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Detection of Transposable Element Insertion Site Polymorphisms by Sequence-Specific Amplification Polymorphism (SSAP)
通过序列特异扩增多态性(SSAP)法检测转座子插入位点的多态性   

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Abstract

Transposable elements represent a major part of any eukaryotic genomes. Notably in plants they can account for more than 80% of the whole genomic sequence (such as in maize). Due to their mobility across the genome, they can act as mutagens but can also be considered as an important source of genetic diversity. It has been shown that they may be activated following various stresses, and it has been assumed that they may contribute to genome evolution and adaptation. Molecular methods have thus been proposed to allow identification of new transposition events, or more generally to tag transposable element insertion site polymorphisms. Sequence-Specific Amplification Polymorphism (SSAP) is a high throughput method derived from AFLP, which has been first tested on the barley genome (Waugh et al., 1997). Its efficiency in tagging TEs in comparison to AFLP is based on the use of specific primers anchored in the TE sequences of interest, requiring the TEs under survey to be previously characterized. SSAP can thus be used to identify any genomic reorganization in the vicinity of TE insertion sites, and still represents an efficient approach to analyse evolutionary dynamics of TEs.

Keywords: Allopolyploidy(异源多倍体), Transposon(转座子), PCR-based polymorphism(基于PCR的基因多态性), LTR-retrotransposon(转座子)

Materials and Reagents

  1. Restriction of genomic DNA using endonucleases
    1. Genomic DNAs from the samples to be analysed at concentrations of 100 ng/µl.
    2. Endonuclease enzyme insensitive to DNA methylation, generating cohesive ends
      Note: TE sequence ends have to be devoid of any corresponding restriction sites.

  2. Adapter ligation
    1. Two oligonucleotides which form a double-stranded adapter, with an overhang complementary to the overhang left by the restriction enzyme used. In the case of EcoRI (Thermo Fisher Scientific, Fermentas) the 5’ overhang is AATT and the oligonucleotide sequences are 5’-CTCGTAGACTGCGTACC-3’ and 5’-AATTGGTACGCAGTCTAC-3’. Annealing of the two oligonucleotides will constitute the adapter (purple rectangles in Figure 1).
    2. T4 DNA ligase (1-3 U/μl) with the corresponding ligase buffer (Promega Corporation, catalog number: M1801 )
    3. Sterile ultrapure water

  3. Pre-amplification
    1. PCR reagents
      1. Homemade Taq DNA polymerase is of sufficient quality for the PCR reactions but Taq Promega has also been used (Promega corporation, catalog number: M8301 ) with the corresponding Taq polymerase buffer.
      2. 25 mM MgCl2
      3. dNTPs (10 µM each)
    2. Pre-amplification primers (10 μM each)
      1. One primer is anchored in the adapter: 5’-GACTGCGTACCAATTC-3’ (primer P1 in Figure 1).
      2. The other primer is anchored in the TE of interest, close to its end to limit the size of amplicon (primer P2 in Figure 1).

  4. Selective amplification
    1. PCR reagents (see pre-amplification step)
    2. Selective amplification primers (10 μM each)
      1. One primer is anchored in the adapter, with the addition of 3 nucleotides at the 3’ end (primer P3 in Figure 1) to limit the number of bands to be visualized and analyzed by gel electrophoresis. Several combinations of A, T, G and C should be used to target different genomic localizations of TE insertion sites.
      2. The other primer is defined to perform a nested PCR in comparison with the pre-amplification step (primer P4 in Figure 1); this primer is labelled with a 5’-IRDye. DNA labelled with IRDye (infrared dye) has to be stored in the dark at -20 °C. To minimize exposure to light, wrap the tube in aluminium foil.

