CN104278022B - A kind of preparation method and application of DNA capture probes - Google Patents

Abstract

The present invention provides a preparation method and application of a DNA capture probe, which adopts a probe preparation method for amplifying a target gene-randomly interrupting-biotin labeling, and replaces the traditional tile-type covering with the design idea of random covering, Compared with the synthesis of a large amount of DNA covering the target region in the traditional probe preparation method, the present invention only needs to synthesize the amplification primers of the target region, avoiding the cost increase problem caused by the direct synthesis of probes, thus greatly reducing the preparation cost and capturing sensitive There is no difference in the specificity and specificity compared with the probe prepared by the traditional probe preparation method.

Description

A kind of preparation method and application of DNA capture probe

technical field

The invention belongs to the field of nucleic acid probe preparation, and in particular relates to a preparation method and application of a DNA capture probe.

Background technique

Traditional Sanger sequencing utilizes a DNA polymerase to extend a primer bound to a template to be sequenced until a chain-terminating nucleotide is incorporated. Each reaction contains all four deoxynucleotide triphosphates (dNTPs) mixed with limited amounts of fluorescently labeled dideoxynucleoside triphosphates (ddNTPs) of different colors. Since ddNTPs lack the 3-OH group required for elongation, elongated oligonucleotides are selectively terminated at G, A, T, or C. The termination point is determined by the corresponding dideoxygen in the reaction. They have a common starting point, but end at different nucleotides. Fragments of different sizes can be separated by capillary electrophoresis, and the sequence can be deduced by observing fluorescent labels of different sizes.

The second-generation sequencing technology mainly adopts the method of sequencing while synthesizing. First, the DNA sample to be tested is broken into small fragments by ultrasonic waves, and different adapters are added to the two ends of these small fragments to construct a single-stranded DNA library. The adapters in these libraries are attached to a solid surface and bridge PCR amplification is performed on the surface. After continuous amplification and denaturation cycles, each DNA fragment is finally concentrated into a bundle at its own position, and each bundle contains many copies of a single DNA template. The purpose of this process is to achieve the signal intensity of the base Amplify to achieve the signal requirements required for sequencing. During the sequencing reaction, DNA polymerase, adapter primers, and four kinds of dNTPs with base-specific fluorescent labels are simultaneously added to the reaction system (as in the Sanger sequencing method). The 3'-OH of these dNTPs are chemically protected so that only one dNTP can be added at a time. After the dNTPs are added to the synthetic strand, all unused free dNTPs and DNA polymerase are eluted. Next, add the buffer required to excite fluorescence, use laser to excite the fluorescence signal, and complete the recording of the fluorescence signal by optical equipment, and finally use computer analysis to convert the optical signal into sequencing bases. In this way, after the fluorescent signal recording is completed, chemical reagents are added to quench the fluorescent signal and remove the dNTP 3'-OH protecting group, so that the next round of sequencing reaction can be performed.

Since its birth in 2005, the second-generation sequencing technology has shown broad application prospects. Through sequencing, scientists can see important information such as single nucleotide polymorphisms, mutation hotspots, genome structural variations, and population polymorphisms. In the field of evolution, scientists explore the evolutionary patterns between species through population polymorphism analysis; in the field of microorganisms, DNA sequencing typing has been proved to be fast and accurate; and in the field of medicine, genome resequencing plays an important role in discovering the relationship between SNPs and major diseases meaning.

Since we are often only interested in a specific region of the genome (such as the exome) rather than the whole genome, we use gene capture probes to capture specific regions and sequence the captured DNA sequences. Probe preparation is a key link in capture sequencing. The current commonly used probe preparation method mainly adopts the method of "tile coverage design and artificial base synthesis". The specific method is: design a certain length of probe sequence for the region of interest, and each sequence moves a certain distance along the gene position. Then, the above-mentioned sequences were synthesized in large quantities by means of artificial synthesis. A typical representative is the Chinese patent "A Screening Method for Mitochondrial Disease Genes" (CN 101270390 A). A 60bp probe sequence is designed for the non-repeated region in the region of interest, and each sequence is designed by moving along the gene position. Move 3bp between the probes to achieve tile-like coverage of the target area; then use in situ synthesis technology to synthesize a large number of probes. Other literature [Calvo SE, Compton AG, Hershman SG, Lim SC, Lieber DS, Tucker EJ, Laskowski A, Garone C, Liu S, Jaffe DB et al: Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Science translational medicine 2012,4(118):118ra110] also adopted a similar idea, but the probe length and moving distance were changed.

