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Jun 2021

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A Molecular Cloning and Sanger Sequencing-based Protocol for Detecting Site-specific DNA Methylation
一种基于分子克隆和Sanger测序的位点特异性DNA甲基化检测方案    

Wei GuoWei Guo*Anthony CannonAnthony Cannon*Damon LischDamon Lisch  (*共同第一作者)
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Abstract

DNA methylation is a conserved chemical modification, by which methyl groups are added to the cytosine of DNA molecules. Methylation can influence gene expression without changing the sequence of a particular gene. This epigenetic effect is an intriguing phenomenon that has puzzled biologists for years. By probing the temporal and spatial patterns of DNA methylation in genomes, it is possible to learn about the biological role of cytosine methylation, as well as its involvement in gene regulation and transposon silencing. Advances in whole-genome sequencing have led to the widespread adoption of methods that examine genome-wide patterns of DNA methylation. Achieving sufficient sequencing depth in these types of experiments is costly, particularly for pilot studies in organisms with large genome sizes, or incomplete reference genomes. To overcome this issue, assays to determine site-specific DNA methylation can be used. Although often used, these assays are rarely described in detail. Here, we describe a pipeline that applies traditional TA cloning, Sanger sequencing, and online tools to examine DNA methylation. We provide an example of how to use this protocol to examine the pattern of DNA methylation at a specific transposable element in maize.

Keywords: Epigenetics (表观遗传学), Molecular cloning (分子克隆), Sanger sequencing (Sanger 测序), DNA methylation (DNA甲基化), Transposon (转座子), Applications (应用程序)

Background

DNA methylation is a conserved chemical modification at the 5’ position in the deoxyribose ring of cytosine that is catalyzed by methyltransferases (Figure 2A and Schultz et al., 2012). In plants, DNA methylation occurs in three sequence contexts: CG, CHG, and CHH, where H represents A, T, or C (Zhang et al., 2018). DNA methylation plays an important role in regulating both genes and transposable elements (TEs) (Schultz et al., 2012; Zhang et al., 2018; Guo et al., 2021). Using a maize MuDR reporter system to study TE silencing, we have demonstrated that DNA methylation at a terminal inverted region (TIR) in one of the MuDR elements, mudrA, is associated with silencing (Woodhouse et al., 2006; Guo et al., 2021).


Bisulfite sequencing involves bisulfite treatment of genomic DNA, resulting in the conversion of unmethylated cytosines into uracil, while methylated cytosines remain unchanged. After treatment, the region of interest is amplified, cloned, and sequenced. Investigating DNA methylation at a gene or a TE requires high-quality genomic DNA, and the conversion of unmethylated cytosines into uracil, while retaining methylated cytosines using sodium bisulfite treatment (Gruntman et al., 2008; Zhang et al., 2018). After this treatment, the region of interest is amplified via polymerase chain reaction (PCR), which is followed by TA-cloning and Sanger sequencing (Foerster, 2010). Because PCR amplification of the converted cytosines will replace uracil with thymine, determining the methylation profile at a region of interest can be achieved by comparing the sequence of the bisulfite-treated DNA with that of untreated DNA sequences (Gruntman et al., 2008). To minimize the noise created by incomplete conversion of unmethylated cytosine to uracil, examining DNA methylation using an internal control gene, and assaying multiple individual colonies of PCR products are needed.


Here, we provide a case study with step-by-step procedures, to examine the pattern of DNA methylation at a silenced MuDR element in maize, which is a well-documented system to validate our protocol. We described a site-specific DNA methylation pipeline, using traditional TA cloning and Sanger sequencing and provide procedures for sample preparation, treatment, cloning work, and data analysis. We highlight the applicability of this traditional DNA methylation pipeline as an inexpensive method for pilot studies, important for decision-making, or for use when an organism’s genome is not fully sequenced. Additionally, this pipeline describes the basis of molecular cloning and epigenetic analysis, and how it can be used as a method for teaching.

Materials and Reagents

  1. Eppendorf tubes (Dot Scientific, catalog number: RA1700-GMT)

  2. Glass beads (MO-SCI, catalog number: GL0191B4/38-53)

  3. Axygen 96-well PCR Microplates (Fisher Scientific, catalog number: 14-222-326)

  4. Tissue Culture Treated Petri Dishes (dot scientific, catalog number: 667621)

  5. Competent E. coli cells (Invitrogen, catalog number: 18265017)

  6. Minimal MuDR line (Damon Lisch lab, Purdue University)

  7. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: BP2482100)

  8. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-1)

  9. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: 28312)

  10. Potassium acetate (Fisher Scientific, catalog number: A16321.36)

  11. Isopropanol (Fisher Scientific, catalog number: 040983.M1)

  12. 100% Ethanol (Fisher Scientific, catalog number: T038181000)