  5. Acrylamide gel for SSAP profile analysis
    1. Kimwipes
    2. Urea (Merck KGaA, catalog number: 1.08488.1000 )
    3. Long Ranger Acrylamide (50%) (Lonza, catalog number: 50611 )
    4. APS (Bio-Rad Laboratories, catalog number: 161-0700 )
    5. Temed (Bio-Rad Laboratories, catalog number: 161-0801 )
    6. Absolute ethanol
    7. Formamide (Sigma-Aldrich, catalog number: F9037 )
    8. 10x TBE buffer (Tris/Borate/EDTA) (see Recipes)
    9. 5.5% acrylamide gel (see Recipes)
    10. Loading buffer (see Recipes)

Equipment

  1. 1.5 ml Eppendorf tubes
  2. Centrifuge
  3. Parafilm
  4. Incubator or oven
  5. Water bath
  6. 96-well PCR plates
  7. 0.45 μm filters
  8. LI-COR DNA analyzer (LI-COR) and corresponding equipment (plates, combs, spacers...)
  9. Thermal cycler
  10. Horizontal shaker

Procedure

Note: Adapted from Waugh et al. (1997) and Pouilly et al. (2008); as used in Sarilar et al. (2013).

  1. Targeting TE insertion sites
    1. Restriction of genomic DNAs
      1. Make a restriction reaction mixture that will be distributed equally into individual Eppendorf tubes. The reaction mixture combines (in order) sterile ultrapure water (with a final volume of 36 µl per reaction), the 1x enzyme buffer and the selected endonuclease (6 U per reaction), taking into account that 5 µl of genomic DNA will be added into the mix.
      2. Distribute 31 µl of the reaction mixture into each Eppendorf tube.
      3. Add 5 µl of each DNA solution to be analysed (which correspond to 500 ng of genomic DNA) to the corresponding tube.
      4. Mix gently the solution in each tube by pipeting, and centrifuge shortly.
      5. Incubate at least 3 h at 37 °C.
      6. Stop the enzyme activity by incubating the tubes 20 min at 65 °C.
      7. Put the tubes on ice, centrifuge briefly and replace them on ice. Proceed immediately to the ligation step.
    2. Prepare the adapters and ligate them to the restricted DNAs
      1. In a 0.5 ml Eppendorf tube, mix the two oligonucleotides (100 µM each) in equal proportions to make the adapter solution (final concentration of 50 µM).
      2. Incubate the mix at 95 °C for 5 min (in a thermocycler) and let cool it down on the bench at room temperature.
      3. Make a ligation reaction mixture, by combining (in order) sterile ultrapure water (with a final volume of 50 µl per reaction), 1x ligase buffer, adapter solution (50 pmol) and T4 DNA ligase (1 U per reaction).
      4. Distribute 14 µl of the reaction mixture into the Eppendorf tubes containing each 36 µl of restricted DNAs.
      5. Mix gently using a pipette and then centrifuge shortly.
      6. Incubate at 20-25 °C overnight (on the bench) to allow ligation.
      7. Dilute the ligation products to 1/10 in 96-well PCR plates: 90 µl of sterile ultrapure water + 10 µl of the restriction-ligation products.
        Note: Store non-diluted restriction-ligation products at -20 °C, but keep 1/10 diluted restriction-ligation products at 4 °C to avoid DNA degradation because of freezing - thawing cycles.
    3. Pre-amplification
      1. Use the first primer pair P1/P2 (Figure 1b) for the pre-amplification.
      2. Make a pre-amplification PCR reaction mixture by combining (in order) sterile ultrapure water (with a final volume of 20 µl per reaction), 1x Taq polymerase buffer, MgCl2 (2.5 mM), dNTP solution (200 µM), primer P1 anchored in the adapter (8 pmol), primer P2 anchored in the TE under study (8 pmol) and Taq DNA polymerase (3-4 U).
      3. Distribute 17 µl of the pre-amplification reaction mixture into the wells of a 96-well PCR plate.
      4. Add 3 µl of the 1/10 diluted restriction-ligation products. Mix gently for each reaction by pipeting 2-3 times.
      5. Centrifuge.
      6. PCR program
        1. 1 cycle at 72 °C for 2 min
        2. 1 cycle at 94 °C for 3 min
        3. 25 cycles of (94 °C for 30 sec, 56 °C for 30 sec and 72 °C for 1 min)
        4. 1 cycle at 72 °C for 5 min
        5. Keep at 10 °C
      7. Verification of the pre-amplification products can be performed by agarose gel electrophoresis: Load 4 µl of the reaction product (with loading buffer) and run the electrophoresis for ~20 min at 100 V.
        Note: A smear should be obtained (an example of the gel is shown in Figure 1b).
      8. Dilute the pre-amplification product in 96-well PCR plates according to the plate layout to carry out the separation of fragments using the LI-COR DNA analyzer.
        Note: The factor of dilution is usually 1/10 (36 µl of sterile ultrapure water + 4 µl of pre-amplification product) but it has to be adapted (1/2, 1/5, 1/10, or no dilution) according to the intensity of the smear observed after electrophoresis, in order to get homogeneous selective amplification products.
    4. Selective amplification
      1. Use the second primer pair P3/P4 (Figure 1c) for the selective amplification to perform a nested PCR (to increase the specificity of the PCR reaction).
      2. Make a selective PCR reaction mixture by combining (in order) sterile ultrapure water (with a final volume of 20 µl per reaction), 1x Taq polymerase buffer, MgCl2 (2.5 mM), dNTP solution (200 µM), selective primer P3 anchored in the adapter with 3 additional selective nucleotides (4 pmol), selective primer P4 anchored in the TE under study and labelled with IRDye (4 pmol) and Taq DNA polymerase (1.5-2 U).
        Note: The primer labelled with the IRDye is sensitive to the light; the PCR plate has to be covered with aluminium foil to avoid degradation.
      3. Distribute 15 µl of the selective PCR reaction mixture into the wells of a 96-well PCR plate.
      4. Add 5 µl of the diluted pre-amplification products. Mix gently for each reaction by pipeting 2-3 times.
      5. Centrifuge.
      6. PCR program: a typical touchdown program is performed (Vos et al., 1995) to increase the specificity of the PCR amplification.
        1. 1 cycle at 94 °C for 4 min
        2. 13 cycles of [94 °C for 30 sec, 65 °C (then -0.7 °C per cycle) for 30 sec and 72 °C for 1 min]
        3. 25 cycles of (94 °C for 30 sec, 56 °C for 30 sec and 72 °C for 1 min)
        4. 1 cycle at 72 °C for 5 min
        5. Keep at 10 °C
          Note: Maintain the 96-well PCR plates out of the light by covering them with aluminium foil and once the program is finished keep them at -20 °C.

