CN113528616A - Detection method for capturing cfDNA5hmC fragment - Google Patents
Detection method for capturing cfDNA5hmC fragment Download PDFInfo
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Abstract
The invention discloses a detection method for capturing cfDNA5hmC fragments, which comprises the following steps: the method comprises the following steps: preparing a reagent to be used in advance, putting the reagent on ice, and establishing a system at the temperature of 37 ℃ for 2 hours; step two: adding 2.5 μ l DBCO-PEG4-biotin, incubating for 2h at 37 deg.C; step three: then using MicroBio-spin30column (Bio-Rad) for purification; step four: 5 μ lCI magnetic beads were pipetted evenly onto a magnetic rack, the supernatant was aspirated after clarification, and 50 μ l of 2 × buffer1 was added and incubated on the rotating rack for 3 min. According to the detection method for capturing the cfDNA5hmC fragment, the streptavidin-coated magnetic beads are used for amplifying the biotinylated DNA fragment. The magnetic beads can be subjected to 'pull-down' analysis, DNA molecules with 5hmC and DNA molecules without biomarkers are separated, and the target fragments are subjected to sequencing and bioinformatics analysis to finally obtain epigenetic markers, so that the capture efficiency of 5hmC is improved to a great extent.
Description
Technical Field
The invention relates to the technical field of capturing cfDNA5hmC fragments, in particular to a detection method for capturing cfDNA5hmC fragments.
Background
5-hydroxymethylcytosine (5-hydroxymethynylcytosine, 5hmC) was first discovered in phage DNA in 1952. It can be modified by glycosyltransferase mediated glycosylation, thus making the phage DNA resistant to host restriction enzyme degradation after entering the host. However, the function of 5hmC has not received much attention. Until 2009, the inventor reports that 5hmC is abundant in human and mouse brain and embryonic stem cell DNA in the journal of Science of contemporary respectively, so that more researchers can learn about the modified base again, and with the research depth, the important biological effect of 5hmC is gradually discovered, and meanwhile, the detection and quantification method of 5hmC is promoted to be developed continuously. We therefore improved this by proposing a detection method that captures cfDNA5hmC fragments.
Disclosure of Invention
In order to solve the technical problems, the invention provides the following technical scheme:
the invention relates to a detection method for capturing cfDNA5hmC fragments, which comprises the following steps:
the method comprises the following steps: preparing a reagent to be used in advance, putting the reagent on ice, and establishing a system at the temperature of 37 ℃ for 2 hours;
step two: adding 2.5 μ l DBCO-PEG4-biotin, incubating for 2h at 37 deg.C;
step three: then using MicroBio-spin30column (Bio-Rad) for purification;
step four: taking 5 mu lCI magnetic beads, blowing and beating the magnetic beads uniformly, placing the magnetic beads on a magnetic frame, sucking supernatant after clarification, adding 50 mu l of 2 buffer1, incubating the magnetic frame for 3min, placing the magnetic frame, sucking the supernatant after clarification, and then adding 50 mu l of 2 buffer1, blowing and beating the uniformly resuspended magnetic beads;
step five: adding the uniform resuspended magnetic beads into a 1.5ml tube which has a volume of 50 μ l, and incubating on a rotating rack for 30 min;
step six: the column was eluted with 100. mu.l of buffer1(1X), buffer2(1X), buffer3 and andbuffer4, twice each buffer wash, each wash being placed on a spin stand for 5 min.
In a preferred embodiment of the present invention, in the first step, a mixture of reagents 10 × buffer, 60 μ MUDP-6-N3-Gle, 12.5UBGT, DNA and water is selected as a system, and the mass of the reagents is 2.5 μ l, 0.5 μ l, 1.25 μ l and 21 μ l, respectively.
