阅读说明：本技术 一种唾液dna提取的方法 (Saliva DNA extraction method ) 是由 张伟 于 2021-09-02 设计创作，主要内容包括：本发明公开一种提取纯化唾液DNA的方法,包括以下步骤：1)预处理：将唾液与预处理裂解液接触,去除生物细胞所粘附的固体物质,得到粗裂解液；2)核酸吸附：将步骤1)得到的粗裂解液与裂解结合液以及磁珠悬浮液混合,形成含有磁性纳米微球-DNA的复合体的溶液体系,收集所述溶液体系中的磁性纳米微球-DNA的复合体；3)洗涤：将步骤2)得到的磁性纳米微球-DNA的复合体依次用洗涤液I、洗涤液II洗涤,然后用洗脱液溶出DNA。本发明提供的方法提取DNA的效率可达90％,提取的DNA可应用于STR复合扩增、DNA测序、DNA定量等下游。(The invention discloses a method for extracting and purifying saliva DNA, which comprises the following steps: 1) pretreatment: contacting saliva with the pretreatment lysate to remove solid matters adhered to biological cells to obtain crude lysate; 2) nucleic acid adsorption: mixing the crude cracking solution obtained in the step 1) with a cracking binding solution and a magnetic bead suspension to form a solution system containing a magnetic nano microsphere-DNA complex, and collecting the magnetic nano microsphere-DNA complex in the solution system; 3) washing: washing the magnetic nano microsphere-DNA complex obtained in the step 2) by using a washing solution I and a washing solution II in sequence, and then dissolving out the DNA by using an eluent. The efficiency of extracting DNA by the method provided by the invention can reach 90%, and the extracted DNA can be applied to STR multiplex amplification, DNA sequencing, DNA quantification and other downstream.)

1. A method for extracting saliva DNA is characterized by comprising the following steps:

(1): pretreatment: adding saliva into a centrifuge tube, adding lysis solution and proteinase K solution, mixing, and incubating the centrifuge tube in a 56 deg.C water bath for 15 min;

(2) nucleic acid adsorption: adding the magnetic bead suspension and the DNA binding solution into a centrifugal tube, and standing at room temperature for 5min after vortex oscillation;

(3) washing: and (3) placing the mixture obtained in the step (2) on a magnetic frame, standing for 2min, after the magnetic beads are completely adsorbed on the side wall of the centrifugal tube, discarding the solution, washing with a washing solution 1 and a washing solution 2, and finally dissolving out the DNA by using an eluent to obtain the purified DNA.

2. The extraction method according to claim 1, wherein the lysate in step (1) comprises Tris 40-60mM, EDTA 10-20mM, NaCl 0.1-0.9M, dodecyl-N-betaine 0.1-0.3%, GuHCl 60-75%, NLS 0.6-0.9%, TritonX 1003-5%, NH4Cl 0.15-0.24%, mercaptoethanol 0.5-2.5%.

3. The method of extraction according to claim 2, wherein the lysis solution comprises Tris 55mM, EDTA 15mM, NaCl 0.3M, dodecyl-N-betaine 0.2%, GuHCl 75%, NLS 0.6%, triton x 1003%, NH4Cl 0.18%, mercaptoethanol 1%.

4. The method of claim 1, wherein the concentration of proteinase K solution in step (1) is 1.7-2.2 mg/mL.

5. The method of extraction according to claim 4, wherein the concentration of proteinase K is 2 mg/mL.

6. The method of claim 1, wherein the DNA binding solution in step (2) is ethanol, isopropanol or ethylene glycol.

7. The extraction method of claim 1, wherein the magnetic bead suspension in step (2) is an aqueous solution of silica-coated magnetic microspheres with a diameter of 50-1000 nm.

8. The extraction method according to claim 1, wherein in step (3), washing solution 1 is 50mM Tris-HCl, 200mM NaCl 50% ethanol solution, pH 8.0.

9. The extraction method according to claim 1, wherein the washing solution 2 in the step (3) is a 70-80% ethanol solution.

10. The method of claim 1, wherein the eluent in step (3) is TE buffer of pH8.0 containing 10mM Tris-HCl, 1mM EDTA.

Technical Field

The invention belongs to the field of DNA detection, and particularly relates to a saliva DNA extraction method.

Background

Nucleic acid extraction is the basis of various molecular biology detection methods, and efficient and complete extraction of DNA is the basis of PCR amplification, library construction, sequencing and other works. Except tissue and organ samples, the most commonly used in molecular biology detection is blood samples, but blood sampling requires professionals to use sterile blood sampling equipment, so that the cost and the complexity are high, and on the other hand, blood sampling brings direct pain and fear to patients and is rejected by many people; for the above reasons, molecular biological tests performed on blood samples have been inconvenient in many cases, particularly in large-scale screening or identification. There is a need to develop methods for extracting DNA for molecular biological assays using readily available samples, such as hair, saliva, and the like.

