CN109371010A - A kind of paramagnetic particle method plasma DNA extracts kit and extracting method - Google Patents
A kind of paramagnetic particle method plasma DNA extracts kit and extracting method Download PDFInfo
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- CN109371010A CN109371010A CN201811415357.0A CN201811415357A CN109371010A CN 109371010 A CN109371010 A CN 109371010A CN 201811415357 A CN201811415357 A CN 201811415357A CN 109371010 A CN109371010 A CN 109371010A
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
The present invention relates to a kind of paramagnetic particle method plasma DNA extracts kits, including following components: magnetic bead, cracking combine liquid, cleaning solution and eluent.A kind of method for extracting plasma DNA is additionally provided, is extracted using using kit, comprising the following steps: S1: the magnetic bead being added into plasma sample and cracking combines liquid, 5-10min is stood after mixing, solution is discarded on magnetic frame；S2: magnetic bead is washed using the cleaning solution, solution is discarded on magnetic frame；S3: the DNA adsorbed on the elution magnetic bead is used.Kit provided by the invention and method can quickly and easily extract dissociative DNA from blood plasma, and avoid the interference of genomic DNA, so that the diagnostic assays for the later period provide good episomal DNA sample, significantly reduce the generation of false positive.
Description
Technical field
The present invention relates to dissociative DNA in blood detection fields, more specifically it relates to which a kind of paramagnetic particle method plasma DNA extracts
Kit and the method for extracting plasma DNA.
Background technique
Free Circulating DNA is that one kind is present in extracellular free state nucleic acid in animals and plants and human body fluid.Exist at present
It can be detected in blood plasma, saliva, urine, cerebrospinal fluid, gastric juice, peritoneal fluid and excrement.
Hong Kong Chinese University professor Lu Yuming has found that there is tires in maternal blood in work in 1997 and 1998
The dissociative DNA of youngster, it was demonstrated that the feasibility and practical property for being carried out using fetus dissociative DNA diagnosing hereditary disease are finally started
The new way of Down's syndrome is detected using second generation gene sequencing, and is applied in more than 90 a countries, and nothing is opened
Create pre-natal diagnosis technology.
The genetic test of free Circulating DNA is more next in control and prevention of disease, the noninvasive Prenatal Screening of fetus and tumor research
One of more important gene tester.
Dissociative DNA extraction mainly is carried out to human plasma serum using the first step of the genetic test of free Circulating DNA,
The quality for extracting product directly influences the preparatory and reliability of testing result, in blood plasma and serum, dissociative DNA segment
Small, content is low, and often has the residual of genomic DNA, and the residual of genomic DNA will cause pole to dissociative DNA testing result
Big influence, so as to effectively extract dissociative DNA, and the kit that can efficiently separate genomic DNA shows particularly important.Mesh
Preceding dissociative DNA extracts kit on the market more focuses on yield, and can not effectively remove genomic DNA.
Summary of the invention
Paramagnetic particle method uses magnetic bead for the isolation technics of carrier, belongs to solid phase extraction method.Since paramagnetic particle method extracts nucleic-acid manipulation
Simply, quickly, reproducible, yield is high, without contacting the toxic reagents such as phenol/chloroform, it can be achieved that automatic operation, compared to biography
System DNA extraction has incomparable advantage.
Based on this, the present invention provides a kind of paramagnetic particle method plasma DNA extracts kits, including following components: magnetic bead,
Cracking combines liquid, cleaning solution and eluent.
In a preferred embodiment, it is described cracking combine liquid include trishydroxymethylaminomethane, ethylenediamine tetra-acetic acid,
Sodium chloride, triton x-100, guanidinium isothiocyanate, guanidine hydrochloride and isopropanol.
In a preferred embodiment, the concentration of trishydroxymethylaminomethane is 20-80mmol/L in the lysate,
The concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, and the concentration of sodium chloride is 50-500mmol/L, the concentration of triton x-100
For 1-10% volume fraction, the concentration of guanidinium isothiocyanate is 1-6mol/L, and the concentration of guanidine hydrochloride is 1-8mol/L, isopropanol
Concentration is 20-60% volume fraction, pH value 3-5.
In a preferred embodiment, the cleaning solution includes cleaning solution I and II, the cleaning solution I include guanidine hydrochloride,
Trishydroxymethylaminomethane, ethylenediamine tetra-acetic acid, sodium chloride, dehydrated alcohol, isopropanol and triton x-100；
The cleaning solution II includes trishydroxymethylaminomethane, ethylenediamine tetra-acetic acid and dehydrated alcohol.
In a preferred embodiment, the concentration of guanidine hydrochloride is 1-8mol/L, trihydroxy methyl amino in the cleaning solution I
The concentration of methane is 20-80mmol/L, and the concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, and the concentration of sodium chloride is 50-
500mmol/L, the concentration of dehydrated alcohol are 30-70% volume fraction, and the concentration of isopropanol is 20%-50% volume fraction, bent
The concentration for drawing logical X-100 is 1-5% volume fraction；
In the cleaning solution II, the concentration of trishydroxymethylaminomethane is 20-80mmol/L, the concentration of ethylenediamine tetra-acetic acid
For 5-20mmol/L, the concentration of dehydrated alcohol is 30-80% volume fraction.
In a preferred embodiment, the eluent includes trishydroxymethylaminomethane and ethylenediamine tetra-acetic acid.
In a preferred embodiment, the concentration of trishydroxymethylaminomethane is 10-50mmol/L in the eluent,
The concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, pH 5-10.
The present invention also provides a kind of methods for extracting plasma DNA, are extracted using mentioned reagent box, including with
Lower step:
S1: the magnetic bead being added into plasma sample and cracking combines liquid, 5-10min is stood after mixing, on magnetic frame
Discard solution.Preferably, 20 μ L magnetic beads are added in every 700 μ L blood plasma and 1mL cracking combines liquid, and oscillation mixes, and are stored at room temperature 5-
10min is then transferred to absorption magnetic bead 3-5min on magnetic frame, discards solution.
S2: magnetic bead is washed using the cleaning solution, solution is discarded on magnetic frame.Preferably, 600 μ L cleaning solutions are first added
I, oscillation mix, and solution is discarded on magnetic frame, add 600 μ L cleaning solution IIs, and oscillation is mixed, discarded on magnetic frame molten
Liquid.
S3: the DNA adsorbed on the elution magnetic bead is used.Preferably, 20-30 μ L eluent is added to be washed
It is de-.
Kit provided by the invention and method can quickly and easily extract dissociative DNA from blood plasma, and avoid gene
The interference of group DNA significantly reduces false positive so that the diagnostic assays for the later period provide good episomal DNA sample
Generation.
Detailed description of the invention
Fig. 1 is the electrophoresis photographs for extracting DNA in embodiment 2 with kit of the invention and commercial reagent box, wherein swimming lane
M is Marker+ genome, is labeled as the DNA that 1 two swimming lanes extract for this kit, and it is commercially available for being labeled as 2 two swimming lanes
The DNA that certain brand kit extracts is labeled as the DNA that 3 two swimming lanes extract for the kit of another commercially available brand；
Fig. 2 is the Agilent that the DNA in the plasma sample that number is TF18062078 is extracted using kit of the invention
2100 maps；
Fig. 3 is the Agilent that the DNA in the plasma sample that number is TF18061893 is extracted using kit of the invention
2100 maps；
Fig. 4 is the Agilent that the DNA in the plasma sample that number is TF18070965 is extracted using kit of the invention
2100 maps；
Fig. 5 is the Agilent that the DNA in the plasma sample that number is TF18070999 is extracted using kit of the invention
2100 maps.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
1. embodiment 1
Kit forms:
Include following components in the kit of the present embodiment: magnetic bead, cracking combine liquid, cleaning solution and elution
It includes trishydroxymethylaminomethane that the cracking, which combines liquid, ethylenediamine tetra-acetic acid, sodium chloride, triton x-100, different
Guanidine thiocyanate, guanidine hydrochloride and isopropanol, the concentration of trishydroxymethylaminomethane is 20-80mmol/L, second two in the lysate
The concentration of amine tetraacethyl is 5-20mmol/L, and the concentration of sodium chloride is 50-500mmol/L, and the concentration of triton x-100 is 1-
10% volume fraction, the concentration of guanidinium isothiocyanate are 1-6mol/L, and the concentration of guanidine hydrochloride is 1-8mol/L, the concentration of isopropanol
For 20-60% volume fraction, pH value 3-5.
The cleaning solution includes cleaning solution I and II, and the cleaning solution I includes guanidine hydrochloride, trishydroxymethylaminomethane, second two
Amine tetraacethyl, sodium chloride, dehydrated alcohol, isopropanol and triton x-100；The concentration of guanidine hydrochloride is 1- in the cleaning solution I
8mol/L, the concentration of trishydroxymethylaminomethane are 20-80mmol/L, and the concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, chlorine
The concentration for changing sodium is 50-500mmol/L, and the concentration of dehydrated alcohol is 30-70% volume fraction, and the concentration of isopropanol is 20%-
50% volume fraction, the concentration of triton x-100 are 1-5% volume fraction.
The cleaning solution II includes trishydroxymethylaminomethane, ethylenediamine tetra-acetic acid and dehydrated alcohol；Trihydroxy methyl amino
The concentration of methane is 20-80mmol/L, and the concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, and the concentration of dehydrated alcohol is 30-
80% volume fraction.
The eluent includes trishydroxymethylaminomethane and ethylenediamine tetra-acetic acid；Trihydroxy methyl amino in the eluent
The concentration of methane is 10-50mmol/L, and the concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, pH 5-10.
Embodiment 2
This example adds 6 μ L Dongsheng DS5000Maker as dissociative DNA in 700 μ L blood plasma, and 300ng genomic DNA,
The effect of the extraction dissociative DNA of mentioned reagent box is verified with this.
Extracting method is as follows:
1) in 2.0ml centrifuge tube, 700 μ l blood plasma, 300ng genomic DNA, 6 μ l Dongsheng DS5000Maker, 20 μ are added
L magnetic bead and 1mL cracking combine liquid, and concussion is mixed, is stored at room temperature 5-10 minutes.
2) it is transferred on magnetic frame, adsorbs magnetic bead 3-5 minutes, abandon solution.
3) 600 μ l cleaning solution I are added, concussion mixes.
4) it is transferred on magnetic frame, adsorbs magnetic bead 3-5 minutes, abandon solution.
5) 600 μ l cleaning solution IIs are added, concussion mixes.
6) it is transferred on magnetic frame, adsorbs magnetic bead 3-5 minutes, abandon solution.
7) 20-30 μ l eluent, eluted dna is added.
It is compareed using commercially available two kinds of brand kits, as a result as shown in Figure 1, using this set invention kit, 100-
The extraction efficiency of 750BPDNA is consistent with brand kit on the market, and 1000BP or more extraction efficiency is lower than market brand reagent
Box, especially genomic DNA are well below brand kit on the market.It can be seen that kit of the invention can reduce as far as possible
The content of genomic DNA in extract, to reduce the probability of false positive.
Embodiment 3
This example directly extracts dissociative DNA that may be present from four plasma samples, and extracting method is as follows:
1) in 2.0ml centrifuge tube, 700 μ L blood plasma are added, 20 μ L magnetic beads and 1ML cracking combine liquid, and concussion mixes, room temperature
Stand 5-10 minutes.
2) it is transferred on magnetic frame, adsorbs magnetic bead 3-5 minutes, abandon solution.
3) 600 μ L cleaning solution I are added, concussion mixes.
4) it is transferred on magnetic frame, adsorbs magnetic bead 3-5 minutes, abandon solution.
5) 600 μ L cleaning solution IIs are added, concussion mixes.
6) it is transferred on magnetic frame, adsorbs magnetic bead 3-5 minutes, abandon solution.
7) 20-30 μ L eluent, eluted dna is added.
As a result as shown in table 1 and Fig. 2-5, the DNA concentration extracted is up to standard, and only detects dissociative DNA peak,
Do not occur genomic DNA and large fragment DNA peak.
The concentration for the DNA that table 1 is extracted from plasma sample
| Sample number | Qubit concentration (ng/ μ L) | Volume (μ L) |
| TF18062078 | 0.202 | 30 |
| TF18061893 | 0.258 | 30 |
| TF18070965 | 0.234 | 30 |
| TF18070999 | 0.238 | 30 |
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of paramagnetic particle method plasma DNA extracts kit, which is characterized in that including following components: magnetic bead, cracking combine
Liquid, cleaning solution and eluent.
2. kit according to claim 1, which is characterized in that it includes trihydroxy methyl amino first that the cracking, which combines liquid,
Alkane, ethylenediamine tetra-acetic acid, sodium chloride, triton x-100, guanidinium isothiocyanate, guanidine hydrochloride and isopropanol.
3. kit according to claim 2, which is characterized in that the concentration of trishydroxymethylaminomethane in the lysate
For 20-80mmol/L, the concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, and the concentration of sodium chloride is 50-500mmol/L, Qula
The concentration of logical X-100 is 1-10% volume fraction, and the concentration of guanidinium isothiocyanate is 1-6mol/L, and the concentration of guanidine hydrochloride is 1-
8mol/L, the concentration of isopropanol is 20-60% volume fraction, pH value 3-5.
4. kit according to claim 1, which is characterized in that the cleaning solution includes cleaning solution I and II, the washing
Liquid I includes that guanidine hydrochloride, trishydroxymethylaminomethane, ethylenediamine tetra-acetic acid, sodium chloride, dehydrated alcohol, isopropanol and Qula lead to X-
100；
The cleaning solution II includes trishydroxymethylaminomethane, ethylenediamine tetra-acetic acid and dehydrated alcohol.
5. kit according to claim 4, which is characterized in that the concentration of guanidine hydrochloride is 1-8mol/ in the cleaning solution I
L, the concentration of trishydroxymethylaminomethane are 20-80mmol/L, and the concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, sodium chloride
Concentration is 50-500mmol/L, and the concentration of dehydrated alcohol is 30-70% volume fraction, and the concentration of isopropanol is 20%-50% body
Fraction, the concentration of triton x-100 are 1-5% volume fraction；
In the cleaning solution II, the concentration of trishydroxymethylaminomethane is 20-80mmol/L, and the concentration of ethylenediamine tetra-acetic acid is 5-
20mmol/L, the concentration of dehydrated alcohol are 30-80% volume fraction.
6. kit according to claim 1, which is characterized in that the eluent includes trishydroxymethylaminomethane and second
Ethylenediamine tetraacetic acid (EDTA).
7. kit according to claim 6, which is characterized in that the concentration of trishydroxymethylaminomethane in the eluent
For 10-50mmol/L, the concentration of ethylenediamine tetra-acetic acid is 5-20mmol/L, pH 5-10.
8. a kind of method for extracting plasma DNA, which is characterized in that use the reagent of any of claims 1-7
Box extracts, comprising the following steps:
S1: the magnetic bead being added into plasma sample and cracking combines liquid, stands 5-10min after mixing, discards on magnetic frame
Solution；
S2: magnetic bead is washed using the cleaning solution, solution is discarded on magnetic frame；
S3: the DNA adsorbed on the elution magnetic bead is used.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750087A (en) * | 2019-03-05 | 2019-05-14 | 温州广立生物医药科技有限公司 | A kind of blood cfDNA extracts kit |
| CN110283818A (en) * | 2019-07-31 | 2019-09-27 | 杭州艾迪康医学检验中心有限公司 | A kind of paramagnetic particle method extracts the kit and method of plasma DNA |
| CN110684764A (en) * | 2019-10-10 | 2020-01-14 | 杭州比格飞序生物科技有限公司 | Lysis solution for nucleic acid extraction, nucleic acid extraction kit and nucleic acid extraction method |
| CN110904097A (en) * | 2019-12-27 | 2020-03-24 | 深圳市海普洛斯生物科技有限公司 | Kit for extracting free DNA (deoxyribonucleic acid) in blood |
| CN111004799A (en) * | 2020-01-09 | 2020-04-14 | 上海奕杉生物科技有限公司 | Method for rapidly extracting cell RNA |
| CN111187767A (en) * | 2020-02-17 | 2020-05-22 | 广州达正生物科技有限公司 | Kit and method for rapidly extracting free DNA of blood plasma and urine |
| CN112011594A (en) * | 2019-05-30 | 2020-12-01 | 苏州海狸生物医学工程有限公司 | Free nucleic acid extraction kit and extraction method thereof |
| CN112553193A (en) * | 2020-12-23 | 2021-03-26 | 苏州中科先进技术研究院有限公司 | Kit for extracting whole blood DNA by paramagnetic particle method and use method thereof |
| CN112662663A (en) * | 2021-01-27 | 2021-04-16 | 苏州赛普生物科技有限公司 | RNA extraction kit suitable for normal-temperature transportation and extraction method |
| CN112980832A (en) * | 2021-04-23 | 2021-06-18 | 南方科技大学 | Nucleic acid extraction method and kit for nucleic acid extraction |
| CN113373141A (en) * | 2021-07-23 | 2021-09-10 | 中国科学院宁波材料技术与工程研究所 | Extraction method of free DNA |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109750087A (en) * | 2019-03-05 | 2019-05-14 | 温州广立生物医药科技有限公司 | A kind of blood cfDNA extracts kit |
| CN112011594A (en) * | 2019-05-30 | 2020-12-01 | 苏州海狸生物医学工程有限公司 | Free nucleic acid extraction kit and extraction method thereof |
| CN110283818A (en) * | 2019-07-31 | 2019-09-27 | 杭州艾迪康医学检验中心有限公司 | A kind of paramagnetic particle method extracts the kit and method of plasma DNA |
| CN110684764A (en) * | 2019-10-10 | 2020-01-14 | 杭州比格飞序生物科技有限公司 | Lysis solution for nucleic acid extraction, nucleic acid extraction kit and nucleic acid extraction method |
| CN110904097A (en) * | 2019-12-27 | 2020-03-24 | 深圳市海普洛斯生物科技有限公司 | Kit for extracting free DNA (deoxyribonucleic acid) in blood |
| CN111004799A (en) * | 2020-01-09 | 2020-04-14 | 上海奕杉生物科技有限公司 | Method for rapidly extracting cell RNA |
| CN111187767A (en) * | 2020-02-17 | 2020-05-22 | 广州达正生物科技有限公司 | Kit and method for rapidly extracting free DNA of blood plasma and urine |
| CN112553193A (en) * | 2020-12-23 | 2021-03-26 | 苏州中科先进技术研究院有限公司 | Kit for extracting whole blood DNA by paramagnetic particle method and use method thereof |
| CN112662663A (en) * | 2021-01-27 | 2021-04-16 | 苏州赛普生物科技有限公司 | RNA extraction kit suitable for normal-temperature transportation and extraction method |
| CN112980832A (en) * | 2021-04-23 | 2021-06-18 | 南方科技大学 | Nucleic acid extraction method and kit for nucleic acid extraction |
| CN113373141A (en) * | 2021-07-23 | 2021-09-10 | 中国科学院宁波材料技术与工程研究所 | Extraction method of free DNA |
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