3. The detection method for capturing cfDNA5hmC fragments as claimed in claim 1, wherein in the first step, the reagent preparation comprises the following steps:
s1: placing AMPureXPbead at room temperature for 30min in advance, and mixing well to obtain magnetic bead suspension;
s2: taking out a new 1.5m low-adsorption centrifuge tube, sequentially transferring the samples connected with the joints into a marked tube, adding 80 mu L (0.8 time of the volume of the sample) of AMPureXPbead, blowing and uniformly mixing, and standing at room temperature for 5 min;
s3: placing the beads on a magnet seat (the tube is not required to be placed too low when the beads are placed on a magnetic frame), and sucking the supernatant by using a large and small gun tip combination with the dosage range larger than the total volume of the solution after the liquid is completely clarified;
s4: adding 200 mul of 80% ethanol into each tube, waiting for 30 seconds, and sucking the supernatant by using a large and small gun tip combination with the dosage range larger than the total volume of the solution;
s5: repeating the step 13, washing with ethanol twice, and sucking out the excess 80% ethanol;
s6: standing at room temperature for 5min to completely volatilize ethanol (the surface of the magnetic beads is not reflective, and the surface has no cracks or micro cracks);
s7: adding 11 μ L of uclease-freewater (for example, 10ng of DNA input is used, the concentration after elution is about 1ng/ul), blowing, mixing, and standing at room temperature for 1 min;
s8: after instantaneous centrifugation, the EP tube is placed on a magnetic frame until the solution becomes clear; sucking the supernatant into a new 1.5mL low adsorption tube for subsequent operation;
s9: half of this was taken for 5hmC and the remaining product was stored at-80 ℃.
As a preferred technical scheme of the invention, in the third step, the method for purifying by using MicroBio-spin30column (Bio-Rad) comprises the following steps:
the method comprises the following steps: shaking up the reagent, removing bubbles, opening the lower seal, placing into a special 2ml collecting tube, standing for 2min to allow liquid to flow through, and pouring out the liquid in the 2ml tube;
step two: 1000g, centrifuging for 2 min;
step three: the column was transferred to a dedicated 1.5ml EP collection tube, centrifuged at 1000g for 4min and made to volume of 50. mu.l.
In the fourth step, a palm centrifuge of D1008 type SCILOGEX is selected to avoid magnetic bead sedimentation during the instantaneous centrifugation operation of magnetic beads (AMPureXP magnetic beads, T1 magnetic beads).
In a preferred embodiment of the present invention, in the sixth step, the cover liquid is prevented from being lost by the instant separation after the rotation.
The invention has the beneficial effects that: according to the detection method for capturing the cfDNA5hmC fragment, the streptavidin-coated magnetic beads are used for amplifying the biotinylated DNA fragment. The magnetic bead can be subjected to pull-down analysis, DNA molecules with 5hmC and DNA molecules without biomarkers are separated, and the target fragments are subjected to sequencing and bioinformatics analysis to finally obtain epigenetic markers, so that the capture efficiency of 5hmC is improved to a great extent;
the specific DNA fragment captures covalently linked biotin, so that the capture process is not limited by the conventional antigen-antibody action affinity, and further the DNA fragment containing 5hmC is captured specifically.
Drawings
Fig. 1 is a flow diagram of a detection method of the invention capturing cfDNA5hmC fragments;
FIG. 2 is a flow chart of reagent preparation for a detection method of capturing cfDNA5hmC fragments according to the present invention;
fig. 3 is a purification flow chart of a detection method of capturing cfDNA5hmC fragments according to the present invention.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
The first embodiment is as follows: as shown in fig. 1-2, the detection method for capturing cfDNA5hmC fragment of the present invention comprises the following steps:
the method comprises the following steps: preparing a reagent to be used in advance, putting the reagent on ice, and establishing a system at the temperature of 37 ℃ for 2 hours;
step two: adding 2.5 μ l DBCO-PEG4-biotin, incubating for 2h at 37 deg.C;
step three: then using MicroBio-spin30column (Bio-Rad) for purification;
step four: taking 5 mu lCI magnetic beads, blowing and beating the magnetic beads uniformly, placing the magnetic beads on a magnetic frame, sucking supernatant after clarification, adding 50 mu l of 2 buffer1, incubating the magnetic frame for 3min, placing the magnetic frame, sucking the supernatant after clarification, and then adding 50 mu l of 2 buffer1, blowing and beating the uniformly resuspended magnetic beads;
step five: adding the uniform resuspended magnetic beads into a 1.5ml tube which has a volume of 50 μ l, and incubating on a rotating rack for 30 min;
step six: the column was eluted with 100. mu.l of buffer1(1X), buffer2(1X), buffer3 and andbuffer4, twice each buffer wash, each wash being placed on a spin stand for 5 min.