Saliva belongs to a biological sample which can be easily extracted without damage, and the components of the saliva are greatly different from those of serum and plasma, so that the DNA extraction is difficult to complete by using the same or similar kit: saliva is colorless, tasteless and odorless liquid; the pH value is about 6 to 7; saliva contains amylase, lysozyme, peroxidase, mucin, mucopolysaccharide, phospholipid, etc.; the large amount of mucin and mucopolysaccharide makes saliva viscosity reach 20 times of that of water, and these components are also the main problems to be solved and the main substances to be removed in the extraction of saliva DNA. Currently, the existing DNA extraction methods such as phenol chloroform method, salting-out method, adsorption column method, immunoaffinity method, magnetic bead method and the like can be mainly used for saliva DNA extraction, wherein the adsorption column method has obvious defects in extraction effect and extracted DNA concentration, so that it is necessary to develop a magnetic bead DNA extraction method with simple operation to improve extraction efficiency and meet the requirement of rapidly processing samples on site in screening, forensic investigation and other applications.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a simple, quick and efficient saliva DNA extraction method, which is suitable for extracting DNA from saliva preserved in a protective solution.

A method for extracting saliva DNA is characterized by comprising the following steps:

(1): pretreatment: adding saliva into a centrifuge tube, adding lysis solution and proteinase K solution, mixing, and incubating the centrifuge tube in a 56 deg.C water bath for 15 min;

(2) nucleic acid adsorption: adding the magnetic bead suspension and the DNA binding solution into a centrifugal tube, and standing at room temperature for 5min after vortex oscillation;

(3) washing: and (3) placing the mixture obtained in the step (2) on a magnetic frame, standing for 2min, after the magnetic beads are completely adsorbed on the side wall of the centrifugal tube, discarding the solution, washing with a washing solution 1 and a washing solution 2, and finally dissolving out the DNA by using an eluent to obtain the purified DNA.

Further, the cracking solution in the step (1) comprises 40-60mM of Tris, 10-20mM of EDTA, 0.1-0.9M of NaCl, 0.1-0.3 percent of dodecyl-N-betaine, 60-75 percent of GuHCl, 0.6-0.9 percent of NLS, 1003-5 percent of TritonX, 0.15-0.24 percent of NH4Cl 0.15 and 0.5-2.5 percent of mercaptoethanol. Preferably, the lysate comprises Tris 55mM, EDTA 15mM, NaCl 0.3M, dodecyl-N-betaine 0.2%, GuHCl 75%, NLS 0.6%, TritonX 1003%, NH4Cl 0.18.18%, mercaptoethanol 1%.

Further, the concentration of the proteinase K solution in the step (1) is 1.7-2.2mg/mL, and the preferable concentration is 2 mg/mL.

Further, the DNA binding solution in the step (2) is ethanol, isopropanol or ethylene glycol. Preferably 65% to 90% isopropanol.

Further, the magnetic bead suspension in the step (2) is an aqueous solution of silicon-coated magnetic microspheres with the diameter of 50-1000 nm.

Further, in the step (3), the washing solution 1 is a 50mM ethanol solution containing 50mM Tris-HCl and 200mM sodium chloride at a concentration of 50%; washing solution 2 is 70-80% ethanol solution.

Further, the eluent of step (3) was TE buffer of pH8.0 containing 10mM Tris-HCl, 1mM EDTA.

In the presence of high salt, DNA binds to the surface of silica-based coated magnetic beads. After rinsing, the high purity DNA is eluted in an eluent or deionized water. The yield of DNA is greatly related to the number of cells in the sample, the storage condition and the time. The purified DNA has good purity and high integrity (more than 15kb), and can be used for downstream experiments such as second-generation sequencing, chip detection, PCR detection and the like.