  2. Electrophoresis run on LI-COR DNA analyzer to separate amplified products
    1. Prepare the DNA analyzer according to manufacturer’s instructions.
    2. Use 0.25 mm spacers between glass plates and prepare 40 ml of 5.5% acrylamide gel.
    3. Do pre-run with autofocus and following parameters: 45 °C; 2,000 W; 25 mA.
    4. Load the gel
      1. Mix the PCR products of selective amplification (2 µl) with equal amount of loading buffer.
      2. Heat the samples at 95 °C for 5 min for DNA denaturation and snap cool on ice before loading. Keep also on ice while loading the samples on the acrylamide gel.
      3. Load 0.5 µl of each sample in each well. Between each deposit, rinse the multi-syringe in water. Load on the same acrylamide gel the samples which have to be compared, and load the controls on each gel if necessary.
    5. Analyze the SSAP multiband fingerprints by scoring polymorphic bands which may correspond to TE insertion site polymorphisms (Figure 1d).
      Note: Wear gloves when working with acrylamide because of its carcinogenic and toxic properties.


      Figure 1. Schematic description of the different steps of the SSAP procedure to detect TE insertion site polymorphisms. (a). The genomic DNAs to be analysed are restricted using an endonuclease (here EcoRI, as used in Sarilar et al., 2013) and then ligated to corresponding adapters. (b). A pre-amplification is performed on the restriction-ligation products using a specific PCR primer pair (to be designed) with one primer (P1) complementary to the adapter and the other primer (P2) anchored in the TE of interest. As illustrated by the picture, the pre-amplification should lead to a PCR product visualized as a smear after agarose gel electrophoresis. (c). The resulting PCR products are then subjected to a selective PCR amplification using another specific PCR primer pair (to be designed) with one primer (P3) complementary to the adapter with 3 additional selective nucleotides and the other primer (P4) anchored in the TE and labeled with an IRDye (at the 5’ end). (d). The resulting amplicons are finally resolved in a denaturing acrylamide gel; polymorphic bands can be visualized and scored as non-additive SSAP bands (being additional + or missing -) when comparing the progeny profiling with the parental ones (as illustrated here for the analysis of resynthesized allotetraploid lines in comparison with their diploid progenitors, Sarilar et al., 2013). Identification of the nature of the polymorphism responsible for non-additivity (e.g. transposition, genomic rearrangement, polymorphism at the endonuclease cutting site) requires further cloning and sequencing of the corresponding SSAP amplicon.

