CN108707602A - A kind of dipped into formalin tissue DNA extracts kit and extracting method - Google Patents
A kind of dipped into formalin tissue DNA extracts kit and extracting method Download PDFInfo
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Abstract
The present invention provides a kind of dipped into formalin tissue DNA extracts kit and extracting methods, belong to DNA extractive techniques field, and the kit includes following components：SDS buffer solutions, combination buffer, Proteinase K, DNA purifying magnetic bead, the first washing lotion, the second washing lotion and eluent；The extracting method includes ultrasonication acquisition lysate after 1) mixing dipped into formalin tissue with SDS buffer solutions, and two step digestion are carried out to lysate with Proteinase K；Then purify magnetic bead with DNA to extract, the dipped into formalin tissue DNA that elution obtains.Dipped into formalin tissue DNA extracts kit and extracting method of the present invention can realize the extraction to dipped into formalin tissue DNA, and the DNA for extracting acquisition is high-quality, and the rate of recovery is high.
Description
Technical field
The invention belongs to DNA extractive techniques field more particularly to a kind of dipped into formalin tissue DNA extracts kit and
Extracting method.
Background technology
In recent years, the incidence of cancer is higher and higher in world wide, and the generation of tumour is mostly multiple gene mutations
Result.Compared with a traditional generation is sequenced, it is that one kind can that (next-generation sequencing, NGS), which was sequenced, in two generations
The sequencing technologies of multiple genes are detected simultaneously.The fast development of two generations sequencing comes for the tailored diagnostics of tumour and accurate treatment zone
Gospel, greatly improves the quality of life of cancer patient.But the most important condition of these extensive genetic tests is to obtain
The DNA sample of good quality.It is patient tissue sample that oncogene, which detects most direct object, but due to fresh cancerous tissue sample
Sampling with transport it is relatively difficult, and after preserving for a long time, the degradation of sample DNA be possible to influence genetic test as a result, simultaneously
The final diagnosis for influencing cancer patient.Paraffin-embedded tissue (formalin-fixedparaffin-embedded, FFPE) is conventional
It is stored in hospital pathology department, but is influenced by slice and tissue treatment, is generally difficult to obtain the DNA of high quality.Therefore, paraffin packet
Tissue is buried to be difficult to meet the needs of extensive genetic test from quality and quantity.In general, doctor is carrying out surgical site infections,
Can be by Sample preservation in formalin (10% neutral formalin), these samples become the weight of the accurate diagnosis and treatment of cancer patient individuation
Want the sources DNA.But since formalin is while fixed dna so that cell membrane and nuclear membrane become difficult to rupture, resistance
The release of DNA and RNA are stopped, but also DNA can be caused to have different degrees of crosslinked action with protein or other nucleic acid, has extracted
When DNA, the endless all round reversing of crosslinked action can influence the subsequent experimentals such as DNA sequencing or qPCR.Currently used method is mainly wrapped
Include long-time RNA extraction based on proteinase K digestion and Mechanical Crushing binding protein enzyme K digestion methods etc..At present the extraction time of these methods compared with
It is long, cannot meet clinical the needs of quickly detecting, can not good smudge cells, release nucleic acid-protein crosslinked action, because
This is difficult to the DNA for obtaining high quality.
Invention content
In view of this, the purpose of the present invention is to provide it is a kind of can quickly, the extraction dipped into formalin group of high quality
Knit the extracts kit and extracting method of DNA
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：A kind of dipped into formalin tissue DNA carries
Take kit, including following components：SDS buffer solutions, combination buffer, Proteinase K, DNA purifying magnetic bead, the first washing lotion, second
Washing lotion and eluent；A concentration of 0.03~0.04mol/L of the SDS buffer solutions；The combination buffer include 3.0~
3.5mol/L GuHCl, 0.5~1.0mol/L NaCl, 0.01~0.02mol/L SDS, 0.08~0.15mol/L Triton
X-100,1.25~1.30mol/LEDTA and 0.8~1.2mol/L Tris-HCl；First washing lotion include 0.60~
0.65mol/L GuHCl, 0.40~0.50mol/LNaCl, and 8.0~8.5mmol/LTween 20；Second washing lotion includes
0.8~1.2mol/L Tris and volume fraction are 20~30% ethyl alcohol；The eluent is 0.008~0.012mol/LTris
Solution.
Preferably, a concentration of 0.035mol/L of the SDS buffer solutions；The combination buffer includes 3.3mol/L
GuHCl, 0.75mol/LNaCl, 0.014mol/L SDS, 0.11mol/L Triton X-100,1.28mol/LEDTA and
1.0mol/L Tris-HCl；First washing lotion includes 0.63mol/L GuHCl, 0.45mol/LNaCl, and 8.22mmol/
LTween 20；Second washing lotion includes 1.0mol/L Tris and volume fraction is 26% ethyl alcohol；The eluent is
0.01mol/L Tris solution.
Preferably, further include PBS buffer solution and isopropanol.