In the first step, a system is established to select a mixture of reagents 10 × buffer, 60 μ MUDP-6-N3-Gle, 12.5UBGT, DNA and water, wherein the mass of the reagents is 2.5 μ l, 0.5 μ l, 1.25 μ l and 21 μ l respectively.
Wherein, in the first step, the reagent preparation comprises the following steps:
s1: placing AMPureXPbead at room temperature for 30min in advance, and mixing well to obtain magnetic bead suspension;
s2: taking out a new 1.5m low-adsorption centrifuge tube, sequentially transferring the samples connected with the joints into a marked tube, adding 80 mu L (0.8 time of the volume of the sample) of AMPureXPbead, blowing and uniformly mixing, and standing at room temperature for 5 min;
s3: placing the beads on a magnet seat (the tube is not required to be placed too low when the beads are placed on a magnetic frame), and sucking the supernatant by using a large and small gun tip combination with the dosage range larger than the total volume of the solution after the liquid is completely clarified;
s4: adding 200 mul of 80% ethanol into each tube, waiting for 30 seconds, and sucking the supernatant by using a large and small gun tip combination with the dosage range larger than the total volume of the solution;
s5: repeating the step 13, washing with ethanol twice, and sucking out the excess 80% ethanol;
s6: standing at room temperature for 5min to completely volatilize ethanol (the surface of the magnetic beads is not reflective, and the surface has no cracks or micro cracks);
s7: adding 11 μ L of uclease-freewater (for example, 10ng of DNA input is used, the concentration after elution is about 1ng/ul), blowing, mixing, and standing at room temperature for 1 min;
s8: after instantaneous centrifugation, the EP tube is placed on a magnetic frame until the solution becomes clear; sucking the supernatant into a new 1.5mL low adsorption tube for subsequent operation;
s9: half of this was taken for 5hmC and the remaining product was stored at-80 ℃.
Example two: as shown in fig. 3, the detection method for capturing cfDNA5hmC fragment of the present invention comprises the following steps:
the method comprises the following steps: preparing a reagent to be used in advance, putting the reagent on ice, and establishing a system at the temperature of 37 ℃ for 2 hours;
step two: adding 2.5 μ l DBCO-PEG4-biotin, incubating for 2h at 37 deg.C;
step three: then using MicroBio-spin30column (Bio-Rad) for purification;
step four: taking 5 mu lCI magnetic beads, blowing and beating the magnetic beads uniformly, placing the magnetic beads on a magnetic frame, sucking supernatant after clarification, adding 50 mu l of 2 buffer1, incubating the magnetic frame for 3min, placing the magnetic frame, sucking the supernatant after clarification, and then adding 50 mu l of 2 buffer1, blowing and beating the uniformly resuspended magnetic beads;
step five: adding the uniform resuspended magnetic beads into a 1.5ml tube which has a volume of 50 μ l, and incubating on a rotating rack for 30 min;
step six: the column was eluted with 100. mu.l of buffer1(1X), buffer2(1X), buffer3 and andbuffer4, twice each buffer wash, each wash being placed on a spin stand for 5 min.
The specific DNA fragment captures covalently linked biotin, so that the capture process is not limited by the conventional antigen-antibody action affinity, and further the DNA fragment containing 5hmC is captured specifically.
