CN106754892A - A kind of improved centrifugal column method peripheral blood dissociative DNA extracts reagent and its extracting method - Google Patents
A kind of improved centrifugal column method peripheral blood dissociative DNA extracts reagent and its extracting method Download PDFInfo
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Abstract
The invention discloses a kind of improved centrifugal column method peripheral blood dissociative DNA extracts reagent and its extracting method.The extracts reagent includes 0.2ug/uL~5ug/uL yeast tRNA, and remainder includes ultra-pure water；And the pH value of the DNA extracts reagents is 7.3~8.2.The extracting method includes：Pending peripheral blood is mixed with Proteinase K, the first buffer solution and said extracted reagent is added, it is well mixed to form digestive juice, to absolute ethyl alcohol is added in digestive juice, digestion mixed liquor is formed, digestion mixed liquor centrifugation is obtained into dissociative DNA.Extracts reagent of the invention can improve extraction efficiency, the extraction yield of existing common centrifugal column method is improved 3 10 times, so as to realize that the expensive specific aim of fictitious hosts extracts peripheral blood dissociative DNA centrifugal column method kit, material source is simple, it is with low cost, nucleic acid extraction rate can be improved, substantially improve the problem that scientific research personnel is met with peripheral blood free nucleic acid is extracted, it is high with practicality, the good advantage of generalization.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly, to for DNA technique field, one is particularly related to
Plant improved centrifugal column method peripheral blood dissociative DNA extracts reagent and its extracting method.
Background technology
It is well known that inhereditary material nucleic acid is located in cell mostly, but either healthy crowd or patient groups, all
There is fraction DNA to be located at extracellular, referred to as free nucleic acid (cell-free DNA).Dissociative DNA is there is also in peripheral blood, referred to as
Peripheral Circulation DNA (Circulating DNA in plasma).
In one paper of 1987, researcher is studied 37 malignant tumor patient blood plasma, wherein 10
Dissociative DNA has all been isolated in sample.The plasma DNA of Preliminary Identification is double-strand, and magnitude range distribution is about 40bp-
220bp.In subsequent research, they by a kind of external DNA synthetic tests (In vitro DNA for developing themselves
Synthesis Test), there are basic judgement, the general principle of this test to the characteristic of the DNA in cancer patient's blood plasma
It is that the DNA stability of tumorigenic can decline when carcinogen is added in test reaction.To the nineties, researcher exists
The isogenic mutation of K-ras are detected on the free Circulating DNA of tumor patient blood plasma in succession.
Due to peripheral blood free nucleic acid, content is rare in peripheral blood, the above researcher uses or biography
The phenol chloroform technology of system, also than larger (needing collection 50ml peripheral bloods), experiment difficulty can for the amount of required peripheral blood
Think and know.Therefore, the efficiency for extracting the free nucleic acid in peripheral blood how is improved, becomes what extracts kit on the market ran into
A great problem.With the continuous progress of science and technology, the extraction for also having emerged in large numbers some successively on the market for peripheral blood free nucleic acid is tried
Agent box, can more effectively improve nucleic acid extraction rate, but all expensive, for the more traditional centrifugal column method of cost, be higher by 50-
200 times, Cost Problems become many laboratories process peripheral blood free nucleic acid when restraining factors, therefore improve tradition from
Stem method, improving nucleic acid extraction efficiency has turned into the task of top priority.
The content of the invention
The main object of the present invention solves limitation conventional centrifugal post method and dissociates in extraction peripheral blood aiming above present situation
The bottleneck problem of nucleic acid, there is provided a kind of improved centrifugal column method peripheral blood dissociative DNA extracts reagent and its extracting method, examination used
Agent composition is simple, easy to operate, with low cost, is not required to special installation, and can significantly reduce cost and improve nucleic acid extraction rate.
To realize aforementioned invention purpose, the technical solution adopted by the present invention includes：
A kind of improved centrifugal column method peripheral blood dissociative DNA extracts reagent is the embodiment of the invention provides, it is included
0.2ug/uL~5ug/uL yeast tRNA, remainder includes ultra-pure water；And the pH value of the DNA extracts reagents be 7.3~
8.2。
Among certain preferred embodiments, the pH value of the improved centrifugal column method peripheral blood dissociative DNA extracts reagent
It is 7.5, solvent is ultra-pure water, solute is following final concentration material：Yeast tRNA 1ug/uL.