The present invention also provides a kind of methods for extracting dipped into formalin tissue DNA using the kit, including with
Lower step：
1) ultrasonication obtains lysate after mixing dipped into formalin tissue with SDS buffer solutions；
2) after mixing the lysate with Proteinase K, 54 DEG C~58 DEG C 50~70min of digestion obtain digestive juice；
3) by the digestive juice in 78 DEG C~82 DEG C 40~80min of secondary digestion, cooling, separation of solid and liquid collects liquid phase component
For liquid to be extracted；
4) liquid to be extracted, PBS buffer solution, combination buffer, DNA purifying magnetic beads and isopropanol are mixed, in 54~
After 56 DEG C of 10~15min of heating, Magneto separate collects the magnetic bead in conjunction with DNA；
5) magnetic bead of the combination DNA is rinsed with the first washing lotion and the second washing lotion successively；After the rinsing, it will rinse
The natural air drying that the magnetic bead of combination DNA afterwards carries out 3~5min obtains dry magnetic bead；
6) after the dry magnetic bead and eluent being mixed in 54~56 DEG C of 8~12min of heating, Magneto separate collects liquid phase
Component is the dipped into formalin tissue DNA for extracting and obtaining.
Preferably, the dipped into formalin is organized as the graininess of 0.8~1.2mm of diameter；The dipped into formalin
The mass volume ratio of tissue and SDS buffer solutions is (0.3~1) g:10ml.
Preferably, the duty factor of the ultrasonication is 18~22%；The moment maximum power of the ultrasonication is 70
~80w；The recurring number of each pulse of the ultrasonication is 180~220；The time of the ultrasonication is 120~360s；
The temperature of the ultrasonication is 18~26 DEG C.
Preferably, the volume ratio of the lysate and Proteinase K is (4~6)：1.
Preferably, the liquid to be extracted, PBS buffer solution, combination buffer, DNA purify the volume ratio of magnetic bead and isopropanol
Preferably (100~140)：(350~450)：(450~550)：(80~160)：(35~45).
Preferably, first washing lotion and the volume ratio of DNA purifying magnetic beads are 15：(2~4)；Second washing lotion and DNA
The volume ratio for purifying magnetic bead is 15：(2~4).
Preferably, the volume ratio of the eluent and DNA purifying magnetic beads is 5：(4~8).
Compared with prior art, beneficial effects of the present invention are as follows：
Dipped into formalin tissue DNA extracts kit and extracting method of the present invention, can realize to formalin
The extraction of tissue DNA is impregnated, the DNA for extracting acquisition is high-quality, and the rate of recovery is high.
Dipped into formalin tissue DNA extracting method of the present invention utilizes sonicated cells, the original of ultrasonication
Reason is to utilize ultrasonic wave promotion medium, makes pressure change in liquid and forms numerous bubbles and rapid implosion, and the sharp energy of generation will
Object is broken up to achieve the purpose that broken；Ultrasonication can help broken cell membrane in a short period of time, by intracellular
DNA, RNA, protein etc. release.
Dipped into formalin tissue DNA extracting method of the present invention reverses nucleic acid-by two step protease K digestings
The crosslinked action of protein, being capable of better released dna.Heretofore described DNA purifying magnetic bead can recycle all DNA pieces
Section improves the DNA rate of recovery；The first washing lotion and the second washing lotion repeatedly rinse the magnetic bead in conjunction with DNA in the present invention, and undesired impurities are clear
It washes off, extracts the genomic DNA of high quality.
Description of the drawings
Fig. 1 is the influence that different ultrasonic times extract DNA total amount；
Fig. 2 is different 2100 figures of ultrasonic time DNA；
Fig. 3 is the influence that different ultrasonic temperatures extract DNA total amount
Fig. 4 is different 2100 figures of ultrasonic temperature DNA；
Fig. 5 is the influence that different initial amounts extract DNA total amount；
Fig. 6 is different 2100 figures of initial amount DNA；
Fig. 7 is the influence that different extracts kits extract DNA total amount；
Fig. 8 is different 2100 figures of kit DNA；
Fig. 9 is 2100 figure of mouse lung and hepatic tissue DNA.
Specific implementation mode
The present invention provides a kind of dipped into formalin tissue DNA extracts kits, including following components：SDS buffer solutions,
Combination buffer, Proteinase K, DNA purifying magnetic bead, the first washing lotion, the second washing lotion and eluent；The concentration of the SDS buffer solutions
For 0.03~0.04mol/L；The combination buffer includes 3.0~3.5mol/L GuHCl, 0.5~1.0mol/L NaCl,
0.01~0.02mol/L SDS, 0.08~0.15mol/LTriton X-100,1.25~1.30mol/LEDTA and 0.8~
1.2mol/L Tris-HCl；First washing lotion includes 0.60~0.65mol/L GuHCl, 0.40~0.50mol/L NaCl,
With 8.0~8.5mmol/LTween 20；Second washing lotion include 0.8~1.2mol/L Tris and volume fraction be 20~
30% ethyl alcohol；The eluent is 0.008~0.012mol/LTris solution.
In the present invention, the kit includes SDS buffer solutions, and the concentration of the SDS buffer solutions is preferably 0.032~
0.038mol/L, more preferably 0.035mol/L；In the present invention, the solvent of the SDS buffer solutions is preferably Tris-HCl, described
The pH value of Tris-HCl is preferably 8.1；Heretofore described SDS buffer solutions act as lytic cell.
In the present invention, the kit includes combination buffer, in the present invention, the combination buffer include 3.0~
3.5mol/L GuHCl, 0.5~1.0mol/L NaCl, 0.01~0.02mol/L SDS, 0.08~0.15mol/LTriton
X-100,1.25~1.30mol/LEDTA and 0.8~1.2mol/L Tris-HCl；Include preferably 3.3mol/L GuHCl,
0.75mol/L NaCl, 0.014mol/L SDS, 0.11mol/L Triton X-100,1.28mol/LEDTA and 1.0mol/L
Tris-HCl.In the present invention, the effect of the combination buffer is that DNA is promoted to be combined with DNA purifying magnetic beads.