The saliva DNA extraction method comprises the following specific steps: (1) turning the saliva sample upside down for 5 times, mixing, and standing for 5 min; (2) transferring 500ul of saliva into a new 1.5ml centrifuge tube, adding 20 ul of proteinase K and 300 ul of lysis solution into the new centrifuge tube, vortex, shaking, mixing uniformly, and incubating the centrifuge tube in a 56 ℃ water bath for 15 min; (3) preparing a DNA binding solution with magnetic beads: after the magnetic beads are fully and uniformly mixed, 20 mu L of the mixture is added into 300 mu L of DNA binding solution (the mixture required by the experiment can be prepared in advance according to the proportion); (4) adding 320 mu L of DNA binding solution added with magnetic beads into the mixture obtained in the step (2), and standing at room temperature for 5min after vortex oscillation for 5 s; (5) placing the centrifugal tube on a magnetic frame for standing for 1 minute, and completely removing the solution after the magnetic beads are adsorbed on the side wall of the centrifugal tube; (6) adding 750 mu L of washing solution 1 into the centrifugal tube, taking the centrifugal tube off the magnetic frame for vortex oscillation for 5s, then placing the centrifugal tube on the magnetic frame for standing for 2min, and then absorbing the washing solution while avoiding contacting magnetic beads; (7) adding 750 mu L of washing solution 2 into the centrifugal tube, taking the centrifugal tube off the magnetic frame, carrying out vortex oscillation for 5s, placing the centrifugal tube on the magnetic frame, standing for 2min, and then absorbing the washing solution while avoiding contacting magnetic beads; (8) repeating the step (7) once; (9) standing the magnetic beads combined with the DNA obtained in the step (8) at room temperature for 10min, airing, adding 50 mu L of eluent or deionized water, uniformly mixing by vortex oscillation to completely resuspend the magnetic beads in the eluent, and standing for 5 min; (10) placing the centrifuge tube on the magnetic frame again, standing for 2min, transferring the eluate to a new centrifuge tube, and storing at-20 deg.C for use.

Compared with the conventional DNA extraction method, the saliva DNA extraction method provided by the invention has the following advantages: the whole operation flow method is simple and quick, and the whole extraction flow can be completed within 1 hour; the centrifugal operation is less in steps, and the experimental process can be used for automatic extraction of the workstation.

Drawings

FIG. 1 is the result of agarose gel electrophoresis identification of a portion of DNA samples in examples, which is schematically shown in the drawing, wherein M: DNA marker, 1-7 are samples 1-8 in sequence.

Detailed Description

The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.

Example 1

The specific steps of saliva DNA extraction are as follows:

1. 1mL of saliva of 8 volunteers is put into a collection tube with saliva protective agent, the saliva sample is turned upside down for 5 times and mixed evenly, and the mixture is kept stand for 5 min.

2. 500ul of saliva is taken and transferred into a new centrifugal tube of 1.5ml, then 20 ul of proteinase K and 300 ul of lysate are added into the new centrifugal tube, vortex, shake and mix evenly, and then the centrifugal tube is put into a water bath kettle of 56 ℃ for incubation for 15 min.

3. The tube was removed from the water bath and left to stand at room temperature for 5 minutes before brief centrifugation to reflux the solution to the bottom of the tube 300. mu.l of a well-mixed mixture of isopropanol (300. mu.l) and Magbeads (20. mu.l) was added to the tube.

4. The centrifuge tube was placed on a magnetic rack and allowed to stand for 1 minute, and the solution was completely discarded after Magbeads were adsorbed to the side wall of the centrifuge tube (keeping the centrifuge tube fixed on the magnetic rack).

5. And (3) taking the centrifugal tube off the magnetic frame, adding 750 mu L of cleaning solution 1 into the centrifugal tube, taking the centrifugal tube off the magnetic frame, carrying out vortex oscillation for 5s, standing on the magnetic frame for 2min, and then absorbing the cleaning solution while avoiding contacting magnetic beads.

6. Adding 750 mu L of washing solution 2 into the centrifuge tube, taking the centrifuge tube off the magnetic frame for vortex oscillation for 5s, placing the centrifuge tube on the magnetic frame for standing for 2min, and then absorbing the washing solution while avoiding contacting magnetic beads.

7. Repeating the step (6) once.

8. Keeping the centrifugal tube fixed on the magnetic frame, removing the solution on the tube bottom and the tube cover of the centrifugal tube by using a pipettor, then standing at room temperature for 5-10 minutes to fully volatilize the ethanol, adding 50 mu L of eluent or deionized water, carrying out vortex oscillation and uniform mixing to completely resuspend the magnetic beads in the eluent, and standing for 5 min.

9. And (3) placing the centrifuge tube on a magnetic frame for standing for 2 minutes, and transferring the solution into a new centrifuge tube by using a liquid transfer machine for preservation at-20 ℃ for later use after the Magbeads are fully adsorbed on the side wall of the centrifuge tube.

Example 2

Quality test result of saliva DNA extraction

The quality check of the DNA sample obtained in example 1 was confirmed by ordinary agarose gel electrophoresis.

Carrying out 1% agarose gel electrophoresis, wherein the electrophoresis conditions are as follows: the gel concentration is 0.8%; 0.5 XTAE electrophoresis buffer; electrophoresis was carried out for 20min at a voltage of 150 v. Electrophoresis results show a sharp high molecular weight band, which indicates that the extracted DNA sample has high purity and good quality. The electrophoretogram is shown in FIG. 1.