The embodiment of the present invention additionally provides a kind of kit, its centrifugal column method peripheral blood dissociative DNA for including aforementioned improved
Extracts reagent.
The embodiment of the present invention additionally provides a kind of improved centrifugal column method peripheral blood dissociative DNA extracting method, and it includes：
Pending peripheral blood is mixed with Proteinase K, the first buffer solution and the improved centrifugal column method periphery is added
Blood dissociative DNA extracts reagent or kit, it is well mixed to form digestive juice, then to adding absolute ethyl alcohol in the digestive juice,
Digestion mixed liquor is formed, the digestion mixed liquor centrifugation is obtained into dissociative DNA.
Wherein, first buffer solution is AL buffer solutions, German Qiagen.
Among some embodiments, the pending peripheral blood is 5 with the volume ratio of Proteinase K：1~20：1；It is described
The enzyme activity concentration range of Proteinase K is 5mg/mL~30mg/mL.
Among some more preferred embodiment, the pending peripheral blood is 10 with the volume ratio of Proteinase K：1,
The enzyme activity concentration range of the Proteinase K is 20mg/mL.
Among some embodiments, first buffer solution is 0.5 with the volume ratio of pending peripheral blood：1~2：1；
The pending peripheral blood is with the volume ratio of the improved centrifugal column method peripheral blood dissociative DNA extracts reagent
200：1~5000：1；
The pending peripheral blood is 3 with the volume ratio of absolute ethyl alcohol：1~10：1.
Among some more preferred embodiment, first buffer solution is 1 with the volume ratio of pending peripheral blood：
1；
The pending peripheral blood is with the volume ratio of the improved centrifugal column method peripheral blood dissociative DNA extracts reagent
1000：1；
The pending peripheral blood is 2.5 with the volume ratio of absolute ethyl alcohol：1.
Among some embodiments, the temperature of the digestive juice is 50 DEG C~65 DEG C, and digestion time is 0.2h~20h.
It is more highly preferred to, the temperature of the digestive juice is 56 DEG C, and digestion time is 6h.
Among some embodiments, the extracting method also includes：The digestion mixed liquor is dividedly in some parts centrifugal column
Middle centrifugation, first to the second buffer solution is added in centrifugal column, cleans centrifugal column, then to the 3rd buffer solution is added in centrifugal column, clean
Centrifugal column.
Wherein, second buffer solution is AW1 buffer solutions, German Qiagen；3rd buffer solution is AW2 buffer solutions,
German Qiagen.
Further, the extracting method also includes：After cleaning centrifugal column, by empty centrifugal column be placed into centrifuge from
Heart 2min, and room temperature dries, then to adding the 4th buffer solution, the dissociative DNA for affording in centrifugal column.
Wherein, the 4th buffer solution is AE buffer solutions, German Qiagen.
Among some embodiments, second buffer solution is 0.5 with the volume ratio of the pending peripheral blood：1~
2：1；
3rd buffer solution is 0.5 with the volume ratio of the pending peripheral blood：1~2：1；
The volume ratio of the pending peripheral blood and the 4th buffer solution is 10：1~200：1；
Eluting temperature is 20 DEG C~70 DEG C.
It is more highly preferred to, second buffer solution is 0.7 with the volume ratio of the pending peripheral blood：1；
3rd buffer solution is 0.7 with the volume ratio of the pending peripheral blood：1；
The volume ratio of the pending peripheral blood and the 4th buffer solution is 31.25：1；
Eluting temperature is 55 DEG C.
Compared with prior art, advantages of the present invention includes：
(1) a kind of improved centrifugal column method peripheral blood free nucleic acid extracts reagent that the present invention is provided, only need to use market
Existing whole blood extracts the reagent in nucleic acid reagent box, and material source is simple, and operation difficulty is low, without know-how training and special
Instrument and equipment；
(2) a kind of improved centrifugal column method peripheral blood free nucleic acid extracting method that the present invention is provided, it is existing it is common from
Improved on the basis of stem method, add said extracted reagent to improve extraction efficiency in extraction process, will be existing common
The extraction yield of centrifugal column method improves 3-10 times；
(3) present invention is provided a kind of improved centrifugal column method peripheral blood free nucleic acid extracts reagent and kit, compared with city
On face for existing product kit, it is capable of achieving the expensive specific aim of fictitious hosts and extracts peripheral blood dissociative DNA centrifugal column method
Kit, can by cost reduction by more than 50%, substantially improve scientific research personnel extract peripheral blood free nucleic acid in met with ask
Topic, generalization good advantage high with practicality.