  13. DNA loading dye 10× (NEB, catalog number: 10816015)

  14. 2× PCR Master Mix (Syd Labs, catalog number: MB067-EQ2G-L)

  15. EpiMark Hot Start Taq DNA Polymerase (NEB, catalog number: M0490S)

  16. Bisulfite conversion kit (ZYMO RESEARCH, catalog number: D5005)

  17. Lysogeny broth (LB) medium (Sigma-Aldrich, catalog number: L2897)

  18. T4 ligase (NEB, catalog number: M0202S)

  19. TA cloning vector (Fisher Scientific, catalog number: K1231)

  20. RNase A (Fisher Scientific, catalog number: EN0531)

  21. 8-strip PCR tubes and Caps (dot scientific, catalog number: 503-8PCR-A)

  22. Zymoclean Gel DNA recovery Kits (Zymo catalog number: D4007)

  23. ZymoPURE Plasmid Miniprep Kit (Zymo catalog number: D4210)

Equipment

  1. Pipettes (Fisher Scientific, catalog number: EPPR4331)

  2. Microcentrifuge (Fisher Scientific, catalog number: 5424)

  3. Incubator (VWR, catalog number: 13259-36)

  4. Qubit fluorometer (Fisher Scientific, catalog number: Qubit 2.0)

  5. Gel electrophoresis system (NeoSCI, catalog number: 55-1094)

  6. Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl)

  7. Low speed orbital shaker (Corning LSE, catalog number: 6780-FP)

  8. PCR machine (Bio-Rad, catalog number: 1861096)

Procedure

An overview of procedures can be seen in Figure 1.



Figure 1. An overview of procedures of DNA extraction protocol

  1. Sample collection and DNA extraction

    1. Collect samples and store them at -80°C, or immediately perform DNA extraction using the protocol described previously (Guo et al., 2022).

    2. Digest genomic DNA with RNase.

    Normally, 1 μL of RNase is added to each DNA sample, and incubated at 37°C for 30 min.

    3. Evaluate the quality of DNA.

    The quality of genomic DNA should be evaluated by running gel electrophoresis, and using a Qubit fluorometer or UV spectrophotometer. A large, clear, intact band on top indicates the presence of high quality genomic DNA. In addition to high-quality DNA, a successful bisulfite conversion reaction requires a quantity of DNA suggested by the commercial kit being used.

  2. Primer design

    Primers can be designed using a variety of online tools, such as Bisulfite Primer Seeker (https://www.zymoresearch.com/pages/bisulfite-primer-seeker).

    Note: It is recommended that primers be designed before the bisulfite treatment steps. Primers for an internal control gene should also be designed. Ideally, this should be a gene that has already been determined to be entirely or largely unmethylated.

  3. Bisulfite conversion

    Readers should follow the instructions from the commercial kit being used for bisulfite conversion.

    Note: Converted DNA can be stable at 4°C or -20°C for a short period of time, but it is best to check the manual from the kit you are using. It is recommended to PCR-amplify the region of interest immediately after the conversion is done to ensure the best results.

  4. TA cloning of the PCR amplicons

    Typical cloning PCR conditions are (Table 1):


    Table 1. Overview of cloning PCR conditions

    Component 50 μL Reaction
    5× EpiMark Hot Start

    Taq Reaction buffer

    10 μL
    10 mM dNTPs 1 μL
    10 μM Forward Primer 1 μL
    10 μM Reverse Primer 1 μL

    EpiMark Hot Start

    Taq Polymerase

    0.25 μL
    Converted DNA 1 μL/variable
    Nuclease-free water To 50 μL

    Thermal cycling conditions:

    95°C, 30 s

    35–40 cycles of:

    95°C, 15–30 s

    45–68°C, 15–60 s

    68°C, 1 min per kb

    Final extension:

    68°C, 5 min


Note: Because genomic DNA is treated and converted by the bisulfite reagent, a regular Taq DNA polymerase may not tolerate uracil-containing DNA and high AT targets. Therefore, it is necessary to use the appropriate DNA polymerase to ensure high fidelity, low bias, and sufficient yield. We recommend EpiMark Hot Start Taq DNA Polymerase from the New England Lab (NEB), which generates the PCR products containing dA overhangs at the 3’ end. This allows for a ligation to vector with dT/dU-overhangs. To clone the region of interest using PCR, it’s recommended to run a PCR with a larger volume, to ensure sufficient DNA can be recovered. Usually, 50 μL or more gives better results. One can directly purify the PCR product using a commercial kit. Alternatively, the PCR product can be recovered from the gel slice. If using this method, it’s important to use fresh TAE buffer and a clean razor blade, when performing electrophoresis and recovery.


