CN105112400A - Kit for extracting free DNA - Google Patents
Kit for extracting free DNA Download PDFInfo
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- CN105112400A CN105112400A CN201510551073.4A CN201510551073A CN105112400A CN 105112400 A CN105112400 A CN 105112400A CN 201510551073 A CN201510551073 A CN 201510551073A CN 105112400 A CN105112400 A CN 105112400A
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Abstract
The invention provides a kit for extracting free DNA and belongs to the technical field of high-throughput DNA detecting. The kit uses two-step magnetic bead absorption to extract the free DNA in blood plasma or urine, low-concentration isopropanol is used in the first step, and magnetic beads mainly absorb large DNA fragments above 500bp; the concentration of the isopropanol is increased in the second step, the magnetic beads mainly absorb small DNA fragments below 500bp, and the DNA fragments above and below 500bp can be extracted by the two-step method. The invention further provides standard products suitable for the two-step method, and the standard products are DNA fragments of 127bp and 208bp. The kit is applicable to prenatal diagnosis and early-stage tumor screening and promising in application prospect in clinical medicine.
Description
Technical field
The present invention relates to high-throughput DNA detection technical field, particularly, relate to a kind of test kit and application thereof of extracting dissociative DNA in blood plasma or urine.
Background technology
Dissociative DNA (cellfreeDNA, cfDNA) is a kind of extracellular dna of acellular state, is mainly made up of the mixture of strand or double-stranded DNA and strand and double-stranded DNA, exists with DNA mono-protein complex or dissociative DNA two kinds of forms.Also there is cfDNA in healthy human peripheral blood, but the peripheral blood cfDNA content of tumour patient has increasing of significance compared with healthy human peripheral blood cfDNA, and there are transgenation, restructuring, disappearance etc. in blood of cancer patients DNA.Blood of cancer patients dissociative DNA is mainly the necrosis of tumour cell or apoptosis, micrometastasis stove or the cracking of circulating tumor cell and the tumour cell of molecular marker for increased proliferation and discharges.
Oncogene detects the most direct to liking patient's primary tissue sample, but do not carry out the tumour patient of performing the operation for those, tumor specimen not easily obtains, though and the technology of Biopsy is comparatively ripe, but patient acceptance is relatively low, especially those be there is to the patient of many places transfer.Even if leave sample before, but the local sample preserved a few months ago or even several years ago often, in view of the dynamic of Oncogenome is with heterogeneous, the information of their reflections has been that information that is out-of-date or representative is incomplete.Clinical study shows, cfDNA can reflect the tumor load of entire patient, grade malignancy, transfer ability and real-time transgenation information well, has good consistence with the gene information of tumor tissues.Multicenter study finds, the sudden change of the DNA in nonsmall-cell lung cancer (NSCLC) patients blood plasma in EGF-R ELISA (EGFR) sudden change of the free Tumour DNA (circulating-freetumorDNA) of circulation and specimens is directly related.Research incorporates the 1060 routine EGFR sudden change positives and is in the patient of different steps, analyze and compared for the tumor tissues of patient and the plasma dna of pairing thereof, found that the tumor tissues of 652 patients is consistent with the mutation status of the EGFR in pairing blood plasma, in these patients, the detection sensitivity of individuality of 94% is 66%, and specificity is 100%.Carry out repeated experiment to two parts of blood plasma of same patient, result display repeatability is higher, and sudden change consistence reaches 97%.By being widely used in clinical guidance to human peripheral blood cfDNA abrupt climatic change, the catastrophe of cfDNA is called a mark of tumour, and the examination of blood plasma cfDNA becomes the trend of individualized disease treatment.Thus, select cfDNA as the sample source of gene test, can ensure oncotherapy in sample intelligence comprehensively precisely, and have compared with biopsy and check that Wicresoft is little, no radioactivity pollute, economic dispatch advantage, and allow to carry out Real-Time Monitoring to therapeutic response.