Heretofore described kit further includes Proteinase K, and the source of Proteinase K of the present invention is not particularly limited,
Using commercial protein enzyme K.In the present invention, the vigor of the Proteinase K is preferably 500~700mAU/ml, more preferably
550~650mAU/ml, most preferably 600mAU/ml.In the present invention, the Proteinase K act as vitellophag lysate
In protein；The crosslinked action for reversing nucleic acid-protein simultaneously, makes DNA fully discharge.
In the present invention, the kit further includes DNA purifying magnetic beads, and the present invention purifies the DNA in the source of magnetic bead
It is not particularly limited, magnetic bead is purified using commercially available DNA.In specific implementation process of the present invention, the DNA purifies magnetic bead
Magnetic bead is purified for commercialized DNA.The DNA for acting as with releasing of heretofore described DNA purifying magnetic bead is combined.
In the present invention, the kit further includes the first washing lotion and the second washing lotion, and first washing lotion includes 0.60~
0.65mol/L GuHCl, 0.40~0.50mol/L NaCl and 8.0~8.5mmol/LTween 20 include preferably
0.63mol/L GuHCl, 0.45mol/L NaCl and 8.22mmol/LTween 20.Second washing lotion include 0.8~
1.2mol/L Tris and volume fraction are 20~30% ethyl alcohol, are preferably 26% including 1.0mol/LTris and volume fraction
Ethyl alcohol.In the present invention, the effect of first washing lotion and the second washing lotion is to wash undesired impurities on magnetic bead, improves extraction
The quality of DNA.
In the present invention, the kit further includes eluent, and the eluent is that 0.008~0.012mol/L Tris are molten
Liquid, preferably 0.009~0.011mol/L, more preferably 0.010mol/L.The effect of heretofore described eluent is will to tie
The DNA closed on magnetic bead is eluted.
In the present invention, the kit further includes preferably PBS buffer solution, and the concentration of the PBS buffer solution is preferably
0.01mol/L, pH value are preferably 7.4；The PBS buffer solution act as adjusting pH value in a certain range；It is of the present invention
Kit further includes preferably isopropanol, and heretofore described isopropanol is preferably analytically pure isopropanol, the isopropanol
It act as promoting the precipitation of DNA.
One preferred embodiment of heretofore described kit be include 0.035mol/L SDS buffer solutions, combination buffer
(including 3.3mol/L GuHCl, 0.75mol/LNaCl, 0.014mol/L SDS, 0.11mol/L Triton X-100,
1.28mol/LEDTA and 1mol/LTris-HCl), the first washing lotion (including .63mol/L GuHCl, 0.45mol/LNaCl and
8.2mmol/LTween 20), the second washing lotion (including 1mol/LTris and volume fraction are 26% ethyl alcohol) and eluent
(0.01mol/LTris)。
The present invention also provides a kind of methods for extracting dipped into formalin tissue DNA using the kit, including with
Lower step：1) ultrasonication obtains lysate after mixing dipped into formalin tissue with SDS buffer solutions；2) by the lysate
After being mixed with Proteinase K, 54 DEG C~58 DEG C 50~70min of digestion obtain digestive juice；3) by the digestive juice in 78 DEG C~82 DEG C
40~80min of secondary digestion, it is liquid to be extracted that cooling, separation of solid and liquid, which collects liquid phase component,；4) liquid to be extracted, PBS are delayed
Fliud flushing, combination buffer, DNA purifying magnetic beads and isopropanol mixing, after 54~56 DEG C are heated 10~15min, Magneto separate is collected
In conjunction with the magnetic bead of DNA；5) magnetic bead of DNA is combined with the first washing lotion and the rinsing of the second washing lotion successively；After the rinsing, it will float
The natural air drying that the magnetic bead of combination DNA after washing carries out 3~5min obtains dry magnetic bead；6) by the dry magnetic bead and eluent
After being mixed in 54~56 DEG C of 8~12min of heating, Magneto separate, it is the dipped into formalin group extracted and obtained to collect liquid phase component
Knit DNA.
Ultrasonication obtains lysate after the present invention mixes dipped into formalin tissue with SDS buffer solutions.In the present invention
In, the dipped into formalin tissue is preferably the pathological tissues from human body or animal body；Heretofore described formal
Preferably medical formalin solution for preserving pathological tissues sample can be specifically during mass percentage is 10% to woods
Property formaldehyde.The dipped into formalin group is woven in the present invention preferably carries out chopping processing using preceding, and the present invention is to described
The tool and method of chopping is not particularly limited；Dipped into formalin tissue after heretofore described chopping is preferably diameter
The graininess of 0.8~1.2mm, the more preferably graininess of diameter 1.0mm；Dipped into formalin tissue is being obtained in the present invention
After grain, the dipped into formalin tissue is mixed with SDS buffer solutions；The dipped into formalin tissue and SDS buffer solutions
Mass volume ratio is preferably (0.2~1) g:10ml, more preferably (0.4~0.8) g:10ml.The present invention is by the formal
After woods immersion tissue is mixed with SDS buffer solutions, ultrasonication is carried out to mixed liquor and obtains lysate.In the present invention, described super
The broken Duty Factor of sound are preferably 18~22%, and more preferably 20%；The Peak of the ultrasonication
IncidentPower is preferably 70~80w, more preferably 75w；The Cycles per burst of the ultrasonication are preferably
180~220, more preferably 200；The time of the ultrasonication is preferably 120~360s, more preferably 200~280s, optimal
It is selected as 240s；The temperature of the ultrasonication is preferably 18~26 DEG C, more preferably 19~24 DEG C, most preferably 20 DEG C.At this
Dipped into formalin tissue described in invention carries out ultrasonication after being mixed with SDS buffer solutions, can using sonicated cells
Broken cell membrane is helped in a short period of time, and by intracellular DNA, RNA, protein etc. releases.
