WO2016154992A1

WO2016154992A1 - Controllable method for extracting nucleic acids

Info

Publication number: WO2016154992A1
Application number: PCT/CN2015/075767
Authority: WO
Inventors: 邓亚光; 邓伟平
Original assignee: 武汉格蓝丽富科技有限公司
Priority date: 2015-04-02
Filing date: 2015-04-02
Publication date: 2016-10-06

Abstract

Disclosed is a controllable method for extracting nucleic acids, belonging to the field of biotechnology and medical science. The method uses controllable labeled nucleic acid probes, modified magnetic beads and magnets and other simple devices, can be used for extracting or concentrating the desired nucleic acid components from different bodily fluids, reagent solutions or cell lysates directly, does not use a separation column, does not require centrifugation, is suitable for the extraction of nucleic acid samples of different sizes, and can achieve the best nucleic acid extraction effect, especially when it comes to small bodily fluid samples and small amounts of cell samples. The method uses controllable labeled nucleic acid probes comprising localization deoxyribonucleic acids with a random or specific sequence structure, ribonucleic acids or hybrids of the two, or probes of biological substitutes, which can extract deoxyribonucleic acids, ribonucleic acids, and macromolecule and micromolecule nucleic acids from a sample as required. The nucleic acid extraction process is simple to operate, has a high recovery and a good purity, and is beneficial to nucleic acid detection and analysis with a high sensitivity and a high stability.

Description

Controllable nucleic acid extraction method

Technical field

The invention belongs to the field of biotechnology and medicine, and particularly relates to a controllable nucleic acid extraction method.

Background technique

Nucleic acid analysis in serum or plasma in recent years has shown many advantages in the early detection and application of disease. Traditional serum or plasma nucleic acid extraction method has a lot of restrictions on small amounts of serum or plasma samples. At the same time, the use of proteases in the treatment of samples has caused the instability of sample nucleic acid extraction and the loss of considerable enzymes. The proportion of nucleic acid components is limited in use and is inconvenient. Mutations in some genes are not detected at all in the context of large non-mutated genes, and there is no good method of concentration. The magnetic bead nucleic acid extraction method has been reported, in which the magnetic bead reagent is directly added to the sample, on the one hand, the magnetic beads easily damage the nucleic acid sample, and on the other hand, the combined pretreatment (such as high temperature treatment) destroys and reduces the magnetic beads. The efficiency of the combination. It has been reported that the magnetic bead extraction nucleic acid method easily and randomly loses a large amount of nucleic acid samples, and cannot effectively control the quality and quantity of nucleic acid extraction of the desired detection gene.

Summary of the invention

The object of the present invention is to provide a controllable nucleic acid extraction method for solving the above problems, which can be extracted from serum or plasma or biological fluid using a combination of a controllable probe and a specific magnetic bead. Method of nucleic acid.

The technical solution adopted by the invention is:

A controllable nucleic acid extraction method comprising the following steps:

(1) adding a specially labeled nucleic acid probe to a sample in which the nucleic acid needs to be extracted, and combining the nucleic acid probe with the nucleic acid in the sample;

(2) adding a modified magnetic bead which can be combined with the nucleic acid probe of the above (1) to the sample described in the step (1) to modify the labeling site of the nucleic acid probe and the surface of the magnetic bead Combine;

(3) adsorbing and collecting the magnetic beads with a magnetic field, so that the nucleic acid samples in the sample are also enriched, separated and extracted;

(4) The nucleic acid sample is released from magnetic beads or magnets for various nucleic acid detection and analysis.

Further, the specially labeled nucleic acid probe adds one or more specific chemical molecular structures to a common nucleic acid probe.

Preferably, the specific chemical molecular structure is combined with a modification of the surface of the magnetic bead or the magnetic bead under certain conditions; the specific chemical molecular structure is biotin (Biotin), and the surface of the magnetic bead The avidin (Strepavidin) is stably bound.