Wherein, in the third step, the method for purifying by using MicroBio-spin30column (Bio-Rad) comprises the following steps:
the method comprises the following steps: shaking up the reagent, removing bubbles, opening the lower seal, placing into a special 2ml collecting tube, standing for 2min to allow liquid to flow through, and pouring out the liquid in the 2ml tube;
step two: 1000g, centrifuging for 2 min;
step three: the column was transferred to a dedicated 1.5ml EP collection tube, centrifuged at 1000g for 4min and made to volume of 50. mu.l.
In the fourth step, in order to avoid magnetic bead sedimentation during the instantaneous centrifugal operation of magnetic beads (AMPureXP magnetic beads and T1 magnetic beads), a palm-type centrifuge of D1008 of SCILOGEX type is selected.
In the sixth step, the cover liquid is prevented from being lost by the instant separation after the rotation.
Biotinylated DNA fragments were amplified using streptavidin-coated magnetic beads. The magnetic beads can be subjected to 'pull-down' analysis, DNA molecules with 5hmC and DNA molecules without biomarkers are separated, and the target fragments are subjected to sequencing and bioinformatics analysis to finally obtain epigenetic markers, so that the capture efficiency of 5hmC is improved to a great extent.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A detection method for capturing cfDNA5hmC fragments is characterized by comprising the following steps:
the method comprises the following steps: preparing a reagent to be used in advance, putting the reagent on ice, and establishing a system at the temperature of 37 ℃ for 2 hours;
step two: adding 2.5 μ l DBCO-PEG4-biotin, incubating for 2h at 37 deg.C;
step three: then using MicroBio-spin30column (Bio-Rad) for purification;
step four: taking 5 mu lCI magnetic beads, blowing and beating the magnetic beads uniformly, placing the magnetic beads on a magnetic frame, sucking supernatant after clarification, adding 50 mu l of 2 buffer1, incubating the magnetic frame for 3min, placing the magnetic frame, sucking the supernatant after clarification, and then adding 50 mu l of 2 buffer1, blowing and beating the uniformly resuspended magnetic beads;
step five: adding the uniform resuspended magnetic beads into a 1.5ml tube which has a volume of 50 μ l, and incubating on a rotating rack for 30 min;
step six: the column was eluted with 100. mu.l of buffer1(1X), buffer2(1X), buffer3 and andbuffer4, twice each buffer wash, each wash being placed on a spin stand for 5 min.
2. The detection method for capturing 5hmC fragments of cfDNA as claimed in claim 1, wherein in the first step, a system is established to select the mixture of reagents 10 buffer, 60 μ MUDP-6-N3-Gle, 12.5UBGT, DNA and water, the mass of the reagents is 2.5 μ l, 0.5 μ l, 1.25 μ l and 21 μ l respectively.
3. The detection method for capturing cfDNA5hmC fragments according to claim 1, wherein in the first step, the reagent preparation comprises the following steps:
s1: placing AMPureXPbead at room temperature for 30min in advance, and mixing well to obtain magnetic bead suspension;
s2: taking out a new 1.5m low-adsorption centrifuge tube, sequentially transferring the samples connected with the joints into a marked tube, adding 80 mu L (0.8 time of the volume of the sample) of AMPureXPbead, blowing and uniformly mixing, and standing at room temperature for 5 min;
s3: placing the beads on a magnet seat (the tube is not required to be placed too low when the beads are placed on a magnetic frame), and sucking the supernatant by using a large and small gun tip combination with the dosage range larger than the total volume of the solution after the liquid is completely clarified;
s4: adding 200 mul of 80% ethanol into each tube, waiting for 30 seconds, and sucking the supernatant by using a large and small gun tip combination with the dosage range larger than the total volume of the solution;
s5: repeating the step 13, washing with ethanol twice, and sucking out the excess 80% ethanol;
s6: standing at room temperature for 5min to completely volatilize ethanol (the surface of the magnetic beads is not reflective, and the surface has no cracks or micro cracks);
s7: adding 11 μ L of uclease-freewater (for example, 10ng of DNA input is used, the concentration after elution is about 1ng/ul), blowing, mixing, and standing at room temperature for 1 min;
s8: after instantaneous centrifugation, the EP tube is placed on a magnetic frame until the solution becomes clear; sucking the supernatant into a new 1.5mL low adsorption tube for subsequent operation;
s9: half of this was taken for 5hmC and the remaining product was stored at-80 ℃.