The present invention is after obtaining the lysate, after the lysate is mixed with Proteinase K, 54 DEG C~58 DEG C digestion 50
~70min obtains digestive juice.The volume ratio of the lysate and Proteinase K is preferably (4~6) in the present invention：1, more preferably
It is 5:1.The temperature of the digestion is preferably 55~57 DEG C in the present invention, more preferably 56 DEG C；The time of the digestion is preferred
For 55~65min, more preferably 58~62min.The purpose of the digestion is soluble protein in the present invention, is discharged in cell
DNA。
The present invention is cold by the digestive juice in 78 DEG C~82 DEG C secondary digestion 40~80min after obtaining the digestive juice
But it is liquid to be extracted, to be separated by solid-liquid separation and collect liquid phase component.In the present invention, the temperature of the secondary digestion is preferably 80 DEG C,
The time of the secondary digestion is preferably 45~75min, more preferably 55~65min.The secondary digestion in the present invention
Effect is the crosslinked action for making Proteinase K further reverse nucleic acid-protein, abundant released dna.The present invention secondary disappears described
After change, natural cooling is carried out, is then separated by solid-liquid separation the liquid after the secondary digestion, it is to be extracted to collect liquid phase component
Liquid.In the present invention, the method for the separation of solid and liquid preferably centrifuges, and the rotating speed of the centrifugation is preferably 12000~
18000rpm, more preferably 15000rpm；The time of the centrifugation is preferably 3~8min, more preferably 5min.
The present invention is pure by the liquid to be extracted, PBS buffer solution, combination buffer, DNA after obtaining the liquid to be extracted
Change magnetic bead and isopropanol mixing, after 54~56 DEG C are heated 10~15min, Magneto separate collects the magnetic bead in conjunction with DNA.In the present invention
In, the liquid to be extracted is preferred first to be mixed with PBS buffer solution, then purifies magnetic bead and isopropanol with combination buffer, DNA again
Mixing.In the present invention, the liquid to be extracted, PBS buffer solution, combination buffer, DNA purify the volume ratio of magnetic bead and isopropanol
Preferably (100~140)：(350~450)：(450~550)：(80~160)：(35~45), more preferably (110~130)：
(380~420)：(480~520)：(100~120)：(38~42), most preferably 120:400:500:110:40.The present invention exists
After the mixing, by mixed liquor after 54~56 DEG C are heated 10~15min, Magneto separate collects the magnetic bead in conjunction with DNA.In the present invention
Described in the temperature that heats be preferably 55 DEG C, the time of the heating is preferably 11~14min, more preferably 12~13min.
In the present invention, the Magneto separate is preferably carried out using Magneto separate frame, and the time of the Magneto separate is preferably 1~5min, more excellent
It is selected as 2min.The present invention preferably discards supernatant after the Magneto separate, collects the magnetic bead in conjunction with DNA.
The present invention combines the magnetic of DNA with the first washing lotion and the rinsing of the second washing lotion successively after obtaining the magnetic bead in conjunction with DNA
Pearl.In the present invention, first washing lotion and the volume ratio of DNA purifying magnetic beads are preferably 15：(2~4), more preferably 15：3；
Second washing lotion and the volume ratio of DNA purifying magnetic beads are 15：(2~4), more preferably 15：3.It was embodied in the present invention
Cheng Zhong after the preferred magnetic bead 2~3 times with the rinsing of the first washing lotion in conjunction with DNA, then rinses the magnetic bead 2 for combining DNA with the second washing lotion
~3 times.In the present invention, the rinse cycle preferably slowly blows and beats magnetic bead and the first washing lotion or the second washing lotion with pipettor
Mixed liquor 8~15 times, makes magnetic bead be mixed well with the first washing lotion or the second washing lotion；It is placed at room temperature for 1min, is placed in magnetic separation rack
Upper standing 2min, abandons supernatant.The present invention carries out 3~5min's after the rinsing, by the magnetic bead of the combination DNA after rinsing
Natural air drying obtains dry magnetic bead, and the natural air drying preferably stops to magnetic bead surfaces without apparent gloss in the present invention, this
Drying time described in invention is unsuitable long, and to prevent magnetic bead drying excessive, DNA can not be eluted.
The present invention after obtaining dry magnetic bead, by the dry magnetic bead and eluent be mixed in 54~56 DEG C of heating 8~
After 12min, Magneto separate, it is the dipped into formalin tissue DNA for extracting and obtaining to collect liquid phase component.In the present invention, described
The volume of eluent and the volume ratio of DNA purifying magnetic beads are preferably 5：(4~8), more preferably 5：(5~7).In the present invention
The method and steps of the Magneto separate is consistent with above-mentioned Magneto separate, and details are not described herein.
The dipped into formalin tissue DNA concentration that the method extraction obtains through the invention is high, high-quality, can apply
In experiments such as subsequent PCR amplification sequencing, qPCR.
Technical solution provided by the invention is described in detail with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Dipped into formalin tissue DNA extracts kit
The preparation of 1.SDS buffer solutions：1g SDS are taken, 50mM Tris-HCl (pH8.1) mixing is added, is settled to 100ml.