  1. Ligation. Typical ligation conditions are (Table 2):


    Table 2. Overview of ligation conditions

    Component 20 μL Reaction
    T4 DNA Ligase Buffer (10×) 2 μL
    Vector DNA (3 kbp) 50 ng
    Insert DNA (300 bp) 15 ng
    T4 DNA Ligase 1 μL
    Nuclease-free Water To 20 μL


    Note: After PCR amplification and gel recovery, incubate the PCR products with the cloning vector, following the instructions below (Table 2). Our previous work used the cloning kit from Thermo Fisher (Guo et al., 2021). Ligation can be completed in 5 min at room temperature, but it is recommended to extend this time to 30 min or longer for any PCR products larger than 3,000 bp.


  2. Transformation

    Readers should follow the instructions from the commercial competent cells being used for transformation.

    Note: Once ligation is complete, samples are ready to undergo vector transformation using E. coli competent cells. Note the antibiotic-resistant gene carried by the vector. Typically, the Top10 strain grows faster than DH 5-alpha strain, but there is no significant difference in using either for a regular cloning experiment. After transformation, cells can be spread directly onto an LB medium containing appropriate antibiotics. Be sure to use either sterilized glass beads or a spreader. Incubate the LB plates at 37°C for 10–16 h.

  3. To determine the colonies that carry a positive amplicon, propagate the cells from individual colonies in liquid LB medium, and run a colony-PCR to select the positive colonies that will undergo sequencing. Typically,

    1. Prepare 2.0-mL tubes on a tube rack, and fill them with 2 mL of liquid LB medium containing antibiotics.

    2. Inoculate each tube with an individual colony from each plate, using a sterilized toothpick or pipette tip.

    3. Tubes are then placed onto a shaker set at 200 rpm and 37°C for 10–14 h.

      Note: Due to some unsuccessful ligation and propagation, and depending on the size of the PCR products, it is recommended to inoculate at least two-fold more colonies than the actual number you want to send for Sanger sequencing.

    4. Once transparent liquid LB medium becomes turbid, run a colony-PCR directly, using 1 mL of liquid LB medium containing cells as the DNA template.

      Note: Since the size of the vector is usually small, and this is a direct PCR amplification from the plasmid from E. coli, a strong positive amplification typically results after as few as 20 PCR cycles.


    Typical colony-PCR conditions are (Table 3):


    Table 3. Overview of colony-PCR conditions

    Component 12.5 μL Reaction
    2× Master Mix 6.25 μL
    10 μM Forward Primer 0.25 μL
    10 μM Reverse Primer 0.25 μL
    DNA 1 μL
    Nuclease-free Water 4.75 μL

    Thermal cycling conditions

    95°C, 3 min

    20 cycles or more of:

    95°C, 30 s

    50°C, 30 s

    70°C, 1 min per kb

    Final extension:

    70°C, 5 min


  1. Once positive colonies are identified, plasmids can be extracted from the remaining liquid culture. Extraction can be done using traditional methods, or a commercial kit. Once plasmids are ready, samples can be sent for Sanger sequencing. It’s recommended to send a few to test, before multiple samples are sequenced. Distinct sequences from ten colonies from each genotype or treatment should be obtained for analysis.

  2. Sequences can be trimmed using SnapGene viewer or any available software. Reference sequences and samples sequences are placed in a separate text file in the FASTA format. Be sure that all sequences are in the correct orientation relative to the reference sequence, and primer sequences are excluded. The two files are uploaded into Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl), which provides quantitative data, as well as images for result visualization.

Results

Examining DNA methylation patterns at MuDR elements in the maize minimal MuDR line

Using this protocol, we have demonstrated that DNA methylation at TIRB, the terminal inverted repeats (TIR) adjacent to the mudrB gene in MuDR, is not associated with transcriptional activity of mudrB, and that mudrA activity is associated with DNA hypomethylation at TIRA (Li et al., 2010; Guo et al., 2021). Here, we describe how to examine DNA methylation in a step-by-step manner. First, we collected leaf tissue from 2-week-old seedlings from active and silenced MuDR lines, and then extracted genomic DNA from those tissues. Gel examination indicated that the DNA was of good quality, as indicated by the intact bright band at the top of the gel (Figure 2B). We then measured the concentration of DNA, to determine the right volume of DNA for the bisulfite conversion reaction (Table 4). Next, we performed bisulfite conversion using a commercial kit, and PCR to amplify short fragments from TIRA and TIRB in two separate amplifications, using two different pairs of primers (Figure 2C). Previous experiments demonstrated that TIRA lacks DNA methylation, which can serve as an internal control in the active MuDR/- line, whereas DNA methylation is present at TIRB (Woodhouse et al., 2006; Guo et al., 2021).