The quality that blood plasma cfDNA extracts is the guarantee detected DNA mutation, and the method extracting cfDNA at present mainly contains phenol chloroform isoamyl alcohol method, post formulation, paramagnetic particle method.The traditional extraction of phenol chloroform isoamyl alcohol method to blood plasma cfDNA is by DNA extraction all in blood plasma out, and size fragment in blood plasma can not be separated, extraction efficiency is low, need improve its efficiency by other means.Post formulation extraction efficiency is high, but the DNA fragmentation of different size can not be separated.Paramagnetic particle method is the functional group adsorbs nucleic acid utilizing nano level magnetic bead bead surface, utilizes magnetic bead to carry out sorting from the volume ratio of solution to different size DNA, both can obtain the higher rate of recovery, can carry out sorting again to cfDNA.Research prompting tumour patient periphery dissociative DNA fragment calibration ordinary person dissociative DNA is little, in colorectal cancer patients blood plasma, the dissociative DNA fragment of more than 80% is all less than 145bp, the dissociative DNA possibility small segment large percentage of this prompting tumour cell release, therefore tumour patient periphery dissociative DNA size fragment is separated, to significant based on peripheral circulation tumor cell DNA monitoring oncogene information further.
Dissociative DNA detection is exactly Real-Time Monitoring, prognosis evaluation, the effective liquid biopsy method of personalized medicine guidance to potential high-risk individuals screening, tumour.At present, in tumour patient blood plasma and free serum DNA, find to there is oncogene methylate, suddenly change and the feature of the infantile tumour cell DNA such as microsatellite alteration.The detection of dissociative DNA is considered to the powerful measure of a potential early diagnosis of tumor and Index for diagnosis.
Summary of the invention
The object of the present invention is to provide a kind of simple to operate, with low cost, test kit that can extract the extraction dissociative DNA of large and small DNA fragmentation respectively.
Large and small DNA fragmentation of the present invention refers to the little DNA fragmentation being greater than the large DNA fragmentation of 500bp He being less than 500bp.
A kind of test kit extracting dissociative DNA provided by the invention, it contains magnetic bead, Proteinase K, Virahol, BindingBuffer.
Test kit of the present invention is also containing standard substance, and described standard substance are people's source DNA fragment of 127bp and 208bp.
Described dissociative DNA is from peripheral blood or urine.
Test kit of the present invention, its working routine comprises the following steps:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-1:10 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-1:10 with blood plasma or urine, also add BindingBuffer and Virahol in system, the volume ratio of Virahol in whole system is 2%-12%; Vibration mixing, whole system, in 55 DEG C-60 DEG C heating 10-20min, is placed on magnetic separator and leaves standstill, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again;
(3) secondary bond: add magnetic bead and Virahol respectively by the supernatant liquor in step (2), wherein, the volume ratio of magnetic bead and supernatant liquor is 1:15-1:20, and the volume ratio of Virahol in whole system is 40%-50%; Leave standstill in room temperature after mixing, then leave standstill on magnetic separator, abandon supernatant liquor; Washing magnetic bead, then drying and wash-out obtain the small pieces segment DNA of below the 500bp size of purifying.
Preferably, test kit working routine of the present invention comprises the following steps:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-1:6 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-1:6 with blood plasma or urine, LysisBuffer and Virahol is also added in system, wherein the volume ratio of LysisBuffer and blood plasma or urine is 2:1-1:1, and the volume ratio of Virahol in this system is 4%-6%; Vibration mixing, whole system, in 55 DEG C of heating 10-15min, is placed on magnetic separator and leaves standstill 2min, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again;
(3) secondary bond: add magnetic bead and Virahol respectively by the supernatant liquor in step (2), wherein, the volume ratio of magnetic bead and supernatant liquor is 1:16-17, and the volume ratio of Virahol in whole system is 42%-45%; Leave standstill 10min in room temperature after mixing, be magnetic separator leaves standstill 2min, abandon supernatant liquor; Washing magnetic bead, drier and wash-out obtains the small pieces segment DNA of below the 500bp size of purifying.
In one embodiment of the invention, in above-mentioned steps (2), the washing of magnetic bead, drying, elution process are respectively:
Washing: (1) adds 600 μ lWashingBufferI, slowly blows and beats magnetic bead 10 times with pipettor; (2) make abundant magnetic bead resuspended.Room temperature places 0.5 ~ 1min.Magnetic resolution, sucks supernatant liquor with pipettor.Repeating step (1) once; (3) add 800 μ lWashingBufferII, slowly blow and beat magnetic bead 10 times with pipettor, make magnetic bead fully resuspended.Room temperature places 0.5 ~ 1min.Magnetic resolution, sucks supernatant liquor with pipettor.(4) repeating step (3) once, eliminates washings as far as possible.
