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A detection method and fragment technology, which are used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve high-performance results
Pending Publication Date: 2021-10-29
深圳泰莱生物科技有限公司
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However, existing methylation profiling techniques have obvious deficiencies, so
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Embodiment 1
[0041] Embodiment one: if Figure 1-3 As shown, a detection method for capturing cfDNA5mC fragments of the present invention comprises the following steps:
[0042] Step 1: Prepare reagents;
[0043] Step 2: Prepare Incubation Mix and operate at 4°C;
[0044] Step 3: Dilute the antibody Antibody 1:15, place the antibody at -80°C, 0.5ul antibody + 7.5ulH2O (excess), put it back to -80°C in time after the antibody is used up;
[0046] Step 5: Mix Incubation Mix+Antibody Mix, incubate overnight at 4°C and rotate at 40 for 17 hours;
[0047] Step 6: Ipure Kit v2 reagent preparation;
[0048] Step 7: DNA washing, during the washing process, the magnetic frame and wash buffer should be put back on ice in time;
[0049] Step 8: Ipure kit V2 kit eluted and purified DNA at 40rpm;
[0050] Step 9: XP magnetic bead purification.
[0051] Wherein, in step 1, the reagent preparation process ...
Embodiment 2
[0072] Embodiment two: if figure 1 Shown, a detection method for capturing cfDNA5mC fragments, comprising the following steps:
[0073] Step 1: Prepare reagents;
[0074] Step 2: Prepare Incubation Mix and operate at 4°C;
[0075] Step 3: Dilute the antibody Antibody 1:15, place the antibody at -80°C, 0.5ul antibody + 7.5ulH2O (excess), put it back to -80°C in time after the antibody is used up;
[0077] Step 5: Mix Incubation Mix+Antibody Mix, incubate overnight at 4°C and rotate at 40 for 17 hours;
[0078] Step 6: Ipure Kit v2 reagent preparation;
[0079] Step 7: DNA washing, during the washing process, the magnetic frame and wash buffer should be put back on ice in time;
[0080] Step 8: Ipure kit V2 kit eluted and purified DNA at 40rpm;
[0081] Step 9: XP magnetic bead purification.
[0082] Ipure kit V2 kit eluted and purified DNA (all speeds were 40rpm)
[0083] (1)DNA...
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Abstract
The invention discloses a detection method for capturing a cfDNA5mC fragment. The detection method comprises the following steps: step 1, preparing reagents; step 2, preparing Inculation Mix, and operating at the temperature of 4 DEG C; step 3, diluting an Antibody at 1: 15, placing the antibody at -80 DEG C, adding 0.5 [mu] l of the antibody and 7.5 [mu] l of H2O (excess), and timely placing the antibody at-80 DEG C after the antibody is used up; step 4, preparation of Antibody Mix: the Antibody Mix is excessive, and three amounts of Antibody Mix are generally prepared for two samples; step 5, mixing an Inculation Mix and the Antibody Mix, rotating at the rotating speed of 40 at the temperature of 4 DEG C, and incubating overnight for 17 hours; and step 6, preparing an Ipre Kit v2 reagent. According to the detection method for capturing the cfDNA5mC fragment, methylation of fifth carbon of cytosine (5-methylcytosine: 5mC) is a methylation type excavated in eukaryotes at the earliest, in gene expression, a transcription start site region is generally non-methylated, and when the gene is expressed at a lower level, the methylation level of cytosine in a regulation region is higher, and library correlation generated by different initial quantities and different methods is an effective parameter for evaluating consistency and high performance.
Description
technical field [0001] The invention relates to the technical field of capturing cfDNA5mC fragments, in particular to a detection method for capturing cfDNA5mC fragments. Background technique [0002] The molecular formula of 5-hydroxymethylcytosine 5-hydroxymethylcytosine is C5H7N3O2, which is the hydroxylated form of 5-methylcytosine (5-methylcytosine, 5mC). Methylcytosine (5-mC), known as the "sixth base", can regulate the shutdown of gene expression and is an important epigenetic modification that may be related to the demethylation process, 5 The specific mechanism of action of -hydroxymethylcytosine is still unclear. [0003] Detection of 5-methylcytosine (5mC) in the genome is critical, as methylation modifications affect gene expression. In general, low methylation levels near transcription start sites are associated with higher transcription levels, whereas genes with high levels of cytosine modifications in regulatory regions exhibit lower transcription levels. ...
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