Brief description of the drawings
Fig. 1 is the extracts kit and available reagent box of the improved centrifugal column method peripheral blood dissociative DNA that the present invention is provided
Cost consumption contrast schematic diagram；
Fig. 2 is to the contrast schematic diagram of dissociative DNA extracted amount in embodiment of the present invention 1-4 and control group experiment.
Specific embodiment
More detailed explanation will hereafter be made to technical scheme.It is understood, however, that in model of the present invention
In enclosing, can between above-mentioned each technical characteristic of the invention and each technical characteristic for specifically describing in below (eg embodiment)
It is combined with each other, so as to constitute new or preferred technical scheme.As space is limited, no longer tire out one by one herein and state.
Before being described to example, it is necessary to which some remarks explanations are provided：
The difference of experimental result can be caused using the reagent of different manufacturers, different batches, belongs to normal phenomenon.Carry out it is small
During sweeping experiment, to ensure the repeatability between parallel laboratory test, it is proposed that after configuration reagent, fully mix and dispense, to ensure every time
The homogeneity of experiment reagent.
A kind of improved centrifugal column method peripheral blood dissociative DNA extracts reagent is the embodiment of the invention provides, it is included
0.2ug/uL~5ug/uL yeast tRNA, remainder includes ultra-pure water；And the pH value of the DNA extracts reagents be 7.3~
8.2。
Among certain preferred embodiments, the pH value of the improved centrifugal column method peripheral blood dissociative DNA extracts reagent
It is 7.5, solvent is ultra-pure water, solute is following final concentration material：Yeast tRNA 1ug/uL.
The embodiment of the present invention additionally provides a kind of kit, its centrifugal column method peripheral blood dissociative DNA for including aforementioned improved
Extracts reagent.
The embodiment of the present invention additionally provides a kind of improved centrifugal column method peripheral blood dissociative DNA extracting method, and it includes：
Pending peripheral blood is mixed with Proteinase K, the first buffer solution and the improved centrifugal column method periphery is added
Blood dissociative DNA extracts reagent or kit, it is well mixed to form digestive juice, then to adding absolute ethyl alcohol in the digestive juice,
Digestion mixed liquor is formed, the digestion mixed liquor centrifugation is obtained into dissociative DNA.
Wherein, first buffer solution is AL buffer solutions, German Qiagen.
Among some embodiments, the pending peripheral blood is 5 with the volume ratio of Proteinase K：1~20：1, preferably
It is 10：1.
Among some embodiments, the enzyme activity concentration range of the Proteinase K is 5mg/mL~30mg/mL, preferably
20mg/mL。
Among some embodiments, first buffer solution is 0.5 with the volume ratio of pending peripheral blood：1~2：1,
Preferably 1：1；
The pending peripheral blood is with the volume ratio of the improved centrifugal column method peripheral blood dissociative DNA extracts reagent
200：1~5000：1, preferably 1000：1；
The pending peripheral blood is 3 with the volume ratio of absolute ethyl alcohol：1~10：1, preferably 2.5：1.
Among some embodiments, the temperature of the digestive juice is 50 DEG C~65 DEG C, and digestion time is 0.2h~20h.
It is more highly preferred to, the temperature of the digestive juice is 56 DEG C, and digestion time is 6h.
Among some embodiments, the extracting method also includes：The digestion mixed liquor is dividedly in some parts centrifugal column
Middle centrifugation, first to the second buffer solution is added in centrifugal column, cleans centrifugal column, then to the 3rd buffer solution is added in centrifugal column, clean
Centrifugal column.
Wherein, second buffer solution is AW1 buffer solutions, and German Qiagen, the 3rd buffer solution is AW2 buffer solutions,
German Qiagen.
Further, the extracting method also includes：After cleaning centrifugal column, by empty centrifugal column be placed into centrifuge from
Heart 2min, and room temperature dries, then to adding the 4th buffer solution, the dissociative DNA for affording in centrifugal column.