Recipes

  1. 10x TBE buffer (1 L)
    Tris Base 108 g
    Boric acid 55 g
    40 ml of EDTA (0.5 M, pH 8)
    ddH2O qsp 1 L
    Filter sterilize (0.45 µM)
  2. 5.5% acrylamide gel (40 ml)
    Add 15.6 g urea to 20 ml ddH2O; agitate while heating until obtain a translucent solution
    Add 4.8 ml of 10x TBE buffer
    Filter 0.45 µm to remove any urea crystals
    Add 4.4 ml Long ranger (50%)
    Complete with ddH2O up to 40 ml
    When ready to inject gel solution, add for polymerization
    1. 264 µl of 10% APS
    2. 26.4 µl Temed
    Mix completely to homogenate and draw the gel solution into a 50 ml syringe (with no needle)
  3. Loading buffer
    Mix 40 mg of bromophenol blue with 2 ml of EDTA (0.5 M, pH 8) and 0.5 ml of ddH2O
    Complete with 47.5 ml of formamide
    Note: Loading buffer is carcinogenic because of formamide, it has to be handled with gloves and under the flow hood.

Acknowledgments

This protocol is adapted from Waugh et al. (1997) and Sarilar et al. (2013).

References

  1. Pouilly, N., Delourme, R., Alix, K. and Jenczewski, E. (2008). Repetitive sequence-derived markers tag centromeres and telomeres and provide insights into chromosome evolution in Brassica napus. Chromosome Res 16(5): 683-700.
  2. Sarilar, V., Palacios, P. M., Rousselet, A., Ridel, C., Falque, M., Eber, F., Chevre, A. M., Joets, J., Brabant, P. and Alix, K. (2013). Allopolyploidy has a moderate impact on restructuring at three contrasting transposable element insertion sites in resynthesized Brassica napus allotetraploids. New Phytol 198(2): 593-604.
  3. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and et al. (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23(21): 4407-4414.
  4. Waugh, R., McLean, K., Flavell, A. J., Pearce, S. R., Kumar, A., Thomas, B. B. and Powell, W. (1997). Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol Gen Genet 253(6): 687-694.

简介

转座元件代表任何真核基因组的主要部分。值得注意的是,在植物中,它们可以占全部基因组序列的80%以上(例如在玉米中)。由于它们在基因组上的移动性,它们可以作为诱变剂,但也可以被认为是遗传多样性的重要来源。已经表明,它们可以在各种胁迫之后被激活,并且已经假定它们可以有助于基因组进化和适应。因此已经提出分子方法以允许鉴定新的转座事件,或更通常地标记可转座元件插入位点多态性。序列特异性扩增多态性(SSAP)是源自AFLP的高通量方法,其已经在大麦基因组上首先测试(Waugh等人,1997)。与AFLP相比,其标记TE的效率基于锚定在感兴趣的TE序列中的特异性引物的使用,需要先前表征所调查的TE。 SSAP因此可以用于鉴定TE插入位点附近的任何基因组重组,并且仍然是分析TE的进化动力学的有效方法。

关键字:异源多倍体, 转座子, 基于PCR的基因多态性, 转座子

材料和试剂

  1. 使用核酸内切酶限制基因组DNA
    1. 来自待分析样品的基因组DNA,浓度为100ng /μl
    2. 核酸内切酶对DNA甲基化不敏感,产生粘性末端
      注意:TE序列末端必须没有任何相应的限制位点。

  2. 适配器连接
    1. 两个寡核苷酸形成双链衔接子,具有与使用的限制酶留下的突出端互补的突出端。 在Eco RI(Thermo Fisher Scientific,Fermentas)的情况下,5'突出端是AATT,寡核苷酸序列是5'-CTCGTAGACTGCGTACC-3'和5'-AATTGGTACGCAGTCTAC-3'。 两个寡核苷酸的退火将构成衔接子(图1中的紫色矩形)
    2. T4 DNA连接酶(1-3U /μl)与相应的连接酶缓冲液(Promega公司,目录号:M1801)
    3. 无菌超纯水

