Pyrosequencing Approach for SNP Genotyping in Plants Using a M13 Biotinylated Primer

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Plant Breeding
Oct 2013



Single Nucleotide Polymorphisms (SNPs), which constitute single base-pair variations in the DNA sequence, are the most abundant molecular markers in plant and animal genomes. They are becoming the markers of choice for genotyping in all fields of molecular biology, as they are easily prone to automation and high throughput, for example through pyrosequencing. This technology is accurate, flexible and can be easily automated. However, the need for primers labelled with biotin, promptly rise the cost of any methodology employing a pyrosequencing approach. In this protocol we described an improved, efficient, reliable and cost-effective pyrosequencing protocol, based on a universal M13 biotinylated primer, for SNP genotyping in plants.

Keywords: SNP (SNP), Pyrosequencing (焦磷酸测序), M13 tail (M13尾巴), Marker-assisted selection (分子标记辅助选择), Cost-effective (性价比高)

Materials and Reagents

  1. DNA (25 ng/µl) e.g. from two-weeks old barley seedlings; or according to PCR practice of each particular organism
  2. PCR reagents
    1. 0.5 U Taq DNA polymerase (Solis Biodyne FIREPol®, catalog number: 01-01-01000 ) or HotStart Taq polymerase (Solis Biodyne HOT FIREPol®, catalog number: 01-02-01000 ; any other suppliers should be also satisfactory) with the corresponding 10x PCR buffer (supplied with Taq DNA polymerase)
    2. 25 mM MgCl2 (supplied with Taq DNA polymerase)
    3. dNTPs (10 µM each) (Thermo Fisher Scientific, catalog number: R0182 )
    4. Forward specific primer tailed at the 5´end with a universal M13 tail (5´- CACGACGTTGTAAAACGAC-3´) (desalted) (1 µM)
    5. Reverse specific primer (desalted) (10 µM)
    6. Biotinylated universal primer with a complementary sequence to the M13 tail (10 µM) (Metabion, Planegg/Steinkirchen)
  3. Agarose (Sigma-Aldrich, catalog number: A9539 )
  4. Ethidium bromide (Roche Diagnostics, catalog number: HP46.2 )
  5. Pyrosequencing reagents
    1. Sequencing primer (desalted) (10 µM)
    2. Streptavidin Sepharose HP (GE Healthcare, catalog number: 17-5113-01 )
    3. Annealing buffer (QIAGEN, catalog number: 979009 ) (see Recipes)
    4. Binding buffer (QIAGEN, catalog number: 979006 ) (see Recipes)
    5. Denaturation solution (QIAGEN, catalog number: 979007 ) (see Recipes)
    6. Washing buffer (QIAGEN, catalog number: 979008 ) (see Recipes)
    7. High-purity water (Milli-Q)
    8. 70% ethanol
    9. PyroMark Gold Q96 Reagents (enzyme mixture, substrate mixture and nucleotides) (QIAGEN, catalog number: 972807 )
  6. Gel running buffer (see Recipes)
  7. 6x DNA loading buffer (see Recipes)


  1. Thermal Cycler (Applied Biosystems)
  2. Horizontal Electrophoresis system (Bio-Rad Laboratories)
  3. PyroMark Q96 instrument (QIAGEN, catalog number: 9001525 )
  4. PyroMark Q96 plate (QIAGEN, catalog number: 979002 )
  5. PyroMark Q96 Vacuum Workstation (220 V) (QIAGEN, catalog number: 9001529 )
  6. Vacuum pump (KNF Lab, Typ N816.1.2 KN.18)
  7. Orbital shaker for microtiter plates
  8. Heating block (80 °C)
  9. PyroMark Q96 Cartridge (QIAGEN, catalog number: 979004 )
  10. PCR 96-well plate (Greiner Bio-One, catalog number: 652250 )