The biggest drawback of the "tile coverage design, artificially synthesized bases" method is that the cost is too high, and with the increase of the target area, the cost of probe synthesis rises sharply.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of a DNA capture probe, which can reduce the cost of probe preparation.

In order to achieve the above object, the present invention adopts the following technical solutions.

A method for preparing a DNA capture probe, comprising the following steps:

(1) Design specific amplification primers for regions of interest in the genome, and use the amplification primers to amplify the regions of interest to obtain amplification products;

(2) fragmenting the amplified product into DNA fragments by ultrasonic treatment;

(3) carry out biotin labeling to DNA fragment;

(4) After step (3), the DNA single strand labeled with biotin is separated from the DNA fragment to obtain a DNA capture probe.

The region of interest is human mitochondrial DNA.

The step (2) specifically includes the following steps: diluting the amplification product (recovered human mitochondrial DNA amplification product) to 10 ng/L, then taking 100 L of the diluted amplification product and putting it into an ultrasonic instrument for ultrasonic treatment, ultrasonic The output power of the instrument is 1000Hz, the number of ultrasonic treatment is 90 times, the time of single ultrasonic treatment is 15 seconds, and the interval between two ultrasonic treatments is 15 seconds.

Before carrying out biotin labeling, the DNA fragment obtained in step (2) is purified according to the size of the fragment, and then biotin-labeled. The purification method includes the following steps: amplify to 100L 10ng/L treated by step (2) Add nucleic acid purification magnetic beads (Ampure bead) to the product, mix well, and then let it stand for 5 minutes; after standing still, place it on a magnetic rack. After the nucleic acid purification magnetic beads are absorbed, discard the supernatant; then wash with 70% ethanol Nucleic acid purification magnetic beads, after washing, dry the nucleic acid purification magnetic beads, resuspend the dried nucleic acid purification magnetic beads in water and place them on the magnetic rack. After the nucleic acid purification magnetic beads are absorbed, transfer the supernatant, which contains Purified DNA fragments.

The added amount of the nucleic acid purification magnetic beads is 90L (the added amount of the nucleic acid purification magnetic beads determines the size of the recovered DNA fragments), and the consumption of 70% ethanol is 500-750L for each washing.

Described step (4) specifically comprises the following steps:

(a) Adsorb the successfully biotin-labeled DNA fragments with avidin-coated magnetic beads (invitrogen);

(b) After step (a), the magnetic beads coated with avidin are washed with aqueous sodium hydroxide solution, and the single-strand DNA of unlabeled biotin in the DNA fragment is separated from the avidin-coated magnetic beads removal on magnetic beads;

(c) After step (b), resuspend the avidin-coated magnetic beads with deionized formamide and heat at 95°C for 10 minutes to separate the biotin from the avidin, and then separate to obtain Single-stranded DNA labeled with biotin.

The concentration of the sodium hydroxide aqueous solution is 1.25mM (the alkalinity is too strong or too weak to achieve the purpose of washing).

The application of the DNA capture probe prepared by the method for preparing the DNA capture probe described above is used to capture a human mitochondrial DNA library from a human whole genome sequencing library.

The amount of the DNA capture probe is 2-2.5% of the mass of the sequencing library.

The beneficial effects of the present invention are reflected in:

The present invention adopts the probe preparation method of amplifying the target gene-random interruption-biotin labeling, replaces the traditional tile-type coverage with the design idea of random coverage, and synthesizes a large amount of DNA to cover the target area with the traditional probe preparation method In contrast, the present invention only needs to synthesize the amplification primers of the target region, avoiding the cost increase problem caused by direct synthesis of probes, thus greatly reducing the preparation cost and the sensitivity and specificity of capture are comparable to those of probes prepared by traditional probe preparation methods. There is no difference compared to the needle.

Description of drawings

Fig. 1 is the capture effect diagram of the human mitochondrial DNA capture probe prepared by the present invention, wherein, A-D line: the capture effect of the probe prepared by the present invention (four repetitions), E line: the capture effect of the probe prepared by traditional method .

detailed description

The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

(1) The preparation method of human mitochondrial DNA capture probe comprises the following steps:

1. Amplify the full-length mitochondrial DNA sequence with three pairs of primers (1F and 1R, 2F and 2R, 3F and 3R);

The conditions for the amplification are:

(1) PCR reaction system:

The DNA polymerase used is Toyobo KOD FX, product number KFX-101.

reaction system:

(2) The PCR reaction program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 8 min; 40 cycles; extension at 72°C for 15 min.

The sequences of the primers are shown in Table 1:

Table 1

2. Mix the amplification products of the three pairs of primers equimolarly, and break them into fragments of about 200bp with an ultrasonic instrument;

The steps and conditions of ultrasonic treatment are:

The recovered PCR product (the product mixture of three pairs of primers) was diluted to 10ng/L;

After dilution, take 100L and add it to the EP tube, then use Ningbo Xinzhi 98-Ⅱ cup ultrasonic instrument for ultrasonic treatment, adjust the output power to 1000Hz, 15 seconds on, 15 seconds off, 90 reactions;

After sonication, add 0.9 times the volume (90L) of Beckman Coulter's Ampure bead to the EP tube, mix thoroughly, and let stand for 5 minutes;

Then centrifuge slightly (the hand-held centrifuge used in the laboratory has no speed and time requirements, as long as the liquid sticking to the tube wall and cover can be thrown to the bottom of the tube, the term for this operation is spin-down), after centrifugation, the EP tube Place on a magnetic rack, and discard the supernatant after the magnetic beads (Ampure bead) are fully adsorbed;

Then add 700L 70% ethanol to wash the magnetic beads (Ampure bead) twice (the EP tube is not removed from the magnetic rack), and dry the magnetic beads after washing;

Then add 16L of water to the EP tube to fully resuspend the magnetic beads (Ampure bead);

Then place the EP tube on the magnetic rack, and after the magnetic beads (Ampure bead) are fully adsorbed, transfer the supernatant (the size of the DNA fragment contained is about 200bp) to a new PCR tube for storage;

3. Biotin-label the interrupted fragments;

Refer to Roche High Prime Product Instructions for Use. Item No. 1585649.

Denature the DNA fragment with a size of about 200bp in the PCR tube at 95°C for 5 minutes, and quickly insert it on ice; after cooling completely, add 4L Biotin-high prime, mix well, and incubate overnight at 37°C.

4. Use 100L of avidin-coated magnetic beads to separate biotin-labeled DNA fragments; (at this time, the biotin-labeled DNA fragments are bound to the avidin-coated magnetic beads).

5. Wash the avidin-coated magnetic beads bound with DNA fragments with 50 L of 1.25 mM sodium hydroxide aqueous solution to remove unlabeled single strands; (at this time, biotin-labeled beads are bound to the avidin-coated magnetic beads DNA single strand)

6. Resuspend the magnetic beads in 100L of deionized formamide, and heat at 95°C for 10 minutes to separate the biotin from the avidin (that is, the DNA single strand labeled with biotin is desorbed from the avidin-coated magnetic beads);

7. The Qiagen minElute kit was used to purify the DNA in the system described in 6, and the resulting product was a biotin-labeled DNA probe, about 3 g, which was the capture probe.

8. Capture the human mitochondrial DNA library from the human whole genome sequencing library with a biotin-labeled DNA probe, specifically as follows:

The target captured by this probe is the constructed high-throughput sequencing library;

Mix the prepared probe (2-2.5% of the mass of the sequencing library) with 100-500ng of the sequencing library, and hybridize for 24 hours under the condition of PCR reaction buffer;

Add avidin-coated magnetic beads to fully combine avidin and biotin;

Use wash buffer to wash away the non-specific binding library;

Add 1.25mM NaOH aqueous solution to separate the double-strands of the captured library, and recover the washed single-stranded library;

PCR amplification was performed on single-stranded libraries. The capture effect is shown in Figure 1. Referring to Fig. 1, compared with the probe capture effect prepared by the traditional method, the probe capture effect prepared by the method of the present invention has achieved 100% coverage of the target area and the coverage is relatively uniform, while the cost is less than 1% of the traditional method.

The greatest advantage of the present invention is that it can greatly reduce the cost of probe preparation under the premise of ensuring capture efficiency and specificity. Taking an academic paper published on Science translational medicine in 2012 as an example, the target synthesis region of the synthesized probe in the paper is about 4.1Mbp. According to the description in the article, a rough calculation will only cost more than 12 million yuan for the synthesis of probes. However, with the method of the present invention, less than 100,000 RMB is needed to prepare the same probe, and the capture effect is equivalent to it.

Claims (7)

1. a kind of preparation method of DNA capture probes, it is characterised in that：Comprise the following steps：

(1) special amplimer is designed for area-of-interest in genome, expand the region of interest using amplimer Domain obtains amplified production；

(2) ultrasonically treated method is adopted to interrupt the amplified production for DNA fragmentation

Amplified production is diluted to 10ng/ μ L, the amplified production after 100 μ L dilutions is then taken and is put in Ultrasound Instrument and carries out ultrasonic place Reason, the power output of Ultrasound Instrument is 1000Hz, and ultrasonically treated number of times is 90 times, and the single sonication time is 15 seconds, surpasses twice It is spaced 15 seconds between sonication；

(3) biotin labeling is carried out to DNA fragmentation

DNA fragmentation is rapidly inserted on ice after 95 DEG C of denaturation 5min；After cooling down completely, 4 μ LBiotin-high prime are added, Fully mix, 37 DEG C of overnight incubations；

(4) after step (3), from the DNA fragmentation, the isolated DNA for being marked with biotin is single-stranded, obtains DNA captures and visits Pin；

The area-of-interest is human mitochondrial DNA.

2. a kind of preparation method of DNA capture probes according to claim 1, it is characterised in that：Before carrying out biotin labeling, The DNA fragmentation that step (2) is obtained is purified according to clip size, biotin labeling, the method for the purifying is then carried out Comprise the following steps：Carry out after nucleic acid purification magnetic bead is added in the 100 μ L 10ng/ μ L amplified productions processed through step (2) Mix, then stand 5min；It is placed in after standing on magnet stand, after nucleic acid purification magnetic bead is adsorbed, inhales and abandon supernatant；Then with 70% Ethanol washs nucleic acid purification magnetic bead, dries nucleic acid purification magnetic bead after washing, and the nucleic acid purification magnetic bead after being dried with water is resuspended is rearmounted On magnet stand, after nucleic acid purification magnetic bead is adsorbed, supernatant is shifted, containing DNA fragmentation after purification in the supernatant.

3. a kind of preparation method of DNA capture probes according to claim 2, it is characterised in that：The nucleic acid purification magnetic bead Addition be 90 μ L, the consumption of 70% ethanol uses 500～750 μ L for washing every time.

4. a kind of preparation method of DNA capture probes according to claim 1, it is characterised in that：Step (4) specifically wrap Include following steps：

A () adsorbs the successful DNA fragmentation of biotin labeling with the coated magnetic bead of Avidin；

B () washs the coated magnetic bead of the Avidin after step (a) with sodium hydrate aqueous solution, by the DNA fragmentation The DNA of unmarked biotin is single-stranded to be removed from the coated magnetic bead of the Avidin；

C () heats 10min with the coated magnetic bead of the resuspended Avidin of deionized formamide and in 95 DEG C after step (b), The biotin is made to separate with the Avidin, the DNA for being then peeled off obtaining being marked with biotin is single-stranded.

5. a kind of preparation method of DNA capture probes according to claim 4, it is characterised in that：The NaOH is water-soluble The concentration of liquid is 1.25mM.

6. the DNA capture probes that prepared by a kind of preparation method of DNA capture probes as claimed in claim 1 are from people's full-length genome The application in capture human mitochondrial DNA library in sequencing library.

7. the DNA capture probes that according to claim 6 prepared by a kind of preparation method of DNA capture probes are from people's full genome The application in human mitochondrial DNA library is captured in group sequencing library, it is characterised in that：The consumption of DNA capture probes is the sequencing The 2～2.5% of Library Quality.