  3. 预扩增
    1. PCR试剂
      1. 对于PCR反应,自制的Taq DNA聚合酶具有足够的质量,但是也已经使用Promega(Promega公司,目录号:M8301)和相应的Taq 聚合酶缓冲液。
      2. 25mM MgCl 2·h/v
      3. dNTPs(每种10μM)
    2. 预扩增引物(各10μM)
      1. 一个引物锚定在衔接子中:5'-GACTGCGTACCAATTC-3'(图1中的引物P1)。
      2. 另一个引物锚定在感兴趣的TE中,靠近其末端以限制扩增子的大小(图1中的引物P2)。

  4. 选择性扩增
    1. PCR试剂(见预扩增步骤)
    2. 选择性扩增引物(每种10μM)
      1. 一个引物锚定在衔接子中,在3'末端添加3个核苷酸(图1中的引物P3)以限制待显示的条带数目并通过凝胶分析 电泳。 应使用A,T,G和C的几种组合来靶向TE插入位点的不同基因组定位
      2. 定义另一引物以与预扩增步骤(图1中的引物P4)相比进行巢式PCR; 该引物用5'-IRDye标记。 用IRDye(红外染料)标记的DNA必须在-20℃下避光保存。 为了尽量减少暴露在光线下,用铝箔包裹管子
  5. 丙烯酰胺凝胶进行SSAP谱分析
    1. Kimwipes
    2. 尿素(Merck KGaA,目录号:1.08488.1000)
    3. 长兰丙烯酰胺(50%)(Lonza,目录号:50611)
    4. APS(Bio-Rad Laboratories,目录号:161-0700)
    5. Temed(Bio-Rad Laboratories,目录号:161-0801)
    6. 绝对乙醇
    7. 甲酰胺(Sigma-Aldrich,目录号:F9037)
    8. 10x TBE缓冲液(Tris /硼酸盐/EDTA)(参见配方)
    9. 5.5%丙烯酰胺凝胶(见配方)
    10. 加载缓冲区(参见配方)

设备

  1. 1.5 ml Eppendorf管
  2. 离心机
  3. parafilm
  4. 孵化器或烤箱
  5. 水浴
  6. 96孔PCR板
  7. 0.45μm过滤器
  8. LI-COR DNA分析仪(LI-COR)和相应的设备(板,梳,垫片...)
  9. 热循环仪
  10. 水平振动器