After we harvested the PCR products directly from the gel slices using a commercial kit, we performed ligation and E. coli transformation, following the procedures described above. We obtained plates with several hundred colonies (Figure 2D). To determine the positive colonies and extract plasmids from those for Sanger sequencing, we propagated cell clones in liquid medium, and performed colony-PCR. We found that most colonies carried inserts of the expected size, suggesting that the cloning work was successful. We then sent the plasmids for Sanger sequencing. Reads were trimmed and pasted into a text file and uploaded to Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl).


Table 4. Examining concentration and purity of genomic DNA samples

Sample Concentration (Qubit) A260/A280 A260/A230
MuDR/- 75 ng/μL 1.82 2.12



Figure 2. TA cloning of TIRA and TIRB in MuDR/-.

A. An illustration of methylated and unmethylated cytosine. B. Genomic DNA of MuDR/-. A total volume of 2 mL of DNA mixed with 10× DNA loading dye was loaded into 1% agarose gel, together with 5 mL of the 100 bp DNA ladder that was loaded into the 2nd lane. M denotes the 100-bp DNA ladder. C. PCR amplification of TIRA and TIRB from MuDR/-. M denotes the 100-bp DNA ladder. A negative control of water as DNA template was included. D. Colonies on LB agar plate containing 50 μg/mL ampicillin.


A dot plot with three colors, indicating DNA methylation occurring in three sequence contexts, was generated and visualized for TIRA and TIRB, respectively (Figure 3A). To get data for the ratio of DNA methylation and show an individual value for each colony, each sequence from each plasmid was uploaded, and data were recorded. A histogram plot with a dot standing for each colony was generated (Figure 3B and 3C), of which the results were consistent, and supported the results obtained using the dot plots.



Figure 3. DNA methylation pattern at TIRA and TIRB in the MuDR line.

A. Dot plot of DNA methylation pattern at TIRA and TIRB. B. Total DNA methylation ratio at TIRA and TIRB. C. DNA methylation ratio for cytosines in each sequence context at TIRA and TIRB. Ten individual clones were sequenced from PCR products of bisulfite-treated samples. The cytosines in different sequence contexts are represented by three colors (red: CG, blue: CHG, green: CHH; where H =A, C, or T). Filled cycles stand for methylated cytosines.

Acknowledgments

This work was funded by a NSF grant to DL (IOS-1237931).

Competing interests

The Authors declare that there is no conflict of interest.

References

  1. Foerster, A. S. M. O. (2010). Analysis of DNA Methylation in Plants by Bisulfite Sequencing. Methods Mol Biol 631: 1-11.
  2. Gruntman, E., Qi, Y., Slotkin, R. K., Roeder, T., Martienssen, R. A. and Sachidanandam, R. (2008). Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinformatics 9: 371.
  3. Guo, W., Wang, D. and Lisch, D. (2021). RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays. PLoS Genet 17(6): e1009326.
  4. Guo, W., Binstock, B., Cannon, A. and Lisch, D. (2022). An inexpensive, fast, and robust DNA extraction method for high-quality DNA for use in genotyping and next-generation sequencing applications in plants. Bio-protocol 10.21769/p1516.
  5. Li, H., Freeling, M. and Lisch, D. (2010). Epigenetic reprogramming during vegetative phase change in maize. Proc Natl Acad Sci U S A 107(51): 22184-22189.
  6. Schultz, M. D., Schmitz, R. J. and Ecker, J. R. (2012). 'Leveling' the playing field for analyses of single-base resolution DNA methylomes. Trends Genet 28(12): 583-585.
  7. Woodhouse, M. R., Freeling, M. and Lisch, D. (2006). The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize. Genetics 172(1): 579-592.
  8. Zhang, H., Lang, Z. and Zhu, J. K. (2018). Dynamics and function of DNA methylation in plants. Nat Rev Mol Cell Biol 19(8): 489-506.