Wherein, the 4th buffer solution is AE buffer solutions, German Qiagen.
Among some embodiments, second buffer solution is 0.5 with the volume ratio of the pending peripheral blood：1~
2：1；
3rd buffer solution is 0.5 with the volume ratio of the pending peripheral blood：1~2：1；
The volume ratio of the pending peripheral blood and the 4th buffer solution is 10：1~200：1；
Eluting temperature is 20 DEG C~70 DEG C.
It is more highly preferred to, second buffer solution is 0.7 with the volume ratio of the pending peripheral blood：1；
3rd buffer solution is 0.7 with the volume ratio of the pending peripheral blood：1；
The volume ratio of the pending peripheral blood and the 4th buffer solution is 31.25：1；
Eluting temperature is 55 DEG C.
Among some more specific embodiment, the extracting method specifically includes following steps：
(1) 1mL blood plasma is taken out from -80 DEG C, is placed in mixture of ice and water and melts；
(2) Proteinase K is added in 15mL centrifugation bottom of the tube；
(3) by 1mL blood plasma add 15mL centrifuge tubes in, and add the first buffer solution and foregoing extracts reagent；
(4) digestion in water-bath is placed after fully mixing；
(5) to adding absolute ethyl alcohol in digestive juice；
(6) digestion mixed liquor is dividedly in some parts in centrifugal column and is centrifuged；
(7) to the second buffer solution is added in centrifugal column, centrifugal column is cleaned；
(8) to the 3rd buffer solution is added in centrifugal column, centrifugal column is cleaned.
(9) empty luxuriant stem, and room temperature dries；
(10) to the 4th buffer solution of addition, eluted dna in centrifugal column.
Other NM steps, are carried out (Qiagen) according to QiaAmp Blood Mini Kit operation manuals.
Below in conjunction with the technical solution of the present invention is further explained the explanation of accompanying drawing and some embodiments.
Embodiment 1
1. reagent prepares：
Proteinase K (Tiangeng), QIAamp Mini spin column (Qiagen), buffer A L (Qiagen), buffer solution
AW1 (Qiagen), absolute ethyl alcohol (traditional Chinese medicines), buffer A W2 (Qiagen), buffer A E (Qiagen), Qubit dsDNA HS
Assay(Life Technologies)。
Reagent I：PH is 7.5, and solvent is ultra-pure water, and solute includes following final concentration material, yeast tRNA (Perkin
Elmer)1ug/uL。
2. experimental implementation process：
(1) by the plasma sample after thawing be placed on it is pre- be cooled in 4 DEG C of centrifuge Sigma (3K-15), with 16000 × g
Rotating speed, 4 DEG C centrifugation 10min.
(2) 100ul Proteinase Ks to 15ml centrifugation bottom of the tube are drawn.
(3) plasma sample supernatant 1mL is extremely added with the 15ml centrifuge tubes of Proteinase K after centrifugation is carefully drawn with pipettor
In.
(4) 1mL buffer A L solution and 1uL reagent I are drawn, is added in sample, vortex oscillation is mixed 15 seconds immediately.
(5) mixed liquor is put into 56 DEG C of water-baths and is incubated 4h.
(6) 15ml centrifuge tubes are taken out from water-bath, of short duration centrifugation 5 seconds makes the liquid accumulation on centrifugation lid and tube wall
To ttom of pipe.
(7) 1mL absolute ethyl alcohols are added in sample, vortex oscillation mix 15 seconds after of short duration centrifugation.
(8) carefully draw in thing 700ul to QIAamp Mini spin column mixed above, close the lid, using from
Scheming Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min, abandons the waste liquid in collecting pipe, repeats to add sample straight
Finished to all centrifugations.
(9) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW1, closes the lid, and uses centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min.
(10) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW2, closes the lid, and uses centrifuge Thermo (Micro CL17), 14000 × g normal temperature centrifugation 2min.
(11) QIAamp Mini spin column are transferred in new collecting pipe, lid reversely, is centrifuged at full speed
4min。
(12) QIAamp Mini spin column are transferred in new collecting pipe, lid are opened, as being dried in the air at 55 DEG C
Dry 10min.