2. the preparation of combination buffer:Weigh 31.84g GuHCl, 4.38gNaCl, 0.42g SDS, 70g Triton X-
100,80ml ultra-pure waters are added, 0.5mlEDTA is added in mixing, and 5ml Tris-HCl are settled to 100ml.
3. the preparation of 1.28mol/LEDTA:It weighs in 18.61g EDTA to 50mL beakers, and 40mL ultra-pure waters is added.
Beaker is placed on magnetic stirring apparatus, NaOH solids are added while stirring until solution change clarification, is adjusted to 8.0 by pH, is settled to
50mL。
4. 1mol/LTris-HCl (pH 8.1) solution is prepared:It weighs in 12.11g Tris to 100mL beakers, and is added
80mL ultra-pure waters make it completely dissolved, and measure pH with pH meter, concentrated hydrochloric acid is used in combination to adjust pH value.Solution is transferred to 100mL capacity
In bottle, purified water is added and is settled to 100mL.
5. the preparation of the first washing lotion：Weigh 6g GuHCl, 2.63gNaCl, 1gTween 20 (8.2mmol/L) plus ultra-pure water
It is settled to 100mL.
6. the preparation of the second washing lotion:60ml ultra-pure waters are added in 12.11g Tris, and 26% ethyl alcohol of 26ml is added in mixing, uses
Concentrated hydrochloric acid is adjusted to 8.0 by pH value of solution, and purified water is added and is settled to 100mL.
7. the preparation of eluent:800ml ultra-pure waters are added in 1.2g Tris, after being completely dissolved, concentrated hydrochloric acid are added dropwise and adjusts pH
It is worth to 8.0.
Embodiment 2
Pretreatment
1. choosing the lung tissue for the cancer patient that an example dipped into formalin is crossed, tissue is taken with No. 18 puncture needles, with operation
The graininess of tissue chopping to a diameter of 1mm is divided into the sample that 5 parts of weight are 4.2mg by blade.Open microTUBE
Screw lid, be added 100 μ l SDS Buffer, the tissue of chopping is added in Buffer, tightens lid, respectively number 1-
5。
2. open Ultrasonic Cell Disruptor, be arranged program, it is all identical in other parameters, 1-5 samples it is broken when
Between incremented by successively, respectively 120s, 180s, 240s, 300s, 360s.No. 1 and No. 2 samples can be seen with the presence of a small amount of tissue block,
Not perfectly homogenousization, other three groups complete homogeneity.Ultrasonication parameter is shown in Table 1.
1 ultrasonication parameter of table
3. lid is opened, sample from being drawn onto in microTUBE in 1.5ml EP pipes.
4. 20 μ l Proteinase Ks, mixing are added into sample.
5. 56 DEG C of incubation 1h.
6. 80 DEG C of incubation 1h, room temperature cooling.
7. 15000g centrifuges 5min, takes supernatant.
Magnetic beads for purifying DNA
1. 1 × PBS of 400 μ L, mixing are added in supernatant.
2. combining：Be added 500 μ L combination buffers, the magnetic bead of 120 μ L, 40 μ L isopropanols vibrate mixing, whole system in
55 DEG C of heating 10-15min, are placed on Magnetic rack and stand 2min, abandon supernatant.
3. washing：600 the first washing lotions of μ L are added, magnetic bead are slowly blown and beaten with pipettor 10 times, magnetic bead is made to mix well.Room temperature
Place 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
4. repeating step 3.
5. 600 the second washing lotions of μ L are added, magnetic bead is slowly blown and beaten with pipettor 10 times, magnetic bead is made to mix well.It is placed at room temperature for
1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
6. repeating step 5, cleaning solution is eliminated as possible.
7. drying：Keep centrifuge tube on magnetic frame, opening the air-dried 3~5min of lid, until magnetic bead surfaces are without apparent gloss.
Drying time is unsuitable long, to prevent magnetic bead drying excessive.
8. elution：100 μ l eluents are added.Centrifuge tube is placed in 55 DEG C of heating 10min (it is primary that period overturns mixing).It sets
In standing 2min on Magnetic rack, supernatant is moved to new centrifuge tube, the DNA as purified.
9. being quantified to sample with qubit, 2100 analysis DNA fragmentation sizes.
Different ultrasonic time DNA extractions total amounts are shown in Table 2.
The different ultrasonic time DNA of table 2 extract total amount result
Ultrasonic time extracts the influence of total amount to DNA and DNA2100 figures are shown in Fig. 1 and Fig. 2；A-E represents ultrasonic time in Fig. 2
Respectively 120s, 180s, 240s, 300s, 360s.
Conclusion：When ultrasonic time is 240s, DNA extracted amounts are maximum, and with the increase of ultrasonic time, DNA extracted amounts are instead
It reduces, therefore 240s is ultrasonic optimum time.
Embodiment 3
1. choosing the lung tissue for the cancer patient that an example dipped into formalin is crossed, tissue is taken with No. 18 puncture needles, with operation
The graininess of tissue chopping to a diameter of 1mm is divided into the sample that 5 parts of weight are 3.9mg by blade.Open microTUBE
Screw lid, be added 100 μ l SDS Buffer, the tissue of chopping is added in Buffer, tightens lid, respectively number 1-
5。
2. opening Ultrasonic Cell Disruptor, program disrupting tissue, 1-5 sample all identical in other parameters are set
Breaking temperature it is incremented by successively, respectively 18 DEG C, 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C.It can be seen that 5 groups of sample standard deviations are completely homogeneous
Change.Ultrasonication parameter is shown in Table 3.