4. The detection method for capturing cfDNA5hmC fragments according to claim 1, wherein in the third step, the method for purifying by using MicroBio-spin30column (Bio-Rad) comprises the following steps:
the method comprises the following steps: shaking up the reagent, removing bubbles, opening the lower seal, placing into a special 2ml collecting tube, standing for 2min to allow liquid to flow through, and pouring out the liquid in the 2ml tube;
step two: 1000g, centrifuging for 2 min;
step three: the column was transferred to a dedicated 1.5ml EP collection tube, centrifuged at 1000g for 4min and made to volume of 50. mu.l.
5. The method for detecting 5hmC fragments captured by cfDNA of claim 1, wherein in the fourth step, a palm centrifuge with model number SCILOGEX D1008 is used to avoid the sedimentation of magnetic beads (AMPureXP magnetic beads, T1 magnetic beads) during the instantaneous centrifugation operation.
6. The detection method for capturing cfDNA5hmC fragments as claimed in claim 1, wherein in step six, the spinning is followed by an instant detachment to avoid loss of cap liquid.
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| US20160362438A1 (en) * | 2013-09-27 | 2016-12-15 | Peking University | 5-formylcytosine specific chemical labeling method and related applications |
| WO2017147945A1 (en) * | 2016-03-02 | 2017-09-08 | 上海易毕恩基因科技有限公司 | Sequencing method for genetic mapping of dna 5-methylcytosine and 5-hydroxymethylcytosine |
| CN109312399A (en) * | 2016-04-07 | 2019-02-05 | 斯坦福大学托管董事会 | By the non-invasive diagnosis that 5- methylolation Cell-free DNA is sequenced |
| CN111961729A (en) * | 2020-09-01 | 2020-11-20 | 深圳泰莱生物科技有限公司 | Kit for detecting content of 5-hydroxymethylcytosine and application thereof |
| CN113061652A (en) * | 2021-03-30 | 2021-07-02 | 成都泰莱医学检验实验室有限公司 | Method for determining 5hmC content in gene marker based on glucose modification |
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- 2021-07-26 CN CN202110846330.2A patent/CN113528616A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160362438A1 (en) * | 2013-09-27 | 2016-12-15 | Peking University | 5-formylcytosine specific chemical labeling method and related applications |
| WO2017147945A1 (en) * | 2016-03-02 | 2017-09-08 | 上海易毕恩基因科技有限公司 | Sequencing method for genetic mapping of dna 5-methylcytosine and 5-hydroxymethylcytosine |
| CN109312399A (en) * | 2016-04-07 | 2019-02-05 | 斯坦福大学托管董事会 | By the non-invasive diagnosis that 5- methylolation Cell-free DNA is sequenced |
| CN111961729A (en) * | 2020-09-01 | 2020-11-20 | 深圳泰莱生物科技有限公司 | Kit for detecting content of 5-hydroxymethylcytosine and application thereof |
| CN113061652A (en) * | 2021-03-30 | 2021-07-02 | 成都泰莱医学检验实验室有限公司 | Method for determining 5hmC content in gene marker based on glucose modification |
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| Title |
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| COLM E NESTOR 等: "Investigating 5-hydroxymethylcytosine (5hmC): the state of the art", 《METHODS MOL BIOL 》, vol. 1094, pages 243 - 258 * |
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