Dry: to keep centrifuge tube on magnetic frame, uncap air-dry to magnetic bead surfaces without obvious gloss (3 ~ 5min).Time of drying is unsuitable long, to prevent magnetic bead excessively dry, causes nucleic acid to be difficult to wash-out.
Wash-out: add 50 μ lElutionBuffer.Centrifuge tube is placed in 55 DEG C of heating 10min (period puts upside down mixing once).Magnetic resolution, moves to new centrifuge tube by supernatant liquor, is the dissociative DNA that purifying obtains.
Further, the invention provides a kind of extraction from human peripheral blood or urine and be greater than the test kit of 500bp dissociative DNA, its working routine is:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-10 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-10 with blood plasma or urine, BindingBuffer and Virahol is also added in system, the volume ratio of Virahol in this system is 2%-12%, preferred 4%-6%; Vibration mixing, whole system, in 55 DEG C of heating 10-15min, is placed on magnetic separator and leaves standstill 2min, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again.
The invention provides a kind of method extracting dissociative DNA in blood plasma or urine, comprise the following steps:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-1:10 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-1:10 with blood plasma or urine, also add BindingBuffer and Virahol in system, the volume ratio of Virahol in whole system is 2%-12%; Vibration mixing, whole system, in 55 DEG C-60 DEG C heating 10-20min, is placed on magnetic separator and leaves standstill, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again;
(3) secondary bond: add magnetic bead and Virahol respectively by the supernatant liquor in step (2), wherein, the volume ratio of magnetic bead and supernatant liquor is 1:15-1:20, and the volume ratio of Virahol in whole system is 40%-50%; Leave standstill in room temperature after mixing, then leave standstill on magnetic separator, abandon supernatant liquor; Washing magnetic bead, then drying and wash-out obtain the small pieces segment DNA of below the 500bp size of purifying.
Present invention also offers Virahol and extract the application in blood plasma or urine in dissociative DNA.
Particularly, described application comprises the following steps:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-1:10 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-1:10 with blood plasma or urine, also add BindingBuffer and Virahol in system, the volume ratio of Virahol in whole system is 2%-12%; Vibration mixing, whole system, in 55 DEG C-60 DEG C heating 10-20min, is placed on magnetic separator and leaves standstill, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again;
(3) secondary bond: add magnetic bead and Virahol respectively by the supernatant liquor in step (2), wherein, the volume ratio of magnetic bead and supernatant liquor is 1:15-1:20, and the volume ratio of Virahol in whole system is 40%-50%; Leave standstill in room temperature after mixing, then leave standstill on magnetic separator, abandon supernatant liquor; Washing magnetic bead, then drying and wash-out obtain the small pieces segment DNA of below the 500bp size of purifying.
The invention provides a kind of standard substance detecting mentioned reagent box of the present invention or the present invention extracts application in the organic efficiency of dissociative DNA method in blood plasma or urine, and described standard substance are people's source DNA fragment of 127bp and 208bp.
The invention provides mentioned reagent box or the purposes of aforesaid method in preparation antenatal diagnosis test kit.
The invention provides mentioned reagent box or the purposes of aforesaid method in preparation tumour early screening kit.
The test kit of extraction dissociative DNA of the present invention has the following advantages and beneficial effect:
(1) can extract the dissociative DNA being greater than 500bp and the dissociative DNA being less than 500bp, the rate of recovery is high, reaches 60%-70%.
(2) the inventive method is simple to operate, by the accurate extraction regulating the concentration of Virahol in system just can realize large and small segment DNA, for providing important technical without wound antenatal diagnosis, disease early screening and prognostic monitoring.
Accompanying drawing explanation
Fig. 1 is 1188bpDNA fragment PCR amplification electrophorogram, and swimming lane 1 is for extracting front sample PCR primer electrophorogram; Swimming lane 2 is reclaim DNA large fragment sample PCR primer electrophorogram after once combining; Swimming lane 3 is 1kbDNAladder; Swimming lane 4 is remainder sample PCR primer electrophorogram after once combining.