3 ultrasonication parameter of table
3. lid is opened, sample from being drawn onto in microTUBE in 1.5ml EP pipes.
4. 20 μ l Proteinase Ks, mixing are added into sample.
5. 56 DEG C of incubation 1h.
6. 80 DEG C of incubation 1h, room temperature cooling.
7. 15000g centrifuges 5min, takes supernatant.
Magnetic beads for purifying DNA
1. 1 × PBS of 400 μ L, mixing are added in above-mentioned supernatant.
2. combining：500 μ LBindingBuffer, the magnetic bead of 120 μ L are added, 40 μ L isopropanols vibrate mixing, entire body
55 DEG C of heating 10-15min are lain in, is placed on Magnetic rack and stands 2min, abandon supernatant.
3. washing：600 μ LWashingBufferI are added, magnetic bead are slowly blown and beaten with pipettor 10 times, keep magnetic bead fully mixed
It is even.It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
4. repeating step 3.
5. 600 μ LWashingBufferII are added, magnetic bead is slowly blown and beaten with pipettor 10 times, magnetic bead is made to mix well.
It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
6. repeating step 5, cleaning solution is eliminated as possible.
7. drying：Keep centrifuge tube on magnetic frame, opening the air-dried 3~5min of lid, until magnetic bead surfaces are without apparent gloss.
Drying time is unsuitable long, to prevent magnetic bead drying excessive.
8. elution：100 μ l ElutionBuffer are added.Centrifuge tube is placed in 55 DEG C of heating 10min, and (period is reverse mixed
It is even primary).It is placed on Magnetic rack and stands 2min, supernatant is moved to new centrifuge tube, as purifying obtains
DNA。
9. being quantified to sample with qubit, 2100 analysis DNA fragmentation sizes.
The DNA extractions total amount of different ultrasonic temperatures the results are shown in Table 4.
The DNA of the different ultrasonic temperatures of table 4 extracts total amount result
Ultrasonic temperature extracts the influence of total amount to DNA and DNA2100 figures are shown in Fig. 3 and Fig. 4；A-E respectively represents ultrasound in Fig. 4
Temperature be 18 DEG C, 20 DEG C, 22 DEG C, 24 DEG C, 26.
Conclusion：When ultrasonic temperature is respectively 18 DEG C, 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C, conspicuousness is had no between DNA extracted amounts
Difference, but at 20 DEG C, DNA output is slightly higher, and segment distribution is more concentrated.
Embodiment 4
Pretreatment
1. choosing the mouse lung tissue that an example dipped into formalin is crossed, tissue is taken with No. 18 puncture needles, it will with knife blade
Tissue chopping is divided into 4 parts that weight is 4mg, 6mg, 8mg, 10mg to the graininess of a diameter of 1mm.Open the spiral shell of microTUBE
Spiral cover is added 100 μ l SDS Buffer, the tissue of chopping is added in Buffer, tightens lid, respectively number 1-4.
2. using Ultrasonic Cell Disruptor and following procedure disrupting tissue.The complete homogeneity of 1-3 sample standard deviations, No. 4 it can be seen that have
A small amount of tissue block exists, not perfectly homogenousization.Ultrasonication parameter is shown in Table 5.
5 ultrasonication parameter of table
3. lid is opened, sample from being drawn onto in microTUBE in 1.5ml EP pipes.
4. 20 μ l Proteinase Ks, mixing are added into sample.
5. 56 DEG C of incubation 1h.
6. 80 DEG C of incubation 1h, room temperature cooling.
7. 15000g centrifuges 5min, takes supernatant.
Magnetic beads for purifying DNA
1. 1 × PBS of 400 μ L, mixing are added in above-mentioned supernatant.
2. combining：500 μ LBindingBuffer, the magnetic bead of 120 μ L are added, 40 μ L isopropanols vibrate mixing, entire body
55 DEG C of heating 10-15min are lain in, is placed on Magnetic rack and stands 2min, abandon supernatant.
3. washing：600 μ LWashingBufferI are added, magnetic bead are slowly blown and beaten with pipettor 10 times, keep magnetic bead fully mixed
It is even.It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
4. repeating step 3.
5. 600 μ LWashingBufferII are added, magnetic bead is slowly blown and beaten with pipettor 10 times, magnetic bead is made to mix well.
It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
6. repeating step 5, cleaning solution is eliminated as possible.
7. drying：Keep centrifuge tube on magnetic frame, opening the air-dried 3~5min of lid, until magnetic bead surfaces are without apparent gloss.
Drying time is unsuitable long, to prevent magnetic bead drying excessive, nucleic acid is caused to be difficult to elute.
8. elution：100 μ l ElutionBuffer are added.Centrifuge tube is placed in 55 DEG C of heating 10min, and (period is reverse mixed
It is even primary).It is placed on Magnetic rack and stands 2min, supernatant is moved to new centrifuge tube, as purifying obtains
DNA。
9. being quantified to sample with qubit, 2100 analysis DNA fragmentation sizes.
The DNA extractions total amount of different initial amounts the results are shown in Table 6.