(13) QIAamp Mini spin column are transferred in clean 1.5ml Eppendorf centrifuge tubes, to film
Center adds the preheated buffer A E of 32ul, closes the lid, 55 DEG C of incubation 5min.
(14) using centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 2min.
(15) QIAamp Mini spin column are discarded, lid is carefully covered, mark is carried out on EP pipes.
(16) quantitative work is carried out to the DNA for extracting using Qubit dsDNA HS Assay.
3. testing result：
Finally measuring final product concentrations is：0.32ng/ul.
Embodiment 2
1. reagent prepares：
Proteinase K (Tiangeng), QIAamp Mini spin column (Qiagen), buffer A L (Qiagen), buffer solution
AW1 (Qiagen), absolute ethyl alcohol (traditional Chinese medicines), buffer A W2 (Qiagen), buffer A E (Qiagen), Qubit dsDNA HS
Assay(Life Technologies)。
Reagent I：PH is 7.5, and solvent is ultra-pure water, and solute includes following final concentration material, yeast tRNA (Perkin
Elmer)1ug/uL。
2. experimental implementation process：
(1) by the plasma sample after thawing be placed on it is pre- be cooled in 4 DEG C of centrifuge Sigma (3K-15), with 16000 × g
Rotating speed, 4 DEG C centrifugation 10min.
(2) 100ul Proteinase Ks to 15ml centrifugation bottom of the tube are drawn.
(3) plasma sample supernatant 1mL is extremely added with the 15ml centrifuge tubes of Proteinase K after centrifugation is carefully drawn with pipettor
In.
(4) 1mL buffer A L solution and 1uL reagent I are drawn, is added in sample, vortex oscillation is mixed 15 seconds immediately.
(5) mixed liquor is put into 56 DEG C of water-baths and is incubated 4h.
(6) 15ml centrifuge tubes are taken out from water-bath, of short duration centrifugation 5 seconds makes the liquid accumulation on centrifugation lid and tube wall
To ttom of pipe.
(7) 1mL absolute ethyl alcohols are added in sample, vortex oscillation mix 15 seconds after of short duration centrifugation.
(8) carefully draw in thing 700ul to QIAamp Mini spin column mixed above, close the lid, using from
Scheming Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min, abandons the waste liquid in collecting pipe, repeats to add sample straight
Finished to all centrifugations.
(9) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW1, closes the lid, and uses centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min.
(10) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW2, closes the lid, and uses centrifuge Thermo (Micro CL17), 14000 × g normal temperature centrifugation 2min.
(11) QIAamp Mini spin column are transferred in new collecting pipe, lid reversely, is centrifuged at full speed
4min。
(12) QIAamp Mini spin column are transferred in new collecting pipe, lid are opened, as being dried in the air at 55 DEG C
Dry 10min.
(13) QIAamp Mini spin column are transferred in clean 1.5ml Eppendorf centrifuge tubes, to film
Center adds the preheated buffer A E of 32ul, closes the lid, 55 DEG C of incubation 5min.
(14) using centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 2min.
(15) QIAamp Mini spin column are discarded, lid is carefully covered, mark is carried out on EP pipes.
(16) quantitative work is carried out to the DNA for extracting using Qubit dsDNA HS Assay.
3. testing result：
Finally measuring final product concentrations is：0.29ng/ul.
Embodiment 3
1. reagent prepares：
Proteinase K (Tiangeng), QIAamp Mini spin column (Qiagen), buffer A L (Qiagen), buffer solution
AW1 (Qiagen), absolute ethyl alcohol (traditional Chinese medicines), buffer A W2 (Qiagen), buffer A E (Qiagen), Qubit dsDNA HS
Assay(Life Technologies)。
Reagent I：PH is 7.5, and solvent is ultra-pure water, and solute includes following final concentration material, yeast tRNA (Perkin
Elmer)1ug/uL。
2. experimental implementation process：
(1) by the plasma sample after thawing be placed on it is pre- be cooled in 4 DEG C of centrifuge Sigma (3K-15), with 16000 × g
Rotating speed, 4 DEG C centrifugation 10min.
(2) 100ul Proteinase Ks to 15ml centrifugation bottom of the tube are drawn.
(3) plasma sample supernatant 1mL is extremely added with the 15ml centrifuge tubes of Proteinase K after centrifugation is carefully drawn with pipettor
In.
