

1) Dispense the DNA solution into 10 ml each tube using a 50 ml centrifuge tube.

2) Calculate the amount of DNA solution required according to the DNA concentration a determined by Nanodrop above, according to the amount of 20 ml per tube of 50 ml centrifuge tube and the final concentration of 550 ng / μL DNA solution. For example, if the DNA concentration a is 750 ng/μl, the amount of DNA solution required is X ml = 20 ml X 550 ng / microliter / (750 ng / microliter).

3) Add x ml from the DNA solution of concentration a to a new 50 ml centrifuge tube and fill the total volume to 20 ml with milli-Q water. 100 μl of the undenatured DNA and the prepared cj-1 DNA, which were used as QPCR assays, were added to a 1.5 ml centrifuge tube for comparison. The concentration of the DNA solution in each tube was accurately determined using Nanodrop.

4) Place the 50 ml centrifuge tube containing the DNA solution in 3) into a 100 °C water bath for 10 min, and place the 3 M NaCl solution in a 65 C water bath for later use. The 3M NaCl was preheated to 65 °C in advance so as not to affect the temperature of the liquid when it was added later.

5) Transfer the 50 ml centrifuge tube from 4) to a 65 °C water bath for 5 min.

6) Add 2.22 ml of pre-warmed 3M NaCl to each tube, mix upside down, place in a 65 °C water bath and anneal according to the calculated DNA refolding time T.

7) After the renaturation time has elapsed, quickly remove the 50 ml centrifuge tube into the water in the water tank, add 2.47 ml of 10 X S 1 buffer to each tube, mix upside down, and insert into the water.

8) Wait 2 to 3 minutes, add the calculated amount of S1 enzyme to each tube, mix upside down, and centrifuge quickly to 3000 rpm or more with a centrifuge.

9) Transfer the centrifuge tube to a 37 water bath for 1 hour to digest. The digested DNA solution should be immediately subjected to the next extraction and purification.

4. Purification of DNA after renaturation

1) Add 25 ml of phenol chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1), mix well, place for 3 min to 5 min, and centrifuge at 4700 rpm for 5 min.

2) Transfer the supernatant to a new centrifuge tube, add 25 ml of chloroform, mix well, place for 3 min to 5 min, and centrifuge at 4700 rpm for 5 min.

3) Transfer the supernatant to a new centrifuge tube, add 1/10 volume of 3M sodium acetate, mix well, add -20

An equal volume of isopropanol precooled at ° C, and the precipitate was allowed to stand at -20 ° C for more than 1 hour. 4) After taking out at -20 °C, leave it at room temperature for 10 min, wipe the water on the outer wall of the centrifuge tube with absorbent paper, and centrifuge at 4700 rpm for 10 min.

5) Pay attention to the sediment at the bottom of the centrifuge tube, carefully pour off the supernatant, then transfer the precipitated DNA to a new 50 ml centrifuge tube, add 25 ml of 70% ethanol, and centrifuge at 4700 rpm for 5 min.

6) Remove the supernatant, wash it with 20 mL of absolute ethanol, and centrifuge at 5700 rpm for 5 min.

7) Remove the supernatant and place the DNA in a constant temperature incubator at 50 °C.

8) Add the pH 8.0 TE solution to dissolve the dried DNA, and prepare the cj-l DNA, mark the date and batch, and set aside.

Thus prepared cj-l DNA sample 1. Example 2: Selection of renaturation time

In order to determine the renaturation time of the DNA in Example 1, the inventors have explored at different times as follows: Eight cs were prepared in a similar manner as in Example 1. The renaturation time of DNA samples is shown in Table 2. Dilute 8 samples to 10 ng/μ liter, add primers and SYBR reagent, put them into QPCR special plate, and perform QPCR detection (real-time fluorescent quantitative PCR) on the machine. Sequence bad QPCR provided with primers according NimbleGen Arrays User 's Guide-Sequence Capture Array) e / wry 1 J, synthesized by Invitrogen Corporation, using TaKaRa company SYBR Premix Ex Tag ™ (prefect Real Time) 200 reactions kits, Num. For DRR041A, QPCR detection was performed.

The specific operation is carried out according to the instructions of the kit.

The reaction system is as follows:

Cj-l DNA sample 1 ^: liter

ROX Dye 0.4 microliters

SYBR premix 10 microliters

Primer 1 μL

Water 7.6 μl

Total volume 20 microliters

The reaction procedure is as follows:

95 °C 30s

95 °C 5s '

60 °C 34s J 45 cycles.

The QPCR results are shown in Table 2.