The DNA of the different initial amounts of table 6 extracts total amount result
Initial amount extracts the influence of total amount to DNA and DNA2100 figures are shown in Fig. 5 and Fig. 6；A-D represents dipped into formalin group
It is respectively 4mg, 6mg, 8mg, 10mg to knit initial amount.Conclusion：With the increase of initial amount, DNA concentration is higher, when 8mg initial amounts,
Concentration reaches maximum value.When initial amount increases to 10mg, insufficient due to cracking, DNA concentration declines instead.
Embodiment 5
Pretreatment
1. choosing the hepatic tissue for the cancer patient that an example dipped into formalin is crossed, tissue is taken with No. 18 puncture needles, with operation
Tissue chopping to the graininess of a diameter of 1mm, is equally divided into 2 parts, weight 4.6mg by blade.Number 1,2 respectively.
2. No. 1 sample is broken according to conventional mechanical, RNA extraction based on proteinase K digestion extracts.Qubit is carried out to obtained DNA
The analysis of quantitative and 2100 clip sizes.
3. No. 2 samples use Ultrasonic Cell Disruptor and following procedure disrupting tissue.Broken tissue can homogeneity.Ultrasound
Breakage parameter is shown in Table 7.
The parameter of 7 ultrasonication of table
4. lid is opened, sample from being drawn onto in microTUBE in 1.5ml EP pipes.
5. 20 μ l Proteinase Ks, mixing are added into sample.
6. 56 DEG C of incubation 1h.
7. 80 DEG C of incubation 1h, room temperature cooling.
8. 15000g centrifuges 5min, takes supernatant.
Magnetic beads for purifying DNA
1. 1 × PBS of 400 μ L, mixing are added in supernatant.
2. combining：500 μ L BindingBuffer, the magnetic bead of 120 μ L are added, 40 μ L isopropanols vibrate mixing, entire body
55 DEG C of heating 10-15min are lain in, is placed on Magnetic rack and stands 2min, abandon supernatant.
3. washing：600 μ LWashingBufferI are added, magnetic bead are slowly blown and beaten with pipettor 10 times, keep magnetic bead fully mixed
It is even.It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
4. repeating step 3.
5. 600 μ LWashingBufferII are added, magnetic bead is slowly blown and beaten with pipettor 10 times, magnetic bead is made to mix well.
It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
6. repeating step 5, cleaning solution is eliminated as possible.
7. drying：Keep centrifuge tube on magnetic frame, opening the air-dried 3~5min of lid, until magnetic bead surfaces are without apparent gloss.
Drying time is unsuitable long, to prevent magnetic bead drying excessive.
8. elution：100 μ l ElutionBuffer are added.Centrifuge tube is placed in 55 DEG C of heating 10min, and (period is reverse mixed
It is even primary).It is placed on Magnetic rack and stands 2min, supernatant is moved to new centrifuge tube, as purifying obtains
DNA。
9. being quantified to sample with qubit, 2100 analysis DNA fragmentation sizes.
The DNA extraction total amount comparison results of different kits are shown in Table 8.
The DNA of the different kits of table 8 extracts total amount results contrast
Different extracts kits extract the influence of total amount to DNA and DNA2100 figures are shown in Fig. 7 and Fig. 8；A represents common in Fig. 8
Kit, B represent the present invention.General reagent box in the present embodiment is commercially available extraction kit.
Conclusion：Same sample uses general reagent box and the present invention to extract respectively, and general reagent box DNA concentration is very low, and
Segment disperse.The DNA concentration that the present invention extracts is very high, and segment is also concentrated very much.
Embodiment 6
Pretreatment
1. choosing mouse lung tissue and hepatic tissue that an example dipped into formalin is crossed, tissue is taken with No. 18 puncture needles, uses hand
For art blade by tissue chopping to the graininess of a diameter of 1mm, weight is respectively 5.4mg and 4.9mg.Open the spiral shell of microTUBE
Spiral cover is added 100 μ l SDS Buffer, the tissue of chopping is added in Buffer, lid is tightened, respectively number 1,2.
No. 2.1 samples are broken according to conventional mechanical, and RNA extraction based on proteinase K digestion extracts.Qubit is carried out to obtained DNA
The analysis of quantitative and 2100 clip sizes.
3. No. 2 samples use Ultrasonic Cell Disruptor and following procedure disrupting tissue.Broken tissue can homogeneity.Ultrasound
Breakage parameter is shown in Table 9.
9 ultrasonication parameter of table
4. lid is opened, sample from being drawn onto in microTUBE in 1.5ml EP pipes.
5. 20 μ l Proteinase Ks, mixing are added into sample.
6. 56 DEG C of incubation 1h.
7. 80 DEG C of incubation 1h, room temperature cooling.
8. 15000g centrifuges 5min, takes supernatant.
Magnetic beads for purifying DNA
1. 1 × PBS of 400 μ L, mixing are added in supernatant.
2. combining：500 μ LBindingBuffer, the magnetic bead of 120 μ L are added, 40 μ L isopropanols vibrate mixing, entire body
55 DEG C of heating 10-15min are lain in, is placed on Magnetic rack and stands 2min, abandon supernatant.
3. washing：600 μ LWashingBufferI are added, magnetic bead are slowly blown and beaten with pipettor 10 times, keep magnetic bead fully mixed
It is even.It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
4. repeating step 3.
5. 600 μ LWashingBufferII are added, magnetic bead is slowly blown and beaten with pipettor 10 times, magnetic bead is made to mix well.
It is placed at room temperature for 1min.It is placed on Magnetic rack and stands 2min, abandon supernatant.
6. repeating step 5, cleaning solution is eliminated as possible.