Fig. 2 is the recovery DNA concentration determination figure of different concns isopropanol extraction large fragment dissociative DNA.
Fig. 3 is under standard substance different concns, DNA yield result figure after once combining.
Fig. 4 is that after secondary bond, DNA fragmentation 2100 is analyzed.
Fig. 5 is under standard substance different concns, DNA yield result figure after secondary bond.
Fig. 6 is under standard substance different concns, the DNA rate of recovery after secondary bond.
Fig. 7 is the yield that test kit of the present invention extracts tumour patient blood plasma dissociative DNA.
Fig. 8 is 2100 analysis Peripheral Blood from Patients with Malignant dissociative DNAs (after secondary bond, DNA reclaims fragment).
Fig. 9 is 2100 analysis Urine of Patients with Malignant Tumours liquid dissociative DNAs (after secondary bond, DNA reclaims fragment).
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art; Reagent used in embodiment is commercial goods.
The present invention's preparation of reagents used is as follows:
BindingBuffer prepares: 318.4gGuHCl (3.3mol/L), 43.8NaCl (0.75mol/L), 4.2gSDS (0.014mol/L), 70gTritonX-100 (0.11mol/L), add 800ml ultrapure water, add 5ml (1.28mol/L) EDTA, 50ml (1mol/LTris-HCl), add ultrapure water to 1L.
The preparation of 1.28mol/LEDTA: take in 18.61gEDTA to 50mL beaker, and add 40mL ultrapure water.Beaker is placed on magnetic stirring apparatus, stirs and add NaOH solid until solution becomes clarification.Then be transferred in 50mL graduated cylinder by EDTA solution, adding purified water to cumulative volume is 50mL, and pH is adjusted to 8.0.
1mol/LTris-HCl (pH8.0) solution preparation: take in 12.11gTris to 100mL beaker, and add 80mL ultrapure water and make it dissolve completely.Then measure pH with PB-21 type pH meter, and carry out the adjustment of pH with concentrated hydrochloric acid.Finally be transferred in 100mL volumetric flask by solution, continuing to add purified water to cumulative volume is 100mL.
WashingBufferI prepares: 60gGuHCl (0.63mol/L), 26.3gNaCl (0.45mol/L), 10gTween20 (8.2mmol/L) adds ultrapure water to 1L.
WashingBufferII prepares: 121.1gTris (1mol/L), adds 600ml ultrapure water, adds 26% dehydrated alcohol (260ml), with concentrated hydrochloric acid pH value of solution is adjusted to 8.0, adds ultrapure water to 1L.
ElutionBuffer prepares: 1.2gTris (0.01mol/L), adds 800ml ultrapure water, after dissolving completely, drips concentrated hydrochloric acid and regulates pH to 8.0.
Embodiment 1 one kinds extracts the method for large fragment dissociative DNA (being greater than 500bp) in blood plasma
Adopt different Virahol bulk concentration gradient (2%, 4.5%, 7%, 9.5%, 12%), adopt single stage method magnetic bead to combine and extract dissociative DNA large fragment.
Experimental system (100l blood plasma+230 μ l detection reagent), detection reagent comprises Proteinase K, magnetic bead, Virahol and BindingBuffer.Blood plasma is: the DNA fragmentation mixing 127bp and 208bp of concentration known in human normal plasma, with two kinds of fragments for standard substance, the size (140-150bp) of cfDNA in the selection simulating blood plasma of clip size, the final concentration mixing DNA fragmentation is 0ng/ml, 7.5ng/ml, 15ng/ml, 30ng/ml, 60ng/ml, 120ng/ml.Each sample do 3 parallel.
The concrete grammar extracting large fragment DNA (> 500bp) is:
(1) Proteinase K of 10 μ l is often added in pipe 100 μ l blood plasma respectively, the magnetic bead of 20 μ l, (6.6 μ l Virahols and 193.6 μ lBindingBuffer; The Virahol of 15 μ l and the BindingBuffer of 185 μ l; 31.5 μ l Virahols and 168.5 μ lBindingBuffer; 23 μ l Virahols and 177 μ lBindingBuffer; 39.6 μ l Virahols and 160.4 μ lBindingBuffer); Vortex oscillation mixes.Centrifuge tube is placed in 55 DEG C of heating 10min (period need put upside down mixing once).Centrifuge tube is placed on magnetic separator and leaves standstill 2min, with pipettor Aspirate supernatant in another new 1.5ml centrifuge tube, collect magnetic bead and wash.