Preferably, the specially labeled nucleic acid probe comprises a deoxyribonucleic acid, a ribonucleic acid probe and a hybrid structure of the two, or a probe of a biological substitute, the probe sequence comprising a random probe sequence and a specific nucleic acid probe sequence .

Preferably, the sample comprises serum, plasma, body fluids, various reagent solutions or cell lysates derived from cells or tissues.

Further, the nucleic acid probe is combined with the nucleic acid in the sample to be characterized by high temperature treatment, acid-base treatment and rapid cooling treatment, and the combination is stable and specific.

Further, the magnetic bead surface modification refers to a molecular structure capable of binding to a probe label, including avidin (Strepavidin), which can be subjected to certain conditions (enzymatic treatment, high temperature treatment, and reagent treatment). ) Separate.

Preferably, the magnetic field comprises the use of an electromagnet, a permanent magnet or both.

Further, the method of releasing the nucleic acid product from the magnet or the magnetic beads in the step (4) comprises a digestion reaction, a heating and a reagent treatment.

Further, in the step (4), when the magnetic beads or the nucleic acid are released from the magnet, a non-magnetic sleeve may be placed on the magnet, so that the magnetic bead body to be captured is first combined on the surface of the plastic sleeve with the magnet inserted therein. After the magnet is removed, the magnetic beads are easily released.

Further, the nucleic acid in the sample comprises extracting whole deoxyribonucleic acid, whole ribonucleic acid and small molecule ribonucleic acid using a labeled random nucleic acid probe, and extracting a specific gene using a localized nucleic acid probe of a specific nucleic acid sequence structure of the label Or a target nucleic acid of a corresponding nucleic acid sequence structure, including macromolecules and small molecule nucleic acid molecules.

Further, the nucleic acid probe of a specific nucleic acid sequence structure can be used to concentrate the desired nucleic acid from the extracted nucleic acid sample.

The core of the inventive principle of the method is to use a labeled nucleic acid probe to be added to the test sample to bind to the nucleic acid in the sample. The label of the nucleic acid probe can be bound to the modification of the surface of the magnetic bead. At this time, the magnetic bead is added to the sample, and the labeled portion of the nucleic acid probe can be combined with the magnetic field adsorption principle to adsorb the magnetic beads. The nucleic acid sample is also extracted from the sample, directly used as a sample detection nucleic acid, or collected and post-treated to obtain a purified nucleic acid of interest.

The invention has the following advantages:

The greatest advantage of the method of the invention is that it is controllable when extracting nucleic acids in different samples. By designing the labeled probe, the quality and quantity of the extracted nucleic acid can be controlled (whole ribonucleic acid, whole deoxyribonucleic acid, specific gene nucleic acid, The amount of nucleic acid to be extracted) and finally the purity of the nucleic acid sample, especially for small samples, can effectively extract the nucleic acid sample, the sample can be a small amount of serum, plasma, various body fluids or a small amount of cell fluid Or cell lysate, etc. Compared with other magnetic bead methods, the first step of the method is to add a labeled nucleic acid probe to the sample, which is suitable for different physical and chemical treatments such as high temperature and acid and alkali, and is not damaged. The performance of the subsequent added magnetic beads can be used to extract nucleic acid samples as well as to concentrate nucleic acid samples of interest. The method is simple, easy to operate, easy to automate the instrument, and does not require protease treatment, which greatly reduces the loss of nucleic acid in the sample, especially the nucleic acid in the nucleic acid binding protein. The method can be used for extraction and concentration of whole deoxyribonucleic acid, whole ribonucleic acid, large and small molecular nucleic acids and target genes.

DRAWINGS

1 is a schematic diagram of a controllable nucleic acid extraction method according to Embodiment 1 of the present invention;

Figure 2 is a diagram showing free DNA in plasma as extracted by the method of the present invention;

Figure 3 is a graph showing the detection of exon 9 and 20 of the phosphatidylinositol 3-kinase gene in Example 4.

detailed description

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

Example 1

This embodiment provides a controllable nucleic acid extraction method.