简介

[摘要] DNA甲基化是一种保守的化学修饰,将甲基添加到DNA分子的胞嘧啶上。甲基化可以在不改变特定基因序列的情况下影响基因表达。这种表观遗传效应是一个有趣的现象,多年来一直困扰着生物学家。通过探索基因组中 DNA 甲基化的时空模式,可以了解胞嘧啶甲基化的生物学作用,以及它在基因调控和转座子沉默中的作用。全基因组测序的进步已导致广泛采用检查全基因组 DNA 甲基化模式的方法。在这些类型的实验中实现足够的测序深度是昂贵的,特别是对于具有大基因组大小或不完整参考基因组的生物体的初步研究。为了克服这个问题,可以使用测定位点特异性 DNA 甲基化的方法。尽管经常使用,但这些检测很少被详细描述。在这里,我们描述了一个应用传统 TA 克隆、Sanger 测序和在线工具来检查 DNA 甲基化的管道。我们提供了一个示例,说明如何使用该协议来检查玉米中特定转座因子的 DNA 甲基化模式。


[背景] DNA 甲基化是胞嘧啶脱氧核糖环 5' 位置的保守化学修饰,由甲基转移酶催化(图 2A 和Schultz等人,2012) 。在植物中,DNA 甲基化发生在三个序列环境中:CG、CHG 和 CHH,其中 H 代表 A、T 或 C (Zhang et al. , 2018) 。 DNA 甲基化在调节基因和转座因子 (TEs) 中发挥重要作用(Schultz等人,2012;Zhang等人,2018;Guo等人,2021) 。使用玉米MuDR报告系统研究 TE 沉默,我们已经证明MuDR元件之一mudrA中末端倒置区域 (TIR) 的 DNA 甲基化与沉默相关(Woodhouse等人,2006 年; Guo等人。 , 2021) 。
亚硫酸氢盐测序涉及对基因组 DNA 进行亚硫酸氢盐处理,导致未甲基化胞嘧啶转化为尿嘧啶,而甲基化胞嘧啶保持不变。处理后,对感兴趣区域进行扩增、克隆和测序。研究基因或 TE 的 DNA 甲基化需要高质量的基因组 DNA,以及将未甲基化的胞嘧啶转化为尿嘧啶,同时使用亚硫酸氢钠处理保留甲基化的胞嘧啶(Gruntman等人,2008 年;Zhang等人, 2018 年) 。在此处理之后,通过聚合酶链式反应 (PCR) 扩增感兴趣的区域,然后进行 TA 克隆和 Sanger 测序(Foerster, 2010) 。因为转化的胞嘧啶的 PCR 扩增将用胸腺嘧啶取代尿嘧啶,所以可以通过将亚硫酸氢盐处理的 DNA 的序列与未处理的 DNA 序列的序列进行比较来确定感兴趣区域的甲基化谱(Gruntman等人,2008) 。为了最大限度地减少未甲基化胞嘧啶向尿嘧啶的不完全转化所产生的噪音,需要使用内部对照基因检查 DNA 甲基化,并分析 PCR 产物的多个单个菌落。
在这里,我们提供了一个分步程序的案例研究,以检查玉米中沉默的MuDR元素的 DNA 甲基化模式,这是一个有据可查的系统来验证我们的协议。我们描述了一个位点特异性 DNA 甲基化流程,使用传统的 TA 克隆和 Sanger 测序,并提供了样品制备、处理、克隆工作和数据分析的程序。我们强调了这种传统 DNA 甲基化管道的适用性,它是一种廉价的试点研究方法,对决策很重要,或者在生物体基因组未完全测序时使用。此外,该管道描述了分子克隆和表观遗传分析的基础,以及如何将其用作教学方法。

关键字:表观遗传学, 分子克隆, Sanger 测序, DNA甲基化, 转座子, 应用程序



材料和试剂


1.Eppendorf 管(Dot Scientific,目录号:RA1700-GMT)
2.玻璃珠(MO-SCI,目录号:GL0191B4/38-53)
3.Axygen 96孔PCR微孔板(Fisher Scientific,目录号:14-222-326)
4.组织培养处理的培养皿(dot science,目录号:667621)
5.感受态大肠杆菌细胞(Invitrogen,目录号:18265017)
6.最小MuDR线(普渡大学 Damon Lisch实验室)
7.乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:BP2482100)
8.氯化钠(NaCl)(Fisher Scientific,目录号:BP358-1)
9.十二烷基硫酸钠(SDS)(Fisher Scientific,目录号:28312)
10.乙酸钾(Fisher Scientific,目录号:A16321.36)
11.异丙醇(Fisher Scientific,目录号:040983.M1)
12.100%乙醇(Fisher Scientific,目录号:T038181000)
13.DNA上样染料10 × (NEB,目录号:10816015)
14.2 × PCR Master Mix(Syd Labs,目录号:MB067-EQ2G-L)
15.EpiMark热启动Taq DNA 聚合酶(NEB,目录号:M0490S)
16.亚硫酸氢盐转化试剂盒(ZYMO RESEARCH,目录号:D5005)
17.溶原肉汤(LB)培养基(Sigma-Aldrich,目录号:L2897)
18.T4连接酶(NEB,目录号:M0202S)
19.TA克隆载体(Fisher Scientific,目录号:K1231)
20.RNase A(Fisher Scientific,目录号:EN0531)
21.8条PCR管和盖子(dot science,目录号:503-8PCR-A)
22.Zymoclean凝胶 DNA 回收试剂盒( Zymo目录号:D4007)
23.ZymoPURE质粒小量制备试剂盒( Zymo目录号:D4210)