程序

注意:改编自Waugh et al。 (1997)和Pouilly et al。 (2008); 如在Sarilar等人 (2013)。

  1. 定位TE插入位点
    1. 基因组DNA的限制
      1. 制备限制性反应混合物,将其平均分配到单独的Eppendorf管中。 反应混合物(按顺序)将无菌超纯水(每次反应的终体积为36μl),1x酶缓冲液和选择的内切核酸酶(每个反应6U)混合,考虑到将加入5μl基因组DNA 进入混合
      2. 将31μl反应混合物分配到每个Eppendorf管中
      3. 将5μl待分析的每种DNA溶液(相当于500 ng的基因组DNA)加入相应的试管中
      4. 通过移液轻轻地混合每个管中的溶液,并短时离心
      5. 在37℃下孵育至少3小时。
      6. 通过将管在65℃下孵育20分钟来停止酶活性
      7. 将管放在冰上,短暂离心,并在冰上更换。 立即进行结扎步骤。
    2. 准备适配器,并将其连接到限制性DNA
      1. 在0.5ml Eppendorf管中,将两种寡核苷酸(每种100μM)以相同比例混合以制备衔接物溶液(最终浓度为50μM)。
      2. 将混合物在95°C孵育5分钟(在热循环仪中),并在室温下在工作台上冷却。
      3. 通过合并(按顺序)无菌超纯水(每个反应终体积为50μl),1x连接酶缓冲液,衔接子溶液(50pmol)和T4 DNA连接酶(每个反应1U)制备连接反应混合物。 />
      4. 将14μl反应混合物分配到含有36μl限制性DNA的Eppendorf管中
      5. 用移液管轻轻混匀,然后短时间离心
      6. 在20-25℃孵育过夜(在工作台上)以允许连接
      7. 在96孔PCR板中将连接产物稀释至1/10:90μl无菌超纯水+10μl限制性连接产物。
        注意:将非稀释的限制性连接产物储存在-20℃,但保持1/10稀释的限制性连接产物在4℃,以避免由于冻融循环而导致DNA降解。
    3. 预扩增
      1. 使用第一对引物对P1/P2(图1b)进行预扩增
      2. 通过组合(按顺序)无菌超纯水(每次反应最终体积为20μl),1×Taq聚合酶缓冲液,MgCl 2缓冲液, (2.5mM),dNTP溶液(200μM),锚定在衔接子中的引物P1(8pmol),锚定在研究中的TE的引物P2(8pmol)和Taq DNA聚合酶(3-4 U)。
      3. 将17μl预扩增反应混合物分配到96孔PCR板的孔中
      4. 加入3μl1/10稀释的限制性连接产物。 通过移液2-3次轻轻混合每次反应
      5. 离心机。
      6. PCR程序
        1. 1个循环,72℃2分钟
        2. 1个循环,94℃3分钟
        3. 25个循环(94℃30秒,56℃30秒和72℃1分钟)
        4. 1个循环,72℃5分钟
        5. 保持在10°C
      7. 预扩增产物的验证可以通过琼脂糖凝胶电泳进行:加载4μl反应产物(加样缓冲液),并在100V电泳约20分钟。
        注意:应该获得涂片(凝胶的实例如图1b所示)。
      8. 根据板布局在96孔PCR板中稀释预扩增产物,使用LI-COR DNA分析仪进行片段的分离。
        注意:稀释因子通常为1/10(36μl无菌超纯水+ 4μl预扩增产物),但必须适应(1/2,1/5,1/10或无稀释),根据电泳后观察到的涂片强度,以获得均匀的选择性扩增产物。
    4. 选择性扩增
      1. 使用第二对引物对P3/P4(图1c)进行选择性扩增以进行嵌套PCR(以提高PCR反应的特异性)。
      2. 通过组合(按顺序)无菌超纯水(每次反应终体积为20μl),1×Taq聚合酶缓冲液,MgCl 2(2.5) (4pmol)锚定在接头中的选择性引物P3,锚定在研究中的TE中并且用IRDye(4pmol)和Taq DNA聚合酶(1.5pmol)标记的选择性引物P4, -2 U)。
        注意:用IRDye标记的引物对光敏感; PCR板必须用铝箔覆盖以避免降解。
      3. 将15μl选择性PCR反应混合物分配到96孔PCR板的孔中
      4. 加入5μl稀释的预扩增产物。 通过移液2-3次轻轻混合每次反应
      5. 离心机。
      6. PCR程序:进行典型的着陆程序(Vos等人,1995)以增加PCR扩增的特异性。
        1. 1个循环,94℃4分钟
        2. 13个循环的[94℃30秒,65℃(然后-0.7℃/循环)30秒和72℃1分钟]。
        3. 25个循环(94℃30秒,56℃30秒和72℃1分钟)
        4. 1个循环,72℃5分钟
        5. 保持在10°C
          注意:通过用铝箔覆盖将96孔PCR板保持在光照下,一旦程序完成,将其保存在-20℃。

  2. 在LI-COR DNA分析仪上进行电泳以分离扩增产物
    1. 根据制造商的说明准备DNA分析仪。
    2. 在玻璃板之间使用0.25mm间隔物,并制备40ml的5.5%丙烯酰胺凝胶。
    3. 使用自动对焦和以下参数进行预运行:45°C; 2,000 W; 25 mA。
    4. 加载凝胶
      1. 将选择性扩增(2μl)的PCR产物与等量上样缓冲液混合
      2. 将样品在95℃加热5分钟以进行DNA变性,并在上样前在冰上快速冷却。 在将样品装载到丙烯酰胺凝胶上时,还要保持在冰上
      3. 在每个孔中加入0.5μl的每个样品。在每次沉积之间,在水中冲洗多注射器。加载在相同的丙烯酰胺凝胶样品,必须进行比较,并加载控制在每个凝胶如有必要
    5. 通过对可能对应于TE插入位点多态性的多态性带进行评分来分析SSAP多带指纹(图1d)。
      注意:使用丙烯酰胺时应戴手套,因为它具有致癌性和毒性。