(4) 1mL buffer A L solution and 1uL reagent I are drawn, is added in sample, vortex oscillation is mixed 15 seconds immediately.
(5) mixed liquor is put into 56 DEG C of water-baths and is incubated 4h.
(6) 15ml centrifuge tubes are taken out from water-bath, of short duration centrifugation 5 seconds makes the liquid accumulation on centrifugation lid and tube wall
To ttom of pipe.
(7) 1mL absolute ethyl alcohols are added in sample, vortex oscillation mix 15 seconds after of short duration centrifugation.
(8) carefully draw in thing 700ul to QIAamp Mini spin column mixed above, close the lid, using from
Scheming Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min, abandons the waste liquid in collecting pipe, repeats to add sample straight
Finished to all centrifugations.
(9) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW1, closes the lid, and uses centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min.
(10) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW2, closes the lid, and uses centrifuge Thermo (Micro CL17), 14000 × g normal temperature centrifugation 2min.
(11) QIAamp Mini spin column are transferred in new collecting pipe, lid reversely, is centrifuged at full speed
4min。
(12) QIAamp Mini spin column are transferred in new collecting pipe, lid are opened, as being dried in the air at 55 DEG C
Dry 10min.
(13) QIAamp Mini spin column are transferred in clean 1.5ml Eppendorf centrifuge tubes, to film
Center adds the preheated buffer A E of 32ul, closes the lid, 55 DEG C of incubation 5min.
(14) using centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 2min.
(15) QIAamp Mini spin column are discarded, lid is carefully covered, mark is carried out on EP pipes.
(16) quantitative work is carried out to the DNA for extracting using Qubit dsDNA HS Assay.
3. testing result：
Finally measuring final product concentrations is：0.37ng/ul.
Embodiment 4
1. reagent prepares：
Proteinase K (Tiangeng), QIAamp Mini spin column (Qiagen), buffer A L (Qiagen), buffer solution
AW1 (Qiagen), absolute ethyl alcohol (traditional Chinese medicines), buffer A W2 (Qiagen), buffer A E (Qiagen), Qubit dsDNA HS
Assay(Life Technologies)。
Reagent I：PH is 7.5, and solvent is ultra-pure water, and solute includes following final concentration material, yeast tRNA (Perkin
Elmer)1ug/uL。
2. experimental implementation process：
(1) by the plasma sample after thawing be placed on it is pre- be cooled in 4 DEG C of centrifuge Sigma (3K-15), with 16000 × g
Rotating speed, 4 DEG C centrifugation 10min.
(2) 100ul Proteinase Ks to 15ml centrifugation bottom of the tube are drawn.
(3) plasma sample supernatant 1mL is extremely added with the 15ml centrifuge tubes of Proteinase K after centrifugation is carefully drawn with pipettor
In.
(4) 1mL buffer A L solution and 1uL reagent I are drawn, is added in sample, vortex oscillation is mixed 15 seconds immediately.
(5) mixed liquor is put into 56 DEG C of water-baths and is incubated 4h.
(6) 15ml centrifuge tubes are taken out from water-bath, of short duration centrifugation 5 seconds makes the liquid accumulation on centrifugation lid and tube wall
To ttom of pipe.
(7) 1mL absolute ethyl alcohols are added in sample, vortex oscillation mix 15 seconds after of short duration centrifugation.
(8) carefully draw in thing 700ul to QIAamp Mini spin column mixed above, close the lid, using from
Scheming Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min, abandons the waste liquid in collecting pipe, repeats to add sample straight
Finished to all centrifugations.
(9) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW1, closes the lid, and uses centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 1min.
(10) carefully QIAamp Mini spin column are transferred in new collecting pipe, add 700ul buffer solutions
AW2, closes the lid, and uses centrifuge Thermo (Micro CL17), 14000 × g normal temperature centrifugation 2min.
(11) QIAamp Mini spin column are transferred in new collecting pipe, lid reversely, is centrifuged at full speed
4min。
(12) QIAamp Mini spin column are transferred in new collecting pipe, lid are opened, as being dried in the air at 55 DEG C
Dry 10min.
(13) QIAamp Mini spin column are transferred in clean 1.5ml Eppendorf centrifuge tubes, to film
Center adds the preheated buffer A E of 32ul, closes the lid, 55 DEG C of incubation 5min.