设备


1.移液器(Fisher Scientific,目录号:EPPR4331)
2.微量离心机(Fisher Scientific,目录号:5424)
3.培养箱(VWR,目录号:13259-36)
4.Qubit 荧光计(Fisher Scientific,目录号:Qubit 2.0)
5.凝胶电泳系统( NeoSCI ,目录号:55-1094)
6.Kismeth ( http://katahdin.mssm.edu/kismeth/revpage.pl )
7.低速轨道振动器(Corning LSE,目录号:6780-FP)
8.PCR机(Bio-Rad,目录号:1861096)


程序


流程概览见图 1。
 
图1. DNA 提取方案程序概述


1.样本采集和 DNA 提取
1. 收集样本并将其储存在 -80 °C 下,或立即使用之前描述的方案进行 DNA 提取(Guo等人,2022 年)
2. 用 RNase 消化基因组 DNA。
通常,1 将μL RNase 添加到每个 DNA 样本中,并在 37 °C 下孵育 30 分钟。
3. 评估 DNA 的质量。
这 基因组 DNA 的质量应通过凝胶电泳和使用 Qubit 荧光计或紫外分光光度计进行评估。顶部的大、清晰、完整的条带表明存在高质量的基因组 DNA。除了高质量的 DNA 之外,成功的亚硫酸氢盐转化反应还需要使用商业试剂盒建议的一定量的 DNA。
2.引物设计
可以使用各种在线工具设计引物,例如 Bisulfite Primer Seeker ( https://www.zymoresearch.com/pages/bisulfite-primer-seeker )。
注意:建议在亚硫酸氢盐处理步骤之前设计引物。还应设计内部对照基因的引物。理想情况下,这应该是一个已经被确定为完全或大部分未甲基化的基因。
3.亚硫酸氢盐转化
读者应遵循用于亚硫酸氢盐转化的商业试剂盒的说明。
注意:转化后的 DNA 可以在 4 °C或 -20 °C下短时间保持稳定,但最好从您使用的试剂盒中查看手册。建议在转换完成后立即对感兴趣的区域进行 PCR 扩增,以确保获得最佳结果。
4.PCR扩增子的TA克隆
典型的克隆 PCR条件是(表 1):


表 1. 克隆 PCR 条件概述
零件50 μl反应_
5 × EpiMark热启动
Taq 反应缓冲液10微升_
10 毫米 dNTP1微升_
10 μ M正向引物1微升_
10 μ M反向引物1微升_
EpiMark热启动
Taq聚合酶0.25微升_
转化的 DNA1μL /变量_
无核酸酶水至50微升


热循环条件:
95°C,30 秒
35-40 个循环:
95°C,15 – 30 秒
45–68°C,15–60 秒
68°C,每 kb 1 分钟
最终扩展:
68°C,5分钟


注意:由于基因组 DNA 由亚硫酸氢盐试剂处理和转化,常规 Taq DNA 聚合酶可能无法耐受含尿嘧啶的 DNA 和高 AT 靶标。因此,需要使用合适的 DNA 聚合酶来保证高保真度、低偏差和足够的产量。我们推荐来自新英格兰实验室 (NEB) 的EpiMark Hot Start Taq DNA 聚合酶,它可以生成在 3' 端包含dA突出端的 PCR 产物。这允许连接到带有 dT/ dU -突出端的载体。要使用 PCR 克隆感兴趣的区域,建议运行更大体积的 PCR,以确保可以回收足够的 DNA。通常,50 μL或更多会产生更好的结果。可以使用商业试剂盒直接纯化 PCR 产物。或者,可以从凝胶切片中回收 PCR 产物。如果使用这种方法,在进行电泳和恢复时,使用新鲜的 TAE 缓冲液和干净的刀片非常重要。


a.结扎。典型的连接条件是(表 2):


表 2. 连接条件概述
零件20 μL反应_
T4 DNA 连接酶缓冲液 (10 × )2微升_
载体 DNA (3 kbp )50 纳克
插入 DNA (300 bp)15 纳克
T4 DNA连接酶1微升_
无核酸酶水至20 μL


