甲基化DNA免疫沉淀

摘要

本视频演示了甲基化DNA免疫沉淀（MeDIP）的实验方案。MeDIP是一个为期两天的实验流程，利用对5-甲基胞嘧啶（anti-5 mC）具有特异性的抗体，从基因组DNA样本中选择性地富集甲基化的DNA片段。

摘要

DNA甲基化模式的鉴定是表观遗传学研究中的常见实验步骤，因为已知甲基化对基因表达具有重要影响，并参与正常发育以及疾病的发生。^1-4因此，能够区分甲基化DNA与非甲基化DNA对于生成此类研究的甲基化谱图至关重要。甲基化DNA免疫沉淀（MeDIP）是一种从目标样本中有效提取甲基化DNA的技术。^5-7. 仅需 200 ng 的 DNA 样品即可满足抗体或免疫沉淀（IP）反应的需求。DNA 经超声处理断裂为 300–1000 bp 大小的片段，并分为免疫沉淀（IP）和输入（IN）两部分。IP DNA 随后经加热变性，再与抗-5'mC 抗体孵育，使单克隆抗体能够结合甲基化的 DNA。接着加入含有可与一抗特异性结合的二抗的磁珠，并进行孵育。这些磁珠连接的二抗将结合第一步中使用的单克隆抗体。利用磁铁将与抗体复合物结合的 DNA（即甲基化 DNA）从溶液中分离出来。随后使用 IP 缓冲液进行多次洗涤，以去除未结合的非甲基化 DNA。然后用蛋白酶 K 消化甲基化 DNA/抗体复合物，降解抗体，仅保留完整的甲基化 DNA。富集后的 DNA 通过苯酚:氯仿抽提法纯化以去除蛋白质成分，再经沉淀并重悬于水中，以备后续使用。可采用 PCR 技术通过分析已知不含甲基化序列和已知含有甲基化序列区域的 IP 与 IN DNA 扩增产物，来验证 MeDIP 实验的效率。纯化后的甲基化 DNA 可用于位点特异性（PCR）或全基因组范围（微阵列和测序）的甲基化研究，尤其适用于与其他研究工具（如基因表达谱分析和芯片比较基因组杂交（CGH））联合应用时。⁸. 对DNA甲基化的进一步研究将有助于发现新的表观遗传学靶点，这些靶点可能在开发针对癌症等以DNA异常甲基化为特征的疾病的新型治疗或预后研究工具方面具有重要价值。^{2, 4, 9-11}.

方案

DNA EXTRACTION AND SAMPLE PREPARATION

DNA from a variety of different samples (cultured cells, fresh frozen as well as formalin-fixed paraffin embedded tissues) can be used for MeDIP. It is important to use highly purified DNA without associated proteins such as histones. It is also important to remove as much RNA as possible from the sample, as it can interfere with both DNA quantitation and antibody binding. The quantity of DNA used for MeDIP can range from 200 ng to 1 µg depending on the amount of DNA available. To demonstrate this protocol, 1 µg of DNA will be used. The following protocol will provide high quality double stranded DNA from cultured cells. Other protocols should be employed for extraction of DNA from other sample types.

1. Add 400 μl of digestion buffer to a cell pellet in a 1.7 ml Eppendorf tube.
2. Add 100 μg of proteinase K to the tube, and incubate overnight at 50°C.
3. Add 500 μl phenol pH 7 and mix gently but thoroughly by inverting.
4. Spin at 13 000 g for 10 mins at room temperature.
5. Remove aqueous (top) fraction to a new tube.
6. Repeat Steps 3 through 5 once.
7. Add 500 µl 1:1 phenol/chloroform pH 7 and mix gently but thoroughly by inverting.
8. Spin at 13 000 g for 10 mins at room temperature.
9. Remove aqueous (top) fraction to a new tube.
10. Add 40 μg of RNase A and incubate 1 hr at 37°C.
11. Add 500 μl phenol pH 7 and mix gently but thoroughly by inverting.
12. Spin at 13 000 g for 10 mins at room temperature.
13. Remove aqueous (top) fraction to a new tube.
14. Repeat Steps 11 through 13 once.
15. Add 500 µl 1:1 phenol/chloroform pH 7 and mix gently but thoroughly by inverting.
16. Spin at 13 000 g for 10 mins at room temperature.
17. Remove aqueous (top) fraction to a new tube.
18. Add 1/10th volume 3 M sodium acetate (40 μl) and mix well.
19. Add 2 volumes (900 μl) of 100% ethanol, mix well, and place at -20°C for 20 mins.
20. Spin at 13 000 g for 20 mins at 4°C.
21. Remove ethanol, pulse spin, and remove residual ethanol.
22. Add 500 μl cold 70% ethanol to wash. Spin at 13 000 g for 20 mins at 4°C.
23. Remove 70% ethanol, pulse spin, and remove residual ethanol by pipetting.
24. Air dry the pellet with the cap open for 10 mins at room temperature to remove all traces of residual ethanol.
25. Resuspend DNA in 50 μl of sterilized dH2O overnight at 4°C.
26. Quantify the DNA using a NanoDrop Spectrophotometer. An A₂₆₀:A₂₈₀ ratio of 1.8 is ideal.
27. Determine DNA quality and size range on an agarose gel with 100 bp ladder. As little as 10 ng of genomic DNA can be run on a 1.7% agarose gel followed by staining using a dye that is highly sensitive to small amounts of DNA such as SYBR Gold.
28. In one siliconized tube per sample, prepare 1 µg of DNA in a total volume of 50 μl with the remainder of the volume made up with sterilized dH₂O.