(14) using centrifuge Thermo (Micro CL17), 6000 × g normal temperature centrifugation 2min.
(15) QIAamp Mini spin column are discarded, lid is carefully covered, mark is carried out on EP pipes.
(16) quantitative work is carried out to the DNA for extracting using Qubit dsDNA HS Assay.
3. testing result：
Finally measuring final product concentrations is：0.41ng/ul.
Control group embodiment
1. reagent prepares：
Proteinase K (Tiangeng), buffer A CL (Qiagen), buffer A CB (Qiagen), Circulating Column
(Qiagen), buffer A CW1 (Qiagen), buffer A CW2 (Qiagen), absolute ethyl alcohol (traditional Chinese medicines), buffer A VE
(Qiagen)。
2. experimental implementation process：
(1) to 100ul Proteinase Ks are added in every pipe, then 0.8ml buffer A CL are added thereto to, vortex oscillation 30s, 56
DEG C water-bath takes out after being incubated 30min, adds 1.8ml buffer A CB, vortex oscillation 30s, is placed in 5min on ice.
(2) pumping and filtering device is used, solution mixed above is added into Circulating column, after draining, respectively
With 600ul buffer A CW1, buffer solution 750ul ACW2, buffer solution 750ul 100% ethanol is washed post, is removed after draining.
(3) it is placed in new collecting pipe, maximum (top) speed centrifugation 5min takes out, and abandons collecting pipe.
(4) Circulating column are placed in new collecting pipe, 56 DEG C of baking oven 10min, buffer A VE is simultaneously pre-
Heat.
(5) take out, Circulating column are placed in new collecting pipe, 30ul eluents are slowly added into film center
AVE。
(6) close the lid, after 56 DEG C are incubated 5min, maximum (top) speed centrifugation 2min.
(7) quantitative work is carried out to the DNA for extracting using Qubit dsDNA HS Assay.
3. testing result：
Finally measuring final product concentrations is：0.35ng/ul.
Fig. 1 is the extracts kit and available reagent box of the peripheral blood dissociative DNA based on centrifugal column method that the present invention is provided
Cost consumption contrast schematic diagram.Wherein, kit A is in available reagent box：MagMAX Cell-Free DNA
Isolation Kit (Thermo), kit B are：NextPrep-MagTM cfDNA Isolation Kit(Bioo
Scientific), kit C is：QIAamp Circulating Nucleic Acid Kit(Qiagen).
The improved centrifugal column method peripheral blood free nucleic acid high-quality extraction that the present invention is used is can be seen that from Fig. 1 and Fig. 2
Method, compared with the extracting method of control group, agents useful for same composition is simple, easy to operate, with low cost, is not required to special installation,
And cost can be significantly reduced and nucleic acid extraction rate is improved, the yield of peripheral blood free nucleic acid is extracted in the case of equal plasma volume
The requirement of market high price kit is reached.
In sum, the present invention can effectively improve the extraction yield of peripheral blood free nucleic acid, effectively reduce cost, significantly
Improve the problem that scientific research personnel is met with peripheral blood free nucleic acid is extracted.
It should be appreciated that above-described embodiment is only explanation technology design of the invention and feature, this is familiar with its object is to allow
The personage of item technology will appreciate that present disclosure and implement according to this that it is not intended to limit the scope of the present invention.It is all
According to the equivalent change or modification that spirit of the invention is made, should all be included within the scope of the present invention.
Claims (13)
1. a kind of improved centrifugal column method peripheral blood dissociative DNA extracts reagent, it is characterised in that include：0.2ug/uL~5ug/uL
Yeast tRNA, remainder includes ultra-pure water；And the pH value of the DNA extracts reagents is 7.3~8.2.
2. improved centrifugal column method peripheral blood dissociative DNA extracts reagent according to claim 1, it is characterised in that described
The pH value of improved centrifugal column method peripheral blood dissociative DNA extracts reagent is 7.5, and solvent is ultra-pure water, and solute is following final concentration
Material：Yeast tRNA 1ug/uL.
3. a kind of kit, it is characterised in that comprising the improved centrifugal column method peripheral blood any one of claim 1-2
Dissociative DNA extracts reagent.