      图1.检测TE插入位点多态性的SSAP程序的不同步骤的示意图描述(a)。使用内切核酸酶(此处为EcoRI,如在Sarilar等人,2013中使用的)限制待分析的基因组DNA,然后连接到相应的衔接头。 (b)。使用使用与衔接子互补的一个引物(P1)和锚定在感兴趣的TE中的另一个引物(P2)的特异性PCR引物对(待设计)对限制性连接产物进行预扩增。如图所示,预扩增应导致PCR产物在琼脂糖凝胶电泳后显现为涂片。 (C)。然后使用另一个特异性PCR引物对(待设计)对所得PCR产物进行选择性PCR扩增,其中一个引物(P3)与具有3个另外的选择性核苷酸的衔接子互补,另一个引物(P4)锚定在TE和用IRDye标记(在5'端)。 (d)。所得扩增子最终在变性丙烯酰胺凝胶中分离;当比较子代谱系与亲​​本谱系时,多态谱带可以可视化并作为非加性SSAP谱带(是额外的+或缺失的)记录(如本文所述的与二倍体祖细胞相比的再合成的异源四倍体系的分析, em> et al。,2013)。负责非加性(例如,转座,基因组重排,核酸内切酶切割位点处的多态性)的多态性的性质的鉴定需要进一步克隆和测序相应的SSAP扩增子。

食谱

  1. 10x TBE缓冲液(1 L)
    Tris碱108 g
    硼酸55 g
    40ml EDTA(0.5M,pH8) ddH sub 2 O qsp 1 L
    过滤灭菌(0.45μM)
  2. 5.5%丙烯酰胺凝胶(40ml) 加入15.6g尿素至20ml ddH 2 O; 搅拌同时加热直到获得半透明溶液
    加入4.8ml 10×TBE缓冲液
    过滤器0.45μm,以除去任何尿素结晶体
    添加4.4 ml长护林员(50%)
    用高达40ml的ddH <2> O完成
    当准备注入凝胶溶液时,加入聚合
    1. 264μl10%APS
    2. 26.4μlTemed
    混合均匀,并将凝胶溶液吸入50ml注射器(无针)
  3. 加载缓冲区
    将40mg溴酚蓝与2ml EDTA(0.5M,pH 8)和0.5ml ddH 2 O混合。
    用47.5ml甲酰胺完成 注意:加载缓冲液是致癌的,因为甲酰胺,它必须用手套和流动罩下处理。

致谢

该协议改编自Waugh等人(1997)和Sarilar等人(2013)。

参考文献

  1. Pouilly,N.,Delourme,R.,Alix,K.and Jenczewski,E。(2008)。 重复序列衍生标记标记着丝粒和端粒,并提供了对欧洲油菜的染色体进化的了解 Chromosome Res 16(5):683-700。
  2. 这些研究结果表明,这些研究结果表明,这些研究结果表明, 。 Allopolyploidy 在再合成的欧洲油菜同源四倍体中的三个对比的转座因子插入位点处的重组具有适度的影响。新植物 198(2):593-604。
  3. Vos,P.,Hogers,R.,Bleeker,M.,Reijans,M.,van de Lee,T.,Hornes,M.,Frijters,A.,Pot,J.,Peleman,J.,Kuiper,M 和等人(1995)。 AFLP:一种新的DNA指纹识别技术。 Nucleic Acids Res < em> 23(21):4407-4414。
  4. Waugh,R.,McLean,K.,Flavell,A.J.,Pearce,S.R.,Kumar,A.,Thomas,B.B.and Powell,W。(1997)。 通过序列特异性扩增多态性揭示的大麦基因组中Bare-1样反转录转座因子的遗传分布 (S-SAP)。 Mol Gen Genet 253(6):687-694。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sarilar, V., Palacios, P. . and Alix, K. (2014). Detection of Transposable Element Insertion Site Polymorphisms by Sequence-Specific Amplification Polymorphism (SSAP). Bio-protocol 4(5): e1054. DOI: 10.21769/BioProtoc.1054.
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