注意:PCR 扩增和凝胶回收后,按照以下说明(表 2)将 PCR 产物与克隆载体一起孵育。我们之前的工作使用了 Thermo Fisher ( Guo et al. , 2021 )的克隆试剂盒。室温下连接可在 5 分钟内完成,但对于任何大于 3,000 bp 的 PCR 产物,建议将此时间延长至 30 分钟或更长。


b.转型
读者应遵循用于转化的商业感受态细胞的说明。
注意:一旦连接完成,样品就可以使用大肠杆菌感受态细胞进行载体转化。注意载体携带的抗生素抗性基因。通常,Top10 菌株的生长速度比 DH 5-α 菌株快,但在常规克隆实验中使用任何一种都没有显着差异。转化后,细胞可以直接扩散到含有适当抗生素的 LB 培养基上。请务必使用经过消毒的玻璃珠或吊具。将 LB 板在 37°C 下孵育 10-16 小时。
c.要确定携带阳性扩增子的菌落,请在液体 LB 培养基中从单个菌落中繁殖细胞,并运行菌落 PCR 以选择将进行测序的阳性菌落。通常,
1.在管架上准备 2.0 mL 管,并用 2 mL 含有抗生素的液体 LB 培养基填充它们。
2.使用消毒过的牙签或移液器尖端,从每个盘子中接种一个单独的菌落。
3.然后将试管置于设置为 200 rpm 和 37°C 的振荡器上 10-14 小时。
注意:由于某些连接和增殖不成功,根据 PCR 产物的大小,建议接种的菌落至少比您要发送的 Sanger 测序的实际数量多 2 倍。
4.一旦透明液体 LB 培养基变得浑浊,使用 1 mL 含有细胞的液体 LB 培养基作为 DNA 模板,直接运行菌落-PCR。
注意:由于载体的大小通常很小,而且这是从大肠杆菌质粒中直接进行的 PCR 扩增,因此通常在 20 个 PCR 循环后会产生强阳性扩增。


典型的菌落 PCR 条件是(表 3):


表 3.菌落 PCR 条件概述
零件12.5 μL反应_
2 ×主混音6.25微升_
10 μ M 正向引物0.25微升_
10 μ M 反向引物0.25微升_
脱氧核糖核酸1微升_
无核酸酶水4.75微升_


热循环条件
95°C,3 分钟
20 个循环或更多:
95°C,30 秒
50℃ ,30秒
70°C,每 kb 1 分钟
最终扩展:
70°C,5 分钟


5.一旦鉴定出阳性菌落,就可以从剩余的液体培养物中提取质粒。可以使用传统方法或商业试剂盒进行提取。一旦质粒准备好,就可以将样品送去进行 Sanger 测序。建议先发送几个进行测试,然后再对多个样本进行测序。应获得来自每个基因型或处理的十个菌落的不同序列用于分析。
6.可以使用SnapGene查看器或任何可用软件修剪序列。参考序列和样本序列以 FASTA 格式放置在单独的文本文件中。确保所有序列相对于参考序列的方向正确,并排除引物序列。这两个文件被上传到Kismeth ( http://katahdin.mssm.edu/kismeth/revpage.pl ),它提供定量数据,以及用于结果可视化的图像。


结果


玉米最小MuDR系中MuDR元件的DNA 甲基化模式
使用该协议,我们已经证明了 TIRB 的 DNA 甲基化,即MuDR中与mudrB基因相邻的末端反向重复 (TIR)与转录无关 mudrB的活性,并且mudrA活性与 TIRA 的 DNA 低甲基化有关(Li等人,2010;Guo等人,2021) 。在这里,我们描述了如何逐步检查 DNA 甲基化。首先,我们从活跃和沉默的MuDR系中收集 2 周龄幼苗的叶组织,然后从这些组织中提取基因组 DNA。凝胶检查表明 DNA 质量良好,如凝胶顶部完整的亮带所示(图 2B)。然后我们测量了 DNA 的浓度,以确定适合亚硫酸氢盐转化反应的 DNA 体积(表 4)。接下来,我们使用商业试剂盒进行亚硫酸氢盐转化,并使用两对不同的引物进行 PCR 以在两个单独的扩增中扩增来自 TIRA 和 TIRB 的短片段(图 2C)。先前的实验表明,TIRA 缺乏 DNA 甲基化,可作为活性MuDR /-系的内部对照,而 TIRB 存在 DNA 甲基化(Woodhouse等人,2006;Guo等人,2021) 。
在我们使用商业试剂盒直接从凝胶切片中收获 PCR 产物后,我们按照上述程序进行了连接和大肠杆菌转化。我们获得了具有数百个菌落的板(图 2D)。为了确定阳性菌落并从那些用于 Sanger 测序的菌落中提取质粒,我们在液体培养基中繁殖细胞克隆,并进行菌落-PCR。我们发现大多数菌落都带有预期大小的插入片段,这表明克隆工作是成功的。然后我们将质粒送去进行 Sanger 测序。读取被修剪并粘贴到文本文件中并上传到Kismeth ( http://katahdin.mssm.edu/kismeth/revpage.pl )。