Referring to Figure 1, there are various free nucleic acid molecules in the plasma of normal and disease patients (Step 1 of Figure 1 : a, b, c, single or double stranded, protein bound or unbound), and biotin is added (Biotin After the nucleic acid probe (1 in Figure 1), mix well, heat it, immediately placed on ice, and rapidly cool down, the nucleic acid probe will be randomly combined with the nucleic acid sample (Figure 2, step 2); The modified magnetic beads (2 in Fig. 1) are mixed with the above sample, and biotin is combined with avidin (Fig. 1 step 3); under the action of a magnetic field (3 in Fig. 1), the magnetic beads are adsorbed. Enrichment (Step 4 of Figure 1); at this point, remove the magnet or simultaneously add the release magnet (4 in Figure 1), and the enriched magnetic bead product is released from the sleeve of the magnet (Figure 5, step 5), which can be collected. To the pure magnetic bead product (5 in Figure 1, step 6). Warming treatment (60-90 degrees), the binding of biotin and avidin is broken, and the random probe will lose its binding to the corresponding nucleic acid sample at high temperature, thereby releasing the nucleic acid sample with biotin or more pure, for subsequent For nucleic acid analysis. The free DNA extracted from the plasma of breast cancer patients, as indicated by the arrows in Figure 2 (Figure 2).

Example 2

Free nucleic acids in plasma are extracted using random RNA probes.

2.1 Take 100 nanoliters of plasma and add 10 nanoliters of Biotin-labeled RNA random probe. After mixing well, heat, cool, and then place on ice.

2.2 Add 2 nanoliters of Strepavidin-modified magnetic beads, mix and place at room temperature.

2.3 Separate and collect magnetic beads by magnetic field adsorption.

2.4 The collected magnetic bead product is directly used as a sample for genetic analysis, or 1 nanoliter of RNaseH enzyme is added to release a nucleic acid sample for use in gene analysis.

Example 3

A specific free nucleic acid in the plasma is extracted with a DNA-labeled random probe.

3.1 Take the required amount of plasma sample and add an appropriate amount of Biotin-labeled DNA random probe. After mixing well, heat, cool, and then place on ice.

3.2 Add the appropriate amount of Strepavidin-modified magnetic beads, mix and place at room temperature.

3.3 Separate and collect the magnetic beads by magnetic field adsorption.

3.4 The collected magnetic bead product is directly used as a sample for genetic analysis or heat treatment to release a nucleic acid sample for use in gene analysis.

Example 4

Extraction of phosphatidylinositol 3-kinase gene-associated free nucleic acids in plasma by labeling phosphatidylinositol 3-kinase gene probe with specific DNA and RNA hybridization structures

4.1 Design of DNA and RNA hybrid structures Biotinylated phosphatidylinositol 3-kinase probes.

Design phosphatidylinositol 3-kinase gene exon 9 probe: *ctgtgaatcCAGAGGGGA* (consisting of DNA and RNA hybrids with biotinylated ends) and exon 20 probe: *ggaatgccagAACTACAATCTTTTG* (by DNA and The RNA hybrid is composed of biotin-labeled ends. The probe is characterized in that the nucleic acid sequence of the probe consists of a DNA (lower case) and RNA (uppercase) hybrid with a biotin end label.

4.2 Take 100 nanoliters of plasma and add 10 nanoliters of Biot in biotinylated DNA and RNA hybridization structure, biotinylated phosphatidylinositol 3-kinase nucleic acid probe. After mixing well, heat, cool, and then place on ice.

4.3 Add 2 nanoliters of Strepavidin-modified magnetic beads, mix and place at room temperature.

4.3 Separate and collect the magnetic beads by magnetic field adsorption (as shown in Figure 1).

4.4 Use the collected magnetic bead product directly as a sample for genetic analysis, or add 1 nanoliter of RNaseH enzyme treatment, heat treatment, apply a magnetic field to the test tube with a magnet to precipitate the magnetic beads, and the solution contains The pure phosphatidylinositol 3-kinase DNA was used for subsequent gene mutation analysis (Fig. 3, PCR detection of exon 9 and exon 20 of the phosphatidylinositol 3-kinase gene).