  1. PyroMark Assay Design Software V.1.0.6. (QIAGEN)


  1. Use the PSQ Assay Design Software for the design of forward, reverse and sequencing primers.
    1. Enter or import the sequence of interest into the “Sequence editor” with the SNP positions represented by IUPAC codes or the following format: C/T.
    2. The amplicon size should be between 150 and 200 bp.
    3. The tailed primer (forward or reverse) should not be longer than 20 bp.
    4. The sequencing primer should be designed between one and five nucleotides of the target SNP.
    5. Select those primers with the highest scores assigned by the software according to their quality for PCR and Pyrosequencing.
    6. Export the primers sequence to an excel file and manually add the universal tail (5´-CACGACGTTGTAAAACGAC-3´) to the 5´end of the forward or reverse primer.
    7. Copy and paste into the excel file the “sequence to analyze”, which will be required later during the run by the PSQ 96 instrument in order to establish the dispensation order for the different nucleotides.
  2. Prepare a PCR mix in a final volume of 25 µl: 1x PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.02 µM of tailed-forward primer, 0.2 µM of reverse primer, 0.18 µM of biotinylated M13 primer, 0.5 U of Taq polymerase and 50 ng of DNA. Even if other DNA source is considered (e.g. PCR product), a minimum of 50 ng should be used as well.
  3. Run the following PCR program: 5 min at 94 °C; 12 cycles with 30 sec at 94 °C, 30 sec at 62 °C (touchdown of 0.5 °C/cycle for initial 12 cycles - final annealing of 56 °C for remaining 35 cycles), 30 sec at 72 °C; and a final extension step of 10 min at 72 °C. At this step, biotin is being incorporated into the PCR product, as described by Schuelke (2000) for fluorescent labelling of PCR fragments.
  4. Run 3 µl of the PCR product in an agarose gel (1.5%) stained with ethidium bromide.
  5. Check the presence of a single and strong DNA fragment with the expected size. If there is any unspecific product, which is highly unusual if the primer design was performed accurately, a HotStart Taq polymerase might be used with the same PCR mix described above, except for MgCl2 (2 mM), dNTPs (0.125 mM) and Taq (0.6 U). The PCR program in this case is as follows: 15 min at 95 °C; 12 cycles with 30 sec at 95 °C, 30 sec at 62 °C (touchdown of 0.5 °C/cycle for initial 12 cycles - final annealing of 56 °C for remaining 45 cycles), 30 sec at 72 °C; and a final extension step of 10 min at 72 °C.
  6. Before starting the pyrosequencing assay, let the buffers reach room temperature.
  7. Add high-purity water to the PCR samples to a final volume of 40 µl.
  8. Prepare a mixture of Streptavidin Sepharose beads (3 µl per sample) and binding buffer (37 µl per sample).
  9. Add 40 µl of the mixture prepared in step 8 to each well of the PCR plate.
  10. Seal the plate and shake the PCR plate constantly at ca. 1,000 rpm for 10 min at room temperature.
  11. Add 40 μl of 0.4 μM sequencing primer in annealing buffer to each well of the PSQ plate that is to be used.
  12. After shaking, place the PCR plate and PSQ plate on the vacuum work station.
  13. Fill each of the five separate troughs in the vacuum workstation with the following solutions: 110 ml ethanol (70%), 90 ml denaturation solution, 110 ml wash buffer, 110 ml high-purity water and 180 ml high-purity water.
  14. Place the vacuum prep tool (VPT) in the water trough and switch on the vacuum pump (Figure 1).

    Figure 1. Vacuum workstation with the vacuum prep tool (VPT) and the different troughs

  15. Flush the filter probes for 20 sec.
  16. Move the VPT into the PCR plate to capture the beads containing immobilized templates
  17. Transfer the VPT sequentially to the troughs containing:
    1. 70% ethanol for 5 sec.
    2. Denaturation solution for 5 sec.
    3. Washing buffer for 5 sec.
  18. Raise the VPT at a 90° angle for 5 sec to drain liquid from the filter probes.
  19. Switch off the vacuum pump and return the VPT the horizontal position.
  20. Place the VPT in the PSQ 96 plate to release the beads. Shake the VPT gently and let it rest for a few seconds on the bottom of the wells.
  21. Move the VPT to the water trough to wash the filters by agitating for 10 sec.
  22. Heat the PSQ plate at 80 °C for 2 min to denature the templates. Cool the PSQ plate at room temperature for at least 5 min to allow the annealing of the sequencing primer.
  23. Select the instrument parameters according to the manufacturer's instructions, including the “sequence to analyze” obtained in the step 1g. Load the cartridge with the estimated amount of PSQ reagents.
  24. Place the plate in the PSQ 96 instrument and insert the cartridge carefully. Start the run
  25. At the end of the run, a pyrogram is shown in a graphical display (Figure 2) and the PSQ software assigns a genotype to each sample by comparison to a previously defined theoretical pattern based on the “sequence to analyze”.

    Figure 2. Pyrograms obtained after the pyrosequencing analysis showing each of two possible genotypes at a T/C SNP


  1. Only tailed forward primers were tested in this protocol. The tailing of reverse primers should work in the same efficient way.
  2. If a clear and strong PCR product is obtained after amplification, the 25 µl of reaction mix might be reduced to only 15 µl, without any negative effect on the subsequent pyrosequencing analysis.
  3. Various primer stoichiometries (1:2, 1:5, 1:10; tailed-forward primer: universal primer) were checked to assess which concentration of universal primer produced the highest amount of biotynilated PCR fragments. No differences were observed in the pyrograms based on the different ratios of tailed forward and universal primers. The 1:10 proportion was selected as it allows to decrease the expense of labelled primer (Silvar et al., 2011).


  1. Annealing buffer
    20 mM Tris-acetate
    2 mM Mg-acetate
    pH 7.6
  2. Binding buffer
    10 mM Tris-HCl
    2 M NaCl
    1 mM EDTA
    0.1% Tween 20
    pH 7.6
  3. Denaturation solution
    0.2 M NaOH
  4. Washing buffer
    10 mM Tris-acetate
    pH 7.6
  5. Gel running buffer
    1x Tris-Borate-EDTA
    89 mM Tris base
    89 mM boric acid
    2 mM EDTA (pH 8.0)
  6. 6x DNA loading buffer
    30% (v/v) glycerol
    0.25% (w/v) bromophenol blue
    0.25% (w/v) xylene cyanol FF


This protocol is adapted from Silvar et al. (2011). The pyrosequencing assay, including annealing plate preparation, immobilization of PCR products to streptavidin beads and the preparation of single-stranded pyrosequencing template DNA were basically carried out as described in the manufacturer´s instructions.


  1. Schuelke, M. (2000). An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18(2): 233-234.
  2. Silvar, C., Perovic, D., Casas, A. M., Igartua, E. and Ordon, F. (2011). Development of a cost effective pyrosequencing approach for SNP genotyping in barley. Plant Breed 130(3): 394-397.


单核苷酸多态性(SNP),其构成DNA序列中的单碱基对变异,是植物和动物基因组中最丰富的分子标记。 它们正成为分子生物学所有领域中基因分型的选择标记,因为它们易于自动化和高通量,例如通过焦磷酸测序。 这种技术是准确,灵活和可以很容易自动化。 然而,对用生物素标记的引物的需要迅速提高了使用焦磷酸测序方法的任何方法的成本。 在这个协议中,我们描述了基于通用M13生物素化引物,用于植物中的SNP基因分型的改进的,有效的,可靠的和成本有效的焦磷酸测序方案。

关键字:SNP, 焦磷酸测序, M13尾巴, 分子标记辅助选择, 性价比高


  1. DNA(25ng /μl)例如来自两周龄的大麦幼苗; 或根据每个特定生物的PCR实践
  2. PCR试剂
    1. 0.5U Taq DNA聚合酶(Solis Biodyne FIREPol ,目录号: 01-01-01000)或HotStart Taq 聚合酶(Solis Biodyne HOT FIREPol 目录号:01-02-01000; 任何其他供应商也应该 令人满意)与相应的10×PCR缓冲液(用Taq DNA聚合酶提供)
    2. 25mM MgCl 2(用Taq DNA聚合酶提供)
    3. dNTP(各10μM)(Thermo Fisher Scientific,目录号:R0182)
    4. 使用通用M13尾(5'-CACGACGTTGTAAAACGAC-3'(脱盐的)(1μM))在5'末端尾标的正向特异性引物
    5. 反向特异性引物(脱盐)(10μM)
    6. 具有与M13尾(10μM)(Metabion,Planegg/Steinkirchen)互补序列的生物素化通用引物,
  3. 琼脂糖(Sigma-Aldrich,目录号:A9539)
  4. 溴化乙锭(Roche Diagnostics,目录号:HP46.2)
  5. 焦磷酸测序试剂
    1. 测序引物(脱盐)(10μM)
    2. 链霉亲和素Sepharose HP(GE Healthcare,目录号:17-5113-01)
    3. 退火缓冲液(QIAGEN,目录号:979009)(参见配方)
    4. 结合缓冲液(QIAGEN,目录号:979006)(参见配方)
    5. 变性溶液(QIAGEN,目录号:979007)(参见配方)
    6. 洗涤缓冲液(QIAGEN,目录号:979008)(参见配方)
    7. 高纯度水(Milli-Q)
    8. 70%乙醇
    9. PyroMark Gold Q96试剂(酶混合物,底物混合物和核苷酸)(QIAGEN,目录号:972807)
  6. 凝胶运行缓冲液(参见配方)
  7. 6x DNA上样缓冲液(见配方)


  1. 热循环仪(Applied Biosystems)
  2. 水平电泳系统(Bio-Rad Laboratories)
  3. PyroMark Q96仪器(QIAGEN,目录号:9001525)
  4. PyroMark Q96板(QIAGEN,目录号:979002)
  5. PyroMark Q96真空工作站(220 V)(QIAGEN,目录号:9001529)
  6. 真空泵(KNF实验室,Typ N816.1.2 KN.18)
  7. 微量滴定板的轨道振动器
  8. 加热块(80℃)
  9. PyroMark Q96 Cartridge(QIAGEN,目录号:979004)
  10. PCR 96孔板(Greiner Bio-One,目录号:652250)


  1. PyroMark Assay设计软件V.1.0.6。 (QIAGEN)


  1. 使用PSQ Assay设计软件设计正向,反向和测序引物
    1. 在"序列编辑器"中输入或导入感兴趣的序列   由IUPAC代码或以下格式表示的SNP位置: C/T。
    2. 扩增子大小应在150至200bp之间。
    3. 尾部引物(正向或反向)不应超过20bp
    4. 测序引物应设计在靶SNP的1至5个核苷酸之间
    5. 根据PCR和焦磷酸测序的质量,选择由软件分配的具有最高分数的引物
    6. 将引物序列导出到excel文件并手动添加 通用尾(5'-CACGACGTTGTAAAACGAC-3')到5'端 正向或反向引物
    7. 复制并粘贴到excel文件中 "序列分析",这将需要以后在运行期间 PSQ 96仪器为了建立分配顺序 不同的核苷酸。
  2. 在25μl的终体积中制备PCR混合物:1×PCR缓冲液,2.5mM MgCl 2,0.2mM dNTP,0.02μM尾部正向引物,0.2μM反向引物,0.18μM生物素化M13引物,0.5U的Taq聚合酶和50ng的DNA。即使考虑