(2) add 600 μ lWashingBufferI, slowly blow and beat magnetic bead 10 times with pipettor, make abundant magnetic bead resuspended.Room temperature places 0.5 ~ 1min.Magnetic resolution, sucks supernatant liquor with pipettor.
(3) repeating step (2) once.
(4) add 800 μ lWashingBufferII, slowly blow and beat magnetic bead 10 times with pipettor, make magnetic bead fully resuspended.Room temperature places 0.5 ~ 1min.Magnetic resolution, sucks supernatant liquor with pipettor.
(5) dry: to keep centrifuge tube on magnetic frame, uncap air-dry to magnetic bead surfaces without obvious gloss (3 ~ 5min).Time of drying is unsuitable long, to prevent magnetic bead excessively dry, causes nucleic acid to be difficult to wash-out.
(6) wash-out: add 50 μ lElutionBuffer.Centrifuge tube is placed in 55 DEG C of heating 10min (period puts upside down mixing once).Magnetic resolution, moves to new centrifuge tube by supernatant liquor, is the free large fragment DNA that purifying obtains.
Result verification: pcr amplification 1188bpDNA electrophorogram as shown in Figure 1, respectively by the plasma sample before DNA extraction, from the DNA sample that magnetic bead reclaims after the present embodiment once combines, after once combining, in supernatant liquor, remainder sample carries out pcr amplification 1188bp fragment, primer sequence (upstream primer: 5 '-TTCACTGCAATCTTTCGAGGC-3 ', downstream primer: 5 '-GTCAGAATTTGAGTTCGTATT-3 ') electrophorogram extracts front as seen and once combines the sample standard deviation after reclaiming and can increase and obtain 1188bp product, and once do not show clear and definite 1188bp fragment in conjunction with remainder sample amplified production electrophoresis in rear supernatant liquor, the DNA fragmentation reclaimed after this prompting once combines is large fragment (being greater than 500bp), residue sample is small segment.
Different isopropyl alcohol concentration (2%, 4.5%, 7%, 9.5%, 12%) extracts DNA large fragment quantitative result, sees Fig. 2.Result illustrates, Virahol volumetric concentration is within the scope of 2%-12%, all can realize the recovery large fragment DNA of greater efficiency.
To above-mentioned once combine after magnetic bead carry out rinsing and wash-out, and it is quantitative to carry out Qubit.Finally by the concentration of the DNA that Qubit Detection and Extraction go out, to calculate the rate of recovery.The results are shown in Figure 3.
Embodiment 2 one kinds extracts the method for small segment dissociative DNA (being less than 500bp) in blood plasma
1, secondary bond is with reference to the method (i.e. a combined techniques) of embodiment 1 record, added and add (magnetic bead of 20 μ l and the Virahol of 189 μ l respectively in the supernatant liquor step (1) in embodiment 1 obtained; The magnetic bead of 20 μ l and the Virahol of 230 μ l; The magnetic bead of 20 μ l and the Virahol of 286 μ l; The magnetic bead of 20 μ l and the Virahol of 350 μ l, the magnetic bead of 20 μ l and the Virahol of 428 μ l) vortex oscillation mixes, and Virahol volumetric concentration is respectively 36%, 41%, 46%, 51%, 56%.Centrifuge tube is left standstill 10min in room temperature.Centrifuge tube is placed on magnetic separator and leaves standstill 2min, suck supernatant liquor with pipettor.
2, wash (1) and add 600 μ lWashingBufferI, slowly blow and beat magnetic bead 10 times with pipettor, make abundant magnetic bead resuspended.Room temperature places 0.5 ~ 1min.Magnetic resolution, sucks supernatant liquor with pipettor.(2) repeated washing step (1) once.(3) add 800 μ lWashingBufferII, slowly blow and beat magnetic bead 10 times with pipettor, make magnetic bead fully resuspended.Room temperature places 0.5 ~ 1min.Magnetic resolution, sucks supernatant liquor with pipettor.(4) repeating step (3) once, eliminates washings as far as possible.