Table 2: QPCR results of cj-l DNA prepared at 8 different renaturation times

τ SYBR Green I 31.15781

τ SYBR Green I 32.52058

2Τ SYBR Green I 27.10918

2Τ SYBR Green I 27.59618

4Τ SYBR Green I 5.503951

4Τ SYBR Green I 5.14319

In Table 2, the Ct value refers to the number of cycles corresponding to the inflection point from the baseline to the exponential growth. Here, Q refers to the Ct value of the sample after the treatment, that is, after the renaturation. The baseline is the horizontal part of the amplification curve.

Table 2 shows that as the renaturation time is extended, the smaller the Q value, the prepared c. The more difficult it is to ensure the quality of DNA, the less time 1/2 has less effect, but this will result in a reduction in reproducible DNA, thus affecting the yield, so time T is preferred as the optimal renaturation time. Example 3: Verification of hybridization effects in sequence capture

First, the sample used in the experiment:

Sample of the danger certificate: cj-1 DNA sample prepared in Example 1.

Control sample: Invitrogen's product cj-l DNA (item number 15279-011).

Hybridization sample: Human genomic library, construction procedure using illumina's Paired-End DNA Sample Prep Kit (PE-102-1001), according to the kit instructions.

Second, the experimental instrument: Roche NimbleGen chip.

Third, the experimental method:

Preparation before the experiment:

Turn on the hybridization unit and adjust the temperature of the hybridization unit to 42 °C. Turn the 2 dry baths to 70 °C and 95 °C respectively. The chip and Mixer were assembled using PAMT (see Figure 4) and the assembled Mixer-array was placed on the hybridizer.

1) The verification sample and the control sample were separately dispensed in a volume of 450 μg per tube, and a hole was punched in a 1.5 ml centrifuge tube cap with a syringe needle hole, and then evaporated to dryness under a reduced pressure in a SpeedVac.

2) Take 5 μg of the hybrid sample separately into the centrifuge tube of step 1), add 11.2 μl of pure water, and transfer to a 70 °C dry bath for 5 minutes.

3) Remove the centrifuge tube from the dry bath, shake it and place it on the centrifuge for full speed centrifugation. Add the following two reagents: 2X SC Hybridiation Buffer 18.5 μl

SC Hybridiation Component A 7.3 ^:L

4) After shaking and mixing, place it on a centrifuge and centrifuge at full speed for 3 _ 30 seconds. The centrifuged sample was transferred to a 95 °C dry bath for 5 minutes.

5) The sample was taken out and shaken, placed in a centrifuge and centrifuged at full speed for 30 seconds, placed on a hybridization instrument, and placed at a position of 42 ° C in a centrifuge tube to prepare for hybridization.

6) Confirm that the chip placement position matches the hybridization instrument, use the M100 pipette to take 37 μl of the sample and carefully pass the Fill Port. The sample in the pipette is injected into the chip.

7) Use Port seals to seal the two sample ports. Press and hold the two sample ports with two fingers. At the same time, use force. Note that the seal is not problematic and hybridize at 42 °C.

8) After the hybridization, elute the hybridized DNA on the NimbleGen sequence capture chip using the Roche NimbleGen Elution System according to the instrument instructions. The sample after the first batch of hybridization was obtained.

The above operation was repeated 9 times, and the hybridized samples of Batches 2 - 10 were respectively obtained, and then the hybridization degree was detected by the QPCR method of Example 2.

Fourth, experimental results and data analysis:

As shown in Table 3 below.

Table 3: Comparison of enrichment before and after hybridization between validation and control samples

Batch 5 sample 1 33.982 25.875 8.108 117.408 127.309

29.462 21.963 7.500 75.521

31.363 23.391 7.972 188.998

In vitro gen products 33.982 25.884 8.098 116.762 126.244

29.462 21.989 7.473 74.385

31.363 23.402 7.961 187.584

Batch 6 Sample 1 33.982 25.949 8.033 112.371 124.861

29.462 22.039 7.423 72.271

31.363 23.383 7.980 189.940

In vitro gen products 33.982 25.965 8.017 111.304 126.680

29.462 22.408 7.054 58.421

31.363 23.228 8.135 210.315

Batch 7 Sample 1 33.982 25.478 8.504 148.205 142.422

29.462 21.867 7.595 79.796

31.363 23.310 8.052 199.264

In vitro gen products 33.982 25.453 8.530 150.443 147.441

29.462 21.954 7.508 75.905

31.363 23.188 8.175 215.974

Batch 8 Sample 1 33.982 26.168 7.814 98.797 116.847

29.462 21.940 7.522 76.505

31.363 23.506 7.857 175.238

In vitro gen products 33.982 25.912 8.070 114.852 119.227

29.462 21.953 7.510 75.957

31.363 23.580 7.783 166.872

Batch 9 Sample 1 33.982 25.845 8.138 119.504 134.531

29.462 21.990 7.473 74.357

31.363 23.233 8.130 209.732

In vitro gen products 33.982 25.762 8.221 125.458 134.155

29.462 21.908 7.554 77.929

31.363 23.312 8.051 199.078

Batch 10 Sample 1 33.982 25.843 8.140 119.634 127.639

29.462 22.045 7.418 72.029

31.363 23.373 7.990 191.254

In vitro gen products 33.982 25.717 8.265 128.771 131.491

29.462 21.998 7.465 74.005 31.363 23.369 •994 191.697

Calculation of enrichment:

The QPCR can measure the Ct value of the sample before hybridization and the Q value of the sample after hybridization, and the enrichment of the sample can be calculated by comparing the two before. The formula for enrichment is (1+E) ^Δα , where Ε represents the amplification efficiency of the primer, ideally 1+Ε=2, ie enrichment = 2 ^Δα . A Ct refers to the difference between the Ct value of the sample before hybridization and the Ct value of the sample after hybridization.

The cj-1 DNA prepared in Example 1 and the cj-1 DNA product produced by Invitrogen (Cat. No. 15279-011) were respectively applied to the Roche NimbleGen chip hybridization by QPCR detection, and the two were compared by enrichment. It can be seen that the cj-1 DNA prepared by the invention can be successfully applied to the sequence capture technology. Industrial applicability

A preparation c of the invention. DNA method, a c. DNA, a method of nucleic acid hybridization, a method of capturing a nucleic acid sequence, a composition, a kit, and the use of cj-1 DNA in preparing a reagent for performing nucleic acid hybridization or capturing a nucleic acid sequence, can be prepared High quality cj-l DNA, and can be effectively applied to nucleic acid hybridization and nucleic acid sequence capture.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

Claim

1. A preparation c. The method of DNA is characterized in that it comprises the following steps:

Fragmenting the genomic DNA to obtain a first DNA fragment mixture, wherein the length of the DNA fragment in the first DNA fragment mixture is from 100 bp to 1000 bp;

The first DNA fragment mixture is subjected to denaturation treatment and renaturation treatment in order to obtain a second DNA fragment mixture, the second DNA fragment mixture comprising single-stranded DNA and double-stranded DNA;

Removing the single-stranded DNA in the second DNA fragment mixture to obtain a DNA fragment mixture from which single-stranded DNA is removed;

The DNA fragment mixture from which the single-stranded DNA is removed is purified to obtain the cj-1 DNA.

2. The method of claim 1 further comprising the step of extracting genomic DNA from the biological sample.

3. Method according to claim 2, characterized in that the biological sample is human tissue or cells.

4. The method according to claim 1, wherein the fragmentation is performed by sonicating the genomic DNA.

The method according to claim 1, wherein the DNA fragment in the first DNA fragment mixture has a length of 200 bp to 600 bp.

The method according to claim 5, wherein the DNA fragment in the first DNA fragment mixture has a length of 300 bp.

7. The method of claim 1, further comprising the step of purifying the first DNA fragment mixture prior to subjecting the first DNA fragment mixture to a variability treatment.

8. The method according to claim 7, wherein the purifying the first DNA fragment mixture is carried out by using a phenol chloroform method, wherein an equal volume of isopropanol is used to precipitate DNA.

9. The method of claim 1 wherein said denaturation treatment is performed at about 100 degrees Celsius.

10. The method of claim 1 wherein the renaturation treatment is performed at about 65 degrees Celsius.

11. The method according to claim 10, wherein during the renaturation treatment, NaCl is added to the first DNA fragment mixture.

The method according to claim 1, wherein the renaturation processing continues for a predetermined time, the predetermined time being calculated according to T=330 / cx 1000, wherein c is a concentration of DNA, a unit Is ng/μ

13. The method according to claim 1, wherein the single-stranded DNA in the mixture of the second DNA fragments is removed using S1 nuclease.

The method according to claim 1, wherein the purification of the DNA fragment mixture for removing single-stranded DNA is carried out by using a phenol chloroform method in which DNA is precipitated using an equal volume of isopropanol.

15. Cot-1 DNA prepared by the method of any one of claims 1 to 14.

A method of hybridizing nucleic acids, comprising the use of c according to claim 15. The step of blocking the DNA.

17. A method according to claim 16 wherein the nucleic acid hybridizes to Northern hybridization, Southern hybridization, or in situ hybridization.

18. The method of claim 17, wherein the in situ hybridization is tissue in situ hybridization, somatic cell in situ hybridization, or fluorescence in situ hybridization.

A method of capturing a nucleic acid sequence, which comprises using the c of claim 15. -J DNA is the step of blocking.

20. A composition comprising c according to claim 15. -J DNA.

A kit comprising the c of claim 15. DNA.

22. A kit according to claim 21 for performing nucleic acid hybridization or capturing nucleic acid sequences.

23. Use of cj-1 DNA according to claim 15 for the preparation of a reagent for nucleic acid hybridization or capture of a nucleic acid sequence.