4. a kind of improved centrifugal column method peripheral blood dissociative DNA extracting method, it is characterised in that including：
Pending peripheral blood is mixed with Proteinase K, the first buffer solution and changing any one of claim 1-2 is added
The kit described in centrifugal column method peripheral blood dissociative DNA extracts reagent or claim 3 for entering, it is well mixed to form digestive juice,
Then to absolute ethyl alcohol is added in the digestive juice, digestion mixed liquor is formed, the digestion mixed liquor centrifugation is dissociated
DNA。
5. improved centrifugal column method peripheral blood dissociative DNA extracting method according to claim 4, it is characterised in that described
Pending peripheral blood is 5 with the volume ratio of Proteinase K：1~20：1；And/or, the enzyme activity concentration range of the Proteinase K is
5mg/mL~30mg/mL.
6. improved centrifugal column method peripheral blood dissociative DNA extracting method according to claim 5, it is characterised in that described
Pending peripheral blood is 10 with the volume ratio of Proteinase K：1, and/or, the enzyme activity concentration range of the Proteinase K is 20mg/mL.
7. improved centrifugal column method peripheral blood dissociative DNA extracting method according to claim 4, it is characterised in that described
First buffer solution is 0.5 with the volume ratio of pending peripheral blood：1~2：1；
And/or, the pending peripheral blood is with the volume ratio of the improved centrifugal column method peripheral blood dissociative DNA extracts reagent
200：1~5000：1；
And/or, the pending peripheral blood is 3 with the volume ratio of absolute ethyl alcohol：1~10：1.
8. improved centrifugal column method peripheral blood dissociative DNA extracting method according to claim 7, it is characterised in that described
First buffer solution is 1 with the volume ratio of pending peripheral blood：1；
And/or, the pending peripheral blood is with the volume ratio of the improved centrifugal column method peripheral blood dissociative DNA extracts reagent
1000：1；
And/or, the pending peripheral blood is 2.5 with the volume ratio of absolute ethyl alcohol：1.
9. improved centrifugal column method peripheral blood dissociative DNA extracting method according to claim 4, it is characterised in that described
The temperature of digestive juice is 50 DEG C~65 DEG C, and digestion time is 0.2h~20h；Preferably, the temperature of the digestive juice is 56 DEG C, is disappeared
The change time is 6h.
10. improved centrifugal column method peripheral blood dissociative DNA extracting method according to claim 7, it is characterised in that also wrap
Include：The digestion mixed liquor is dividedly in some parts in centrifugal column and is centrifuged, first to the second buffer solution is added in centrifugal column, cleaning is centrifuged
Post, then to the 3rd buffer solution is added in centrifugal column, clean centrifugal column.
11. improved centrifugal column method peripheral blood dissociative DNA extracting methods according to claim 10, it is characterised in that also wrap
Include：After cleaning centrifugal column, empty centrifugal column is placed into 2min is centrifuged in centrifuge, and room temperature is dried, then in centrifugal column
The 4th buffer solution is added, the dissociative DNA for affording.
12. improved centrifugal column method peripheral blood dissociative DNA extracting methods according to claim 11, it is characterised in that
Second buffer solution is 0.5 with the volume ratio of the pending peripheral blood：1~2：1；
And/or, the 3rd buffer solution is 0.5 with the volume ratio of the pending peripheral blood：1~2：1；
And/or, the volume ratio of the pending peripheral blood and the 4th buffer solution is 10：1~200：1；
And/or, eluting temperature is 20 DEG C~70 DEG C.
13. improved centrifugal column method peripheral blood dissociative DNA extracting methods according to claim 12, it is characterised in that
Second buffer solution is 0.7 with the volume ratio of the pending peripheral blood：1；
And/or, the 3rd buffer solution is 0.7 with the volume ratio of the pending peripheral blood：1；
And/or, the volume ratio of the pending peripheral blood and the 4th buffer solution is 31.25：1；
And/or, eluting temperature is 55 DEG C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257368A (en) * | 2019-05-16 | 2019-09-20 | 上海臻迪基因科技有限公司 | The method and system of free nucleic acid is separated from the sample containing free nucleic acid |
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| CN110257368A (en) * | 2019-05-16 | 2019-09-20 | 上海臻迪基因科技有限公司 | The method and system of free nucleic acid is separated from the sample containing free nucleic acid |
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