3, the dry centrifuge tube that keeps is on magnetic frame, uncap air-dry to magnetic bead surfaces without obvious gloss (3 ~ 5min).Time of drying is unsuitable long, to prevent magnetic bead excessively dry, causes nucleic acid to be difficult to wash-out.
4, wash-out adds 50 μ lElutionBuffer.Centrifuge tube is placed in 55 DEG C of heating 10min (period puts upside down mixing once).Magnetic resolution, moves to new centrifuge tube by supernatant liquor, is the dissociative DNA that purifying obtains.
DNA fragmentation embodiment 2 obtained respectively carries out sample preprocessing according to AgilentHighSensitivityDNAAssay test kit step, 2100 biological analysers are adopted to detect sample DNA clip size and concentration, the results are shown in Figure 4, the dissociative DNA clip size reclaimed after secondary bond is at below 500bp.
Rinsing and wash-out are carried out to the magnetic bead after above-mentioned secondary bond, and it is quantitative to carry out Qubit.Finally by the concentration of the DNA that Qubit Detection and Extraction go out, to calculate yield and the rate of recovery.The results are shown in Figure 5, Fig. 6, after secondary bond, the DNA rate of recovery is better, all more than 60%.
Embodiment 3 extracts assembling and the application of the test kit of dissociative DNA
Test kit of the present invention contains magnetic bead, Proteinase K, Virahol, BindingBuffer, standard substance and other conventional magnetic beads wash, wash-out reagent.
According to the record of embodiment 1,2, the working routine of test kit of the present invention is as follows:
1, human peripheral blood plasma or urine is separated.
2, once combine, the washing of magnetic bead, drying and wash-out and DNA extraction.
In 100 μ l blood plasma, add the magnetic bead of the Proteinase K of 10 μ l, the BindingBuffer of 185 μ l, the Virahol of 15 μ l and 20 μ l respectively, vortex oscillation mixes.Centrifuge tube is placed in 55 DEG C of heating 10min (period need put upside down mixing once).Centrifuge tube is placed on magnetic separator and leaves standstill 2min, with pipettor Aspirate supernatant in another new 1.5ml centrifuge tube, collecting magnetic bead carries out washing (after the 800 μ l80% ethanol provided for oneself of use wash twice magnetic bead, according to the step (5) of embodiment 1, (6), drying and wash-out are carried out to it again, obtain the large fragment DNA of more than the 500bp of purifying.
The extraction of 3, the washing of secondary bond, magnetic bead, drying and wash-out and DNA
In the supernatant liquor drawn in above-mentioned steps, add the magnetic bead of 20 μ l and the Virahol of 230 μ l respectively, vortex oscillation mixes.Centrifuge tube is left standstill 10min in room temperature, centrifuge tube is placed on magnetic separator and leaves standstill 2min, suck supernatant liquor with pipettor.With reference to embodiment 2 step 2,3, the method for 4 washs magnetic bead, dry and wash-out, obtains the small pieces segment DNA of below the 500bp of purifying.
Embodiment 4 test kit of the present invention extracts the application of peripheral blood dissociative DNA
Adopt the dissociative DNA of the embodiment of the present invention 3 to extract test kit and extract Peripheral Blood from Patients with Malignant dissociative DNA.Signature Informed Consent Form and meet include condition prerequisite under, select malignant tumor patient, record basic clinical data, leave and take peripheral blood 5ml.Be separated tumour patient blood plasma 2000rpm, 10min, 25 DEG C.Be separated 2-2.5ml blood plasma; Get 100 μ l.
According to the working routine of the test kit of embodiment 3, the tumour patient blood plasma of clinical separation is carried out to the extraction of dissociative DNA.
Respectively to after once combining, the dissociative DNA that obtains after secondary bond analyzes.Sample preprocessing is carried out according to AgilentHighSensitivityDNAAssay test kit step, 2100 biological analysers are adopted to detect sample DNA clip size and concentration, yield is shown in Fig. 7, and result shows that the peripheral blood dissociative DNA size of the most of malignant tumor patient adopting two-step approach Beads enrichment to be recovered to is about about 150bp ~ 200bp (the results are shown in Figure 8).