7. drying：Keep centrifuge tube on magnetic frame, opening the air-dried 3~5min of lid, until magnetic bead surfaces are without apparent gloss.
Drying time is unsuitable long, to prevent magnetic bead drying excessive.
8. elution：100 μ l ElutionBuffer are added.Centrifuge tube is placed in 55 DEG C of heating 10min, and (period is reverse mixed
It is even primary).It is placed on Magnetic rack and stands 2min, supernatant is moved to new centrifuge tube, as purifying obtains
DNA。
9. being quantified to sample with qubit, 2100 analysis DNA fragmentation sizes.
The DNA extractions total amount of different kits the results are shown in Table 10.
The DNA of the different kits of table 10 extracts total amount result
Two kinds of organization type DNA2100 figures are shown in that Fig. 9, wherein A represent murine liver tissue, and B represents mouse lung tissue.Conclusion：
The mouse lung and hepatic tissue crossed with present invention extraction dipped into formalin, DNA concentration is all very high, and segment is also concentrated very much.
By above-described embodiment it is found that dipped into formalin tissue DNA extracts kit provided by the invention and extracting method,
It can realize the extraction to dipped into formalin tissue DNA, the DNA for extracting acquisition is high-quality, and the rate of recovery is high.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of dipped into formalin tissue DNA extracts kit, which is characterized in that including following components：SDS buffer solutions, knot
Close buffer solution, Proteinase K, DNA purifying magnetic bead, the first washing lotion, the second washing lotion and eluent；
A concentration of 0.03~0.04mol/L of the SDS buffer solutions；
The combination buffer includes 3.0~3.5mol/L GuHCl, 0.5~1.0mol/L NaCl, 0.01~0.02mol/L
SDS, 0.08~0.15mol/L Triton X-100,1.25~1.30mol/LEDTA and 0.8~1.2mol/L Tris-HCl；
First washing lotion include 0.60~0.65mol/L GuHCl, 0.40~0.50mol/L NaCl and 8.0~
8.5mmol/L Tween 20；
Second washing lotion includes 0.8~1.2mol/L Tris and volume fraction is 20~30% ethyl alcohol；
The eluent is 0.008~0.012mol/L Tris solution.
2. dipped into formalin tissue DNA extracts kit according to claim 1, which is characterized in that the SDS bufferings
A concentration of 0.035mol/L of liquid；
The combination buffer includes 3.3mol/L GuHCl, 0.75mol/L NaCl, 0.014mol/L SDS, 0.11mol/L
Triton X-100,1.28mol/LEDTA and 1.0mol/L Tris-HCl；
First washing lotion includes 0.63mol/L GuHCl, 0.45mol/L NaCl and 8.22mmol/L Tween 20；
Second washing lotion includes 1.0mol/L Tris and volume fraction is 26% ethyl alcohol；
The eluent is 0.01mol/LTris solution.
3. dipped into formalin tissue DNA extracts kit according to claim 1 or 2, which is characterized in that further include
PBS buffer solution and isopropanol.
4. using the method for kit extraction dipped into formalin tissue DNA described in 3 any one of claims 1 to 3, including with
Lower step：
1) ultrasonication obtains lysate after mixing dipped into formalin tissue with SDS buffer solutions；
2) after mixing the lysate with Proteinase K, 54 DEG C~58 DEG C 50~70min of digestion obtain digestive juice；
3) by the digestive juice in 78 DEG C~82 DEG C 40~80min of secondary digestion, it is to wait for that cooling, separation of solid and liquid, which collects liquid phase component,
Extracting solution；
4) liquid, PBS buffer solution, combination buffer, DNA purifying magnetic beads and the isopropanol to be extracted are mixed, in 54~56 DEG C
After heating 10~15min, Magneto separate collects the magnetic bead in conjunction with DNA；
5) magnetic bead of the combination DNA is rinsed with the first washing lotion and the second washing lotion successively；Combination DNA magnetic beads after rinsing are carried out
The natural air drying of 3~5min obtains dry magnetic bead；
6) after the dry magnetic bead and eluent being mixed in 54~56 DEG C of 8~12min of heating, Magneto separate collects liquid phase component
For dipped into formalin tissue DNA.
5. according to the method described in claim 4, it is characterized in that, the dipped into formalin is organized as 0.8~1.2mm of diameter
Particle；The mass volume ratio of the dipped into formalin tissue and SDS buffer solutions is (0.3~1) g:10ml.
6. according to the method described in claim 4, it is characterized in that, the duty factor of the ultrasonication is 18~22%；It is described
The moment maximum power of ultrasonication is 70~80w；The recurring number of each pulse of ultrasonication is 180~220；It is described super
The sound broken time is 120~360s；The temperature of the ultrasonication is 18~26 DEG C.
7. the method according to claim 4 or 6, which is characterized in that the volume ratio of the lysate and Proteinase K be (4~
6)：1.
8. according to the method described in claim 4, it is characterized in that, the liquid to be extracted, PBS buffer solution, combination buffer,
It is preferably (100~140) that DNA, which purifies magnetic bead and the volume ratio of isopropanol,：(350~450)：(450~550)：(80~160)：
(35~45).
9. according to the method described in claim 4, it is characterized in that, the volume ratio that first washing lotion purifies magnetic bead with DNA is
15：(2~4)；Second washing lotion and the volume ratio of DNA purifying magnetic beads are 15：(2~4).
10. according to the method described in claim 4, it is characterized in that, the volume ratio of the eluent and DNA purifying magnetic beads is 5：
(4~8).
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