CN112813062A

CN112813062A - Normal-temperature-stored lysis binding solution for genome extraction and kit

Info

Publication number: CN112813062A
Application number: CN202110375814.3A
Authority: CN
Inventors: 潘超; 杨小岚; 宋建军
Original assignee: Wuhan Naci Biotechnology Co ltd
Current assignee: Wuhan Naci Biotechnology Co ltd
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2021-05-18
Anticipated expiration: 2041-04-08
Also published as: CN112813062B

Abstract

The invention discloses a normal-temperature-stored lysis binding solution for genome extraction and a kit. The lysis binding solution contains 2.5mol/L to 3.5mol/L chaotropic agent, 5mM to 20mM metal chelating agent, 1% to 3% nonionic surfactant, 5% to 10% polyethylene glycol by mass fraction, and 1 to 5mg/mL polyacrylamide in a buffer solution with the sodium ion content of more than or equal to 140mM and less than or equal to 300mM and the pH value of 7 to 9. The kit comprises the lysis binding solution. In the formula of the lysis binding solution, the hydrophobicity of the lysis binding solution is improved by adding a small amount of polyacrylamide, and the DNA is separated out, so that the usage amount of guanidine salt and polyethylene glycol is obviously reduced, the lysis binding solution is integrally stable under a high-salt formula, has good fluidity at normal temperature, is convenient to take and use, does not show separated crystals after being stored for a long time (more than one year) at 4 ℃, and has stable and reliable properties.

Description

Normal-temperature-stored lysis binding solution for genome extraction and kit

Technical Field

The invention belongs to the field of biochemistry, and particularly relates to a normal-temperature cracking binding solution and a kit for saliva genome extraction.

Background

The magnetic bead method is the best method for extracting nucleic acid, has the advantages of high flux, high sensitivity, rapidness, convenience and the like, but the conventional cracking and combination are separately carried out, so that the phenomena of false positive and pollution are easy to occur.

Chinese patent document CN 111808850A, CN109022420A discloses that the splitting and combining are integrated, which greatly improves the working efficiency, reduces the number of times of adding reagents for customers, and prevents the contamination between samples caused by multiple operations. However, the existing lysis binding solution used in the magnetic bead method is very unstable due to the addition of a chaotropic agent and polyethylene glycol with high concentration as a binding agent, and can be crystallized or concentrated at normal temperature or slightly lower temperature, so that the lysis binding solution cannot be used or is difficult to use.

Disclosure of Invention

Aiming at the defects or improvement requirements of the prior art, the invention provides a normal-temperature lysis binding solution and a kit for genome extraction, and aims to reasonably select the type of a chaotropic agent, match polyethylene glycol and a small amount of polyacrylamide as a binding agent, obtain a considerable DNA extraction effect, and keep a stable solution state for a long time at normal temperature, thereby solving the technical problems that the existing lysis binding solution has unstable property at normal temperature due to overhigh concentration, is easy to precipitate or is too thick to be suitable or difficult to take.

To achieve the above object, according to one aspect of the present invention, there is provided a lysis conjugate comprising 2.5 to 3.5mol/L of a chaotropic agent, 5 to 20mM of a metal chelating agent, 1 to 3% of a nonionic surfactant, 5 to 10% by mass of polyethylene glycol, and 1 to 5mg/mL of polyacrylamide in a buffer solution having a pH of 7 to 9 and a sodium ion content of 140mM or more and 300mM or less.

Preferably, the molecular weight of the polyethylene glycol of the lysis binding solution is greater than or equal to 2000, preferably 6000 to 10000, and more preferably 8000.

Preferably, the binding solution is cleaved, and the chaotropic agent is guanidinium isothiocyanate.

Preferably, the splitting and combining solution contains 2.5mg/mL-10mg/L polyacrylamide.

According to another aspect of the invention, a genome extraction kit is provided, which comprises the lysis binding solution provided by the invention.

Preferably, the saliva genome extraction kit comprises, by volume, 40 to 60 parts of the lysis binding solution of any one of claims 1 to 4, 1 to 3 parts of the proteinase K solution with a concentration of between 10mg/ml and 30mg/ml, 16 to 20 parts of the magnetic bead dispersion with a concentration of between 4mg/ml and 8mg/ml, 60 to 100 parts of the first wash, 100 to 140 parts of the second wash, 100 to 140 parts of the third wash, and 6 to 20 parts of the eluent.

Preferably, the saliva genome extraction kit, wherein the pH value of the first washing solution is between 7.8 and 8.5, comprises 100-250mM NaCl, 5-20mM EDTA, 10-30mM Tris, 1-4% Triton in mass fraction, and 0.4-2% SDS in mass fraction.

Preferably, the saliva genome extraction kit comprises the second washing solution containing 100-250mM NaCl and 70-85% of alcohol by mass fraction.

Preferably, the saliva genome extraction kit has a pH value of the third washing solution of 7.0-8.0, 10-30mM Tris, and 70-85% by mass of alcohol.

Preferably, the saliva genome extraction kit, wherein the eluent comprises 10-30mM Tris-HCl, and 0.5-2mM EDTA.

In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:

in the formula of the lysis binding solution, the hydrophobicity of the lysis binding solution is improved by adding a small amount of polyacrylamide, and the DNA is separated out, so that the usage amount of guanidine salt and polyethylene glycol is obviously reduced, the lysis binding solution is integrally stable under a high-salt formula, has good fluidity at normal temperature, is convenient to take and use, does not show separated crystals after being stored for a long time (more than one year) at 4 ℃, and has stable and reliable properties.

The invention is suitable for the cracking of various biological samples of saliva and blood, is matched with an automatic nucleic acid extractor, and can extract by using a magnetic bead method without adding organic solvents including isopropanol, thereby realizing high-quality extraction and accounting. The operation is convenient and simple, and the method is suitable for high-flux extraction.

Drawings

FIG. 1 is a graph showing the results of electrophoresis of saliva sample extraction products in example 1;

FIG. 2 is a graph showing the electrophoresis results of saliva sample extraction products in example 2;

FIG. 3 is a graph showing the result of electrophoresis of saliva sample extraction products in example 3;

FIG. 4 is a graph showing the result of electrophoresis of the blood sample extraction product of example 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The invention provides a lysis binding solution, which contains 2.5mol/L to 3.5mol/L chaotropic agent, 5mM to 20mM metal chelating agent, 1 percent to 3 percent of nonionic surfactant, 5 percent to 10 percent of polyethylene glycol and 1 percent to 5mg/mL polyacrylamide in a buffer solution with the sodium ion content of more than or equal to 140mM and less than or equal to 300mM and the pH value of 7 to 9.

Wherein the polyethylene glycol has a molecular weight of 2000 or more, preferably 6000 to 10000, more preferably 8000; the chaotropic agent is preferably guanidinium isothiocyanate; preferably, the polyacrylamide solution contains 2.5mg/mL-10mg/L of polyacrylamide.

At present, polyethylene glycol is adopted as a binding agent in a cracking binding solution, if high-concentration guanidine salt is used as a chaotropic agent at the same time, the solution is easy to separate out, is thick, and is more unstable and easy to lose efficacy particularly under the condition of containing high-concentration sodium ions. According to the invention, the polyacrylamide is added in a very small amount, so that the hydrophobic property of the solution is obviously improved, and the nucleic acid can be better helped to be separated out and combined with magnetic beads, therefore, the usage amount of guanidine salt and the addition amount of polyethylene glycol can be obviously reduced, the overall solution stability is improved, and the stable state can be maintained at normal temperature. However, the higher concentration of polyacrylamide can cause the fluidity of the whole solution to be obviously reduced, even to be solidified, and the taking and the use are inconvenient, so the dosage of the polyacrylamide needs to be strictly controlled, and the stable cracking property and the reliable cracking effect can be ensured by matching with the selection of the chaotropic agent.

The genome extraction kit provided by the invention comprises, by volume, 40-60 parts of the lysis binding solution provided by the invention, 1-3 parts of proteinase K solution with the concentration of 10-30 mg/ml, 16-20 parts of magnetic bead dispersion with the concentration of 4-8 mg/ml, 60-100 parts of a first washing solution, 100-140 parts of a second washing solution, 100-140 parts of a third washing solution and 6-20 parts of an eluent.

The pH value of the first washing solution is between 7.8 and 8.5, and the first washing solution contains 100-250mM NaCl, 5-20mM EDTA, 10-30mM Tris, 1-4% Triton in mass fraction and 0.4-2% SDS in mass fraction.

The second washing solution contains 100-250mM NaCl and 70-85% alcohol by mass fraction.

The third washing solution has a pH value of 7.0-8.0, and contains 10-30mM Tris and 70-85% of alcohol by mass fraction.

The eluent contains 10-30mM Tris-HCl and 0.5-2mM EDTA.

The following are examples:

1. preparation of cleavage binding solution: the cleavage binding liquids of examples 1 to 3 were prepared according to the raw material component ratios of table 1.

TABLE 1 ingredient Table of lysis binding solution

The cleavage products of examples 1 to 3 were stored at 4 ℃ for one year, and were stable in properties and free from crystal precipitation.

2. Protease K solution: prepared to 20 mg/ml;

3. magnetic bead suspension: prepared to 6mg/mL, the particle size is 400 mm;

4. the first washing liquid comprises the following components in proportion as shown in the table 2;

TABLE 2 ingredient Table of the first washing liquid

Examples

SDS

EDTA

Tris

NaCl

Triton

1

0.5％

5mM

10mM

200mM

2％

2

2％

10mM

20mM

200mM

2％

3

2％

20mM

30mM

200mM

2％

5. The second washing liquid comprises the following components in proportion shown in the table 3;

TABLE 3 second washing liquid composition Table

Examples	Alcohol	NaCl
			1	70％	100mM
2	75％	250mM
			3	85％	250mM

6. A third wash solution, having a dispense ratio as in the following table;

TABLE 4 composition of the third washing liquid

Examples	Alcohol	NaCl
			1	70％	100mM
2	75％	250mM
			3	85％	250mM

7. The eluent comprises the following components: TRIS 10mM, EDTA 1 mM.

The procedure for saliva sample DNA extraction using the kit of the above example was as follows:

(1) the saliva samples were mixed well and 200. mu.L of the saliva sample was pipetted into the corresponding well of a new deep well plate.

(2) 450 mu L of lysis binding solution and 5 mu L of proteinase K are added into each sample well, and then the deep-well plate is placed on a station 1 of an S-96 nucleic acid automatic extraction instrument. In order to ensure the cracking quality, the cracking binding solution can be preheated in advance at 95 ℃.

(3) And (5) turning on the power supply of the instrument, and setting the parameters of the instrument according to the following table after the self-detection of the instrument is completed. And (4) running a program, and automatically pausing the instrument after the first-step cracking is finished.

(4) The magnetic beads (500. mu.L/well), the first wash solution (500. mu.L/well), the second wash solution and the third wash solution (600. mu.L/well), and the eluent (100. mu.L/well) were dispensed into 5 new deep-well plates using an 8-channel or 12-channel pipette, respectively, and then placed on stations 2-6, and the procedure was started as follows.

The results are shown in fig. 1 to 3, and the luminance is high, the band is clear, and the extraction effect is good.

Example 4 DNA extraction of blood samples

(1) And (3) putting 500 mu L of magnetic beads into a 1.5mL centrifuge tube (note: to ensure the magnetic beads are thoroughly resuspended, please shake the magnetic beads thoroughly and mix the mixture uniformly and immediately suck the mixture), placing the mixture on a magnetic frame, standing the mixture for 1min, sucking out all supernatant, and storing the magnetic beads for later use.

(2) A200. mu.L blood sample was taken into a 1.5mL centrifuge tube containing magnetic beads for storage.

(3) Adding 450 mu L of lysis binding solution and 5 mu L of proteinase K (20mg/mL) into each sample hole, shaking, mixing uniformly, and placing at 65 ℃ for lysis for 15-30 min. Mix 5 times with shaking.