Embodiment 5 test kit of the present invention extracts the application of dissociative DNA in urine
Adopt dissociative DNA to extract test kit and extract Urine of Patients with Malignant Tumours liquid dissociative DNA.Signature Informed Consent Form and meet include condition prerequisite under, select malignant tumor patient, record basic clinical data, leave and take urine 50ml.
Urine is carried out packing, every 10ml urine and 6M guanidine thiocyanate, put upside down 8 mixings, add the resin of 1ml, tighten sample lid, room temperature concussion is spent the night, and is placed into by pipe on test-tube stand, leaves standstill, guarantee that resin precipitated is got off, adopt the liquid on strainer wash-out resin, afterwards according to the working routine of the test kit of embodiment 3, the tumour patient urine of clinical separation is carried out to the extraction of dissociative DNA.Respectively to after once combining, the dissociative DNA that obtains after secondary bond analyzes.Sample preprocessing is carried out according to AgilentHighSensitivityDNAAssay test kit step, 2100 biological analysers are adopted to detect sample DNA clip size and concentration, result shows that the urine dissociative DNA size of the most of malignant tumor patient adopting two-step approach Beads enrichment to be recovered to is about about 150bp ~ 200bp, the results are shown in Figure 9.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. extract a test kit for dissociative DNA, it is characterized in that, containing magnetic bead, Proteinase K, Virahol, BindingBuffer.
2. test kit as claimed in claim 1, is characterized in that, it is also containing standard substance, and described standard substance are people's source DNA fragment of 127bp and 208bp.
3. test kit as claimed in claim 1, it is characterized in that, described dissociative DNA is from peripheral blood or urine.
4. the test kit as described in as arbitrary in claim 1-3, it is characterized in that, its working routine comprises the following steps:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-1:10 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-1:10 with blood plasma or urine, also add BindingBuffer and Virahol in system, the volume ratio of Virahol in whole system is 2%-12%; Vibration mixing, whole system, in 55 DEG C-60 DEG C heating 10-20min, is placed on magnetic separator and leaves standstill, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again;
(3) secondary bond: add magnetic bead and Virahol respectively by the supernatant liquor in step (2), wherein, the volume ratio of magnetic bead and supernatant liquor is 1:15-1:20, and the volume ratio of Virahol in whole system is 40%-50%; Leave standstill in room temperature after mixing, then leave standstill on magnetic separator, abandon supernatant liquor; Washing magnetic bead, then drying and wash-out obtain the small pieces segment DNA of below the 500bp size of purifying.
5. test kit as claimed in claim 4, it is characterized in that, its working routine comprises the following steps:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-1:6 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-1:6 with blood plasma or urine, BindingBuffer and Virahol is also added in system, wherein the volume ratio of BindingBuffer and blood plasma or urine is 2:1-1:1, and the volume ratio of Virahol in this system is 4%-6%; Vibration mixing, whole system, in 55 DEG C of heating 10-15min, is placed on magnetic separator and leaves standstill 2min, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again;
(3) secondary bond: add magnetic bead and Virahol respectively by the supernatant liquor in step (2), wherein, the volume ratio of magnetic bead and supernatant liquor is 1:16-17, and the volume ratio of Virahol in whole system is 42%-45%; Leave standstill 10min in room temperature after mixing, be magnetic separator leaves standstill 2min, abandon supernatant liquor; Washing magnetic bead, drier and wash-out obtains the small pieces segment DNA of below the 500bp size of purifying.
6. extract a method for dissociative DNA in blood plasma or urine, it is characterized in that, comprise the following steps:
(1) be separated human peripheral blood blood plasma or extract urine;
(2) once combine: add Proteinase K according to the volume ratio 1:5-1:10 with blood plasma or urine, magnetic bead is added according to the volume ratio 1:5-1:10 with blood plasma or urine, also add BindingBuffer and Virahol in system, the volume ratio of Virahol in whole system is 2%-12%; Vibration mixing, whole system, in 55 DEG C-60 DEG C heating 20-30min, is placed on magnetic separator and leaves standstill, and Aspirate supernatant, in another container, collects the large fragment DNA that magnetic bead carries out washing, dry and wash-out obtains more than the 500bp size of purifying again;
(3) secondary bond: add magnetic bead and Virahol respectively by the supernatant liquor in step (2), wherein, the volume ratio of magnetic bead and supernatant liquor is 1:15-1:20, and the volume ratio of Virahol in whole system is 40%-50%; Leave standstill in room temperature after mixing, then leave standstill on magnetic separator, abandon supernatant liquor; Washing magnetic bead, then drying and wash-out obtain the small pieces segment DNA of below the 500bp size of purifying.