表 4.检查基因组 DNA 样本的浓度和纯度
样本浓度(量子位)A 260 /A 280A 260 /A 230
MUDR /-75 纳克/微升1.822.12




 
图 2。 MuDR /-中 TIRA 和 TIRB 的 TA 克隆。
A. 甲基化和未甲基化胞嘧啶的示意图。 B. MuDR的基因组 DNA /-。将总体积为 2 mL 的 DNA 与 10 × DNA 上样染料混合加载到 1% 琼脂糖凝胶中,连同加载到第 2 条泳道中的 5 mL 100 bp DNA梯。 M 表示 100 bp DNA 梯。 C. 来自MuDR /-的 TIRA 和 TIRB 的 PCR 扩增。 M 表示 100 bp DNA 梯。包括作为 DNA 模板的水的阴性对照。 D. 含有 50 μ g /mL 氨苄青霉素的 LB 琼脂板上的菌落。


分别为 TIRA 和 TIRB 生成并可视化了具有三种颜色的点图,指示在三个序列上下文中发生的 DNA 甲基化(图 3A)。为了获得 DNA 甲基化比率的数据并显示每个菌落的单独值,上传来自每个质粒的每个序列,并记录数据。生成了一个直方图,每个菌落都有一个点代表(图 3B 和 3C),其结果是一致的,并支持使用点图获得的结果。


 
图 3 。 MuDR中TIRA和TIRB的DNA甲基化模式 线。
A. TIRA 和 TIRB 的 DNA 甲基化模式点图。 B. TIRA 和 TIRB 的总 DNA 甲基化比率。 C. TIRA 和 TIRB 的每个序列上下文中胞嘧啶的 DNA 甲基化比率。从亚硫酸氢盐处理的样品的 PCR 产物中对 10 个单独的克隆进行了测序。不同序列上下文中的胞嘧啶由三种颜色表示(红色:CG,蓝色:CHG,绿色:CHH;其中 H = A、C 或 T)。填充循环代表甲基化胞嘧啶。


致谢


这项工作由NSF 授予 DL (IOS-1237931) 资助。


利益争夺


作者声明不存在利益冲突。


参考


1.福斯特,ASMO (2010)。通过亚硫酸氢盐测序分析植物中的 DNA 甲基化。 方法 Mol Biol 631:1-11。
2.Gruntman, E.、Qi, Y.、Slotkin, RK、Roeder, T.、Martienssen, RA 和 Sachidanandam, R. (2008)。 Kismeth:通过亚硫酸氢盐测序分析植物甲基化状态。 BMC 生物信息学9:371。
3.Guo, W.、Wang, D. 和 Lisch, D. (2021)。 RNA 指导的 DNA 甲基化可防止玉米在热应激下转座子沉默的快速和可遗传逆转。 PLoS Genet 17(6): e1009326。
4.Guo, W.、Binstock, B.、Cannon, A. 和 Lisch, D. (2022)。一种廉价、快速、稳健的 DNA 提取方法,可用于植物的基因分型和下一代测序应用中的高质量 DNA 。生物协议10.21769/p1516 。
5.Li, H.、Freeling, M. 和 Lisch, D. (2010)。玉米营养阶段变化过程中的表观遗传重编程。 Proc Natl Acad Sci USA 107(51):22184-22189。
6.Schultz, MD, Schmitz, RJ 和 Ecker, JR (2012)。 “平衡”单碱基分辨率 DNA 甲基化组分析的竞争环境。 趋势基因28(12):583-585。
7.Woodhouse, MR, Freeling, M. 和 Lisch, D. (2006)。 mop1(paramutation1 的介体)突变体逐渐重新激活玉米中由 MuDR 转座子编码的两个基因之一。 遗传学172(1):579-592。
8.Zhang, H.、Lang, Z. 和 Zhu, JK (2018)。植物DNA甲基化的动力学和功能。 Nat Rev Mol 细胞生物学19(8): 489-506。 


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引用:Guo, W., Cannon, A. and Lisch, D. (2022). A Molecular Cloning and Sanger Sequencing-based Protocol for Detecting Site-specific DNA Methylation. Bio-protocol 12(9): e4408. DOI: 10.21769/BioProtoc.4408.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

Anirban Banerjee
University of Waterloo
The article has been written in simple language which will help first-time performers of the workflow. The protocol described is definitely not novel (as suggested by the authors) but it is good to have the different parts of the workflow described in details in one place. I will suggest changing the table titles to something other than 'Overview'. They are recipes or compositions and not overview. Also, 1st row of Table 3 should be in bold.
6/29/2022 2:47:56 PM 回复