(4) Placing the centrifugal tube on a magnetic frame, standing for 1min, repeatedly reversing the magnetic frame for 5-6 times during the standing to adsorb magnetic beads possibly remaining on the tube cover by the magnetic frame, and carefully sucking the tube cover and liquid in the tube by a pipette.

(5) Taking down the centrifugal tube, adding 500 mu L of first washing liquid, oscillating and uniformly mixing the centrifugal tube for 1min, then placing the centrifugal tube on a magnetic frame, standing for 1min, repeatedly reversing the magnetic frame for 5-6 times during the period, and carefully sucking the tube cover and the liquid in the tube by using a pipettor after the magnetic beads are completely adsorbed.

(6) Add 600. mu.L of the second wash and repeat once as in step 5.

(7) Add 600. mu.L of the third wash and repeat once as in step 5.

(8) The tube cover is opened, and the tube is dried at room temperature for 5min until the ethanol is completely volatilized (both ethanol residue and excessive drying can influence the next nucleic acid elution efficiency).

(9) Taking down the centrifuge tube, adding 100 μ L of eluent, shaking and mixing to completely immerse the magnetic beads in the eluent, incubating at 65 deg.C for 10min, and shaking and mixing 3 times. After the incubation is finished, placing the centrifugal tube on a magnetic frame, standing for 1min, after the magnetic beads are completely adsorbed, carefully transferring the liquid into a new clean centrifugal tube by using a liquid transfer device (the magnetic beads are not sucked out), and obtaining the solution which is the nucleic acid sample.

The sample can be directly used for downstream experiments, and if the sample is stored for a long time, the sample is required to be placed in a refrigerator at the temperature of-20 ℃.

The result is shown in fig. 4, which shows high brightness, clear bands, and good extraction effect.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A lysis binding solution comprising 2.5 to 3.5mol/L chaotropic agent, 5 to 20mM metal chelating agent, 1 to 3% nonionic surfactant, 5 to 10% polyethylene glycol by mass fraction, and 1 to 5mg/mL polyacrylamide in a buffer solution having a pH of 7 to 9 and a sodium ion content of 140mM or more and 300mM or less.

2. Lysis binding solution according to claim 1, wherein said polyethylene glycol has a molecular weight of 2000 or more, preferably from 6000 to 10000, more preferably 8000.

3. The lysis conjugate of claim 1, wherein the chaotropic agent is guanidinium isothiocyanate.

4. The lytic binding solution of claim 1, comprising 2.5mg/mL to 10mg/L polyacrylamide.

5. A genome extraction kit comprising the cleavage-binding solution according to any one of claims 1 to 4.

6. A salivary genome extraction kit as claimed in claim 5 comprising, by volume parts, 40 to 60 parts of the split binding solution as claimed in any one of claims 1 to 4, 1 to 3 parts of proteinase K solution at a concentration of 10mg/ml to 30mg/ml, 16 to 20 parts of magnetic bead dispersion at a concentration of 4mg/ml to 8mg/ml, 60 to 100 parts of the first wash, 100 to 140 parts of the second wash, 100 to 140 parts of the third wash, and 6 to 20 parts of the eluent.

7. The saliva genome extraction kit according to claim 6, wherein the first wash solution has a pH of 7.8 to 8.5, and comprises 100 mM NaCl, 5 to 20mM EDTA, 10 to 30mM Tris, 1 to 4% Triton by mass fraction, and 0.4 to 2% SDS by mass fraction.

8. The saliva genome extraction kit according to claim 6, wherein the second washing solution comprises 100-250mM NaCl and 70-85% by mass of alcohol.

9. The salivary genome extraction kit as claimed in claim 6, wherein the third wash solution has a pH of 7.0 to 8.0, comprises 10 to 30mM Tris, and 70 to 85% by mass alcohol.

10. The salivary genome extraction kit of claim 6 wherein the eluent comprises 10 to 30mM Tris-HCl, and 0.5 to 2mM EDTA.