7. Virahol is extracting the application in blood plasma or urine in dissociative DNA.
8. standard substance require the application in the organic efficiency of method described in the arbitrary described test kit of 1-5 or claim 6 in test right, and described standard substance are people's source DNA fragment of 127bp and 208bp.
9. the arbitrary described test kit of claim 1-5 or the purposes of method according to claim 6 in preparation antenatal diagnosis test kit.
10. the arbitrary described test kit of claim 1-5 or the purposes of method according to claim 6 in preparation tumour early screening kit.
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| CN105385682A (en) * | 2015-12-29 | 2016-03-09 | 杭州谷坤生物技术有限公司 | Simple method for fast extracting human fecal bacterium DNA |
| CN105602938A (en) * | 2016-01-22 | 2016-05-25 | 北京圣谷同创科技发展有限公司 | Plasma cfDNA extracting method |
| CN105624798A (en) * | 2016-02-19 | 2016-06-01 | 北京乐普基因科技股份有限公司 | Fetal chromosome library construction kit and method and application thereof |
| CN105671030A (en) * | 2016-02-23 | 2016-06-15 | 苏州摩根基因科技有限公司 | Efficient plasma cell dissociation DNA extraction method based on paramagnetic particle method |
| CN105695450A (en) * | 2016-04-05 | 2016-06-22 | 苏州英芮诚生化科技有限公司 | Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof |
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| CN105602938A (en) * | 2016-01-22 | 2016-05-25 | 北京圣谷同创科技发展有限公司 | Plasma cfDNA extracting method |
| CN105624798A (en) * | 2016-02-19 | 2016-06-01 | 北京乐普基因科技股份有限公司 | Fetal chromosome library construction kit and method and application thereof |
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| CN105695450A (en) * | 2016-04-05 | 2016-06-22 | 苏州英芮诚生化科技有限公司 | Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof |
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| CN106399477A (en) * | 2016-05-17 | 2017-02-15 | 程澎 | Tumor circulation DNA technical detection-cancer early-stage easy-to-occur risk assessment data method |
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| CN107663521A (en) * | 2016-07-28 | 2018-02-06 | 深圳华大基因股份有限公司 | Plasma free nucleic acid extraction kit and its application |
| CN106754880A (en) * | 2016-12-26 | 2017-05-31 | 广州和实生物技术有限公司 | Urine Rapid nucleic acid extraction kit |
| CN106834274A (en) * | 2017-02-17 | 2017-06-13 | 苏州贝斯派生物科技有限公司 | A kind of method for reclaiming fragmentation degradation of dna |
| CN106834274B (en) * | 2017-02-17 | 2019-12-06 | 苏州贝斯派生物科技有限公司 | method for recovering fragmented degraded DNA |
| CN108642047A (en) * | 2018-05-19 | 2018-10-12 | 长沙金域医学检验所有限公司 | The extracting method of DNA in a kind of detection of foetal chromosome aneuploidy |
| CN108707602A (en) * | 2018-08-06 | 2018-10-26 | 臻和（北京）科技有限公司 | A kind of dipped into formalin tissue DNA extracts kit and extracting method |
| WO2020089756A1 (en) | 2018-10-30 | 2020-05-07 | Tubitak | Detection method for halal food and halal food additives |
| CN110846382A (en) * | 2019-11-29 | 2020-02-28 | 北京科迅生物技术有限公司 | Method for enriching free DNA of fetus |
| CN111154750A (en) * | 2020-01-13 | 2020-05-15 | 苏州行知康众生物科技有限公司 | Method for targeted enrichment of plasma target free DNA |
| CN112391381A (en) * | 2020-12-02 | 2021-02-23 | 苏州海狸生物医学工程有限公司 | Urine free DNA extraction kit based on nano magnetic beads and extraction method |
| CN114958825A (en) * | 2022-04-07 | 2022-08-30 | 翌圣生物科技(上海)股份有限公司 | Method for differentially enriching small-fragment nucleic acid molecules |
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