DNA SONICATION

DNA sonication and the MeDIP protocol must be performed in siliconzied tubes to prevent non specific binding of proteins to tube walls. Optimal sonication times for the DNA samples are based on the degree of DNA sample fragmentation as determined from gel electrophoresis (Step 27). For example, DNA extracted from cultured cells should be of very high molecular weight, and will subsequently require more sonication than DNA extracted from archival samples which are often partially degraded. If samples are of uniformly high molecular weight, it is reasonable at this point to proceed with the sonication as described in this protocol without checking each sample individually. If samples are fragmented as with archival samples, it will be necessary to adapt the sonication procedure likely by decreasing sonication times to obtain 300-100bp fragments. If you expect to process samples that are partially degraded, optimization of sonication parameters can be performed on a representative sample from those of interest. Based on the degree of DNA fragmentation observed from gel electrophoresis (Step 27), the experimenter can predetermine the optimal sonication time for the sample. Here we describe a method to obtain 300-1000 bp DNA fragments by sonication with an automated sonicating device (Bioruptor from Diagenode, UCD-200 TM) using high molecular weight DNA.

1. Water in Biorupter must be at 4°C.
2. Sonicate for 7 mins on automatic settings (30 sec on 30 sec off at maximum power).
3. Remove 800 ng (40 μl) of sonicated product and place in siliconized 1.7 ml centrifuge tube for the immunoprecipitation (IP) reaction.
4. Set aside remaining 200 ng (10 μl) to serve as input (IN) reference DNA (store at 4°C).

IMMUNOPRECIPITATON OF METHYLATED DNA

1. Denature the DNA that will be used for IP reaction (800 ng) at 95°C for 10 mins in a water bath.
2. Cool immediately on ice. Let DNA cool completely (approximately 5 mins on ice) before proceeding with next step.
3. Add 5 μg monoclonal antibody.
4. Add IP buffer (used at room temperature throughout this protocol) to a final volume of 500 μl.
5. Incubate for 2 hrs at 4°C in rotating tube holder.
6. Just before step 5 is complete, prepare Dynabeads by washing (Steps 6-11). First, resuspend the beads thoroughly in the vial by vortexing.
7. Transfer 30 μl (~ 2 x 10⁷) of resuspended Dynabeads per reaction plus 1, into a new siliconized tube (for example, if doing 8 reactions, remove enough beads for 9, i.e. 270 μl).
8. Place the tube on the magnetic rack for 2 mins at room temperature.
9. Pipette off the supernatant. When removing supernatant, avoid touching the beads against inside wall (where the beads attract to the magnet) with the pipette tip.
10. Remove the tube from the magnet, and resuspend the beads in an excess volume of IP buffer (750 μl-1000 μl). Place the tube back on the magnetic rack for 2 mins at room temperature.
11. Repeat the wash once more, and then resuspend the washed beads in IP buffer in the original volume removed in Step 7.
12. Add 30 μl of washed Dynabeads to each IP reaction
13. Incubate in a rotating tube holder for 2 hrs at 4°C.
14. After incubation is complete, place the tube on the magnetic rack for 2 mins at room temperature.
15. Pipette off the supernatant. Avoid touching the inside wall of the tube (where the beads attract to the magnet) with the pipette tip. Add 500 μl of IP buffer. Mix the tube contents and put it back on the magnetic rack for 2 mins. Repeat wash with 500 ul IP buffer one time.
16. After removing the supernatant from the last wash, resuspend the beads in 400 μl of digestion buffer.
17. Treat the reaction with 100 μg of Proteinase K and incubate overnight at 50°C.