It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, and are not intended to be limiting, although the present invention will be described in detail with reference to the preferred embodiments. The modifications and equivalents of the present invention are intended to be included within the scope of the appended claims.

Claims

A controllable nucleic acid extraction method comprising the steps of:

(1) adding a specially labeled nucleic acid probe to a sample in which the nucleic acid needs to be extracted, and combining the nucleic acid probe with the nucleic acid in the sample;

(2) adding a modified magnetic bead which can be combined with the nucleic acid probe of the above (1) to the sample described in the step (1) to modify the labeling site of the nucleic acid probe and the surface of the magnetic bead Combine;

(3) adsorbing and collecting the magnetic beads with a magnetic field, so that the nucleic acid samples in the sample are also enriched, separated and extracted;

(4) The nucleic acid sample is released from magnetic beads or magnets for various nucleic acid detection and analysis.
The controllable nucleic acid extraction method according to claim 1, wherein the specially labeled nucleic acid probe adds one or more specific chemical molecular structures to a common nucleic acid probe.
The controllable nucleic acid extraction method according to claim 2, wherein the specific chemical molecular structure is combined with a modification of the surface of the magnetic bead or the magnetic bead under certain conditions; The specific chemical molecular structure is Biotin, which can stably bind to the avidin on the surface of the magnetic beads.
The controllable nucleic acid extraction method according to claim 2 or 3, wherein the specially labeled nucleic acid probe comprises a deoxyribonucleic acid, a ribonucleic acid probe, and a hybrid structure of the two, or a biological substitute Probes, probe sequences include random probe sequences and specific nucleic acid probe sequences.
The controllable nucleic acid extraction method according to claim 1, wherein the sample comprises serum, plasma, body fluid, various reagent solutions or cell lysates derived from cells or tissues.
The controllable nucleic acid extraction method according to claim 1, wherein the nucleic acid probe is combined with the nucleic acid in the sample to be suitable for high temperature treatment, acid-base treatment and rapid cooling treatment, and is combined and stabilized. And has specific characteristics.
The controllable nucleic acid extraction method according to claim 1, wherein the magnetic bead surface modification refers to a molecular structure capable of binding to a probe label, including avidin (Strepavidin), This combination can be separated under certain conditions (enzymatic treatment, high temperature treatment and reagent treatment).
The controllable nucleic acid extraction method according to claim 1, wherein the magnetic field comprises using an electromagnet, a permanent magnet, or both.
The controllable nucleic acid extraction method according to claim 1, wherein the step (4) releases the nucleic acid product from the magnet or the magnetic beads or unbonds the labeling molecule from the surface modification molecule of the magnetic beads. Methods include enzymatic cleavage, warming and reagent treatment.
The controllable nucleic acid extraction method according to claim 1, wherein in the step (4), when the magnetic beads or the nucleic acid are released from the magnet, a non-magnetic sleeve can be placed on the magnet to be captured. of The magnetic beads are first bonded to the surface of the plastic sleeve with the magnet inserted. When the magnet is removed, the magnetic beads are easily released.
The controllable nucleic acid extraction method according to claim 1, wherein the nucleic acid in the sample comprises extracting whole deoxyribonucleic acid, whole ribonucleic acid, and small molecule ribonucleic acid using a labeled random nucleic acid probe. Target nucleic acid probes of specific nucleic acid sequence structures, including macromolecules and small molecule nucleic acid molecules, are extracted using a localized nucleic acid probe of a labeled specific nucleic acid sequence structure.
The controllable nucleic acid extraction method according to claim 1, wherein the desired nucleic acid is concentrated from the extracted nucleic acid sample using a positioning nucleic acid probe having a specific nucleic acid sequence structure.