PURIFICATION OF IMMUNOPRECIPITATED DNA

1. Add 500 μl 1:1 phenol/chloroform pH 7 and vortex thoroughly.
2. Spin at 13 000 g for 10 mins at room temperature.
3. Remove aqueous (top) fraction to a new tube.
4. Repeat Steps 1 through 3 if the interphase between the aqueous and organic layers appears cloudy.
5. Add 1/10^th volume 3 M sodium acetate (40 μl) and vortex.
6. Add 1 μl of glycogen (20 μg/μl) and vortex.
7. Add 2 volumes (1000 μl) of 100% ethanol, vortex, and place at -20°C for 20 mins.
8. Spin at 13 000 g for 20 mins at 4°C.
9. Remove ethanol, pulse spin, and remove residual ethanol.
10. Add 500 μl cold 70% ethanol to wash. Vortex briefly, and spin at 13 000 g for 20 mins at 4°C.
11. Remove 70% ethanol, pulse spin, and remove residual ethanol by pipetting.
12. Air dry the pellet with the cap open for 10 mins at room temperature to remove all traces of residual ethanol.
13. Resuspend DNA pellet in 10 μl sterilized dH₂0.

VALIDATION BY PCR

1. You may test to ensure that your MeDIP procedure is working by performing the MeDIP protocol using normal human DNA (male or female), and subsequently assaying a region known to be enriched for methylation.
2. Remove 30% of MeDIP product to PCR tube, and another 30% of MeDIP product to another PCR tube.
3. Put 10 ng of IN DNA into a PCR tube, and 10 ng of IN DNA into another PCR tube. You should now have four separate PCR reactions to set up.
4. Perform PCR using H19 and CTRL primers as indicated in Table 1.
5. Prepare two mastermixes (one for each primer set) for PCR reactions with 12.5 µl total volume as outlined in Table 2.
6. Thermocycle the PCR using the conditions outlined in Table 3.
7. Run 5 µl of PCR products on a 2% agarose gel for visualization.
8. The expected results for successful MeDIP are shown in Table 4.

Tables

Table 1: H19 and CTRL primers for PCR validation.

Primer Set	Forward Primer	Reverse Primer	Anticipated Product Size
H19	H19_F 5’-cgagtgtgcgtgagtgtgag	H19_R 5’-ggcgtaatggaatgcttgaa	174 bp
CTRL (control)	CTRL_F5’-gagagcattagggcagacaaa	CTRL_R 5’-gttcctcagacagccacattt	139 bp

Table 2. Mastermixes for PCR reactions.

	H19 Mix (per Rxn)	CTRL Mix (per Rxn)
ddH2O	6.875	6.875
10X Buffer	1.25	1.25
dNTP mix (10 mM each)	0.25	0.25
MgCl2 (50 mM)	0.25	0.25
Primers (H19_F/R or CTRL_F/R) (10 µM each F/R)	0.625	0.625
Platinum Taq	0.25	0.25

Table 3. PCR thermocyling conditions.

	95°C	5:00
40X	95°C	0:30
	56°C	0:30
	72°C	0:15

Table 4. Expected PCR results for successful MeDIP.

	Template DNA
Primer used	IN	IP
H19	Positive	Positive
CTRL	Positive	Negative

讨论

人们日益认识到DNA甲基化在疾病中发挥的重要作用，因此开发用于检测这种修饰的分析方法正变得愈发重要^{3, 12, 13}。MeDIP技术是一种适用于全基因组水平和位点特异性水平筛查的可行工具^{6, 7}。该技术仅需少量起始DNA即可快速评估DNA甲基化水平，并便于在不同样本之间进行简单比较。MeDIP产物的下游应用包括多种微阵列技术，如全基因组和CpG岛寡核苷酸微阵列、针对特定基因座的直接PCR检测，以及测序分析。

MeDIP 提供了一种独特的DNA甲基化检测方法，该方法依赖抗体来区分甲基化和非甲基化的DNA⁶。尽管MeDIP比传统的亚硫酸氢盐测序方法更快，且不像限制性酶切分析那样局限于特定序列的分析，但其免疫沉淀效果会受到DNA序列的影响，包括CpG密度、重复元件的存在及其组成。因此，如同所有实验一样，适当的对照对于MeDIP结果的分析与解释至关重要。

在MeDIP技术的多个步骤中需格外小心，包括：使用硅化管以防止DNA非特异性结合到管壁；确保超声处理后DNA充分片段...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
Biorupter 超声破碎仪	工具	Diagenode	UCD-200 TM
1.7 ml SafeSeal 微量离心管	其他	Sorenson BioScience	11510
ND 3300 分光光度计	工具	NanoDrop
一抗：抗-5’-甲基胞嘧啶小鼠单克隆抗体	试剂	Calbiochem	162 33 D3
二抗：Dynabeads M-280 羊抗小鼠 IgG 磁珠	试剂	Invitrogen	112-01D
磁力管架	工具	Invitrogen	CS15000
Mini LabRoller 混匀仪	工具	Labnet International	H5500
IP 缓冲液				10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100，室温保存
消化缓冲液				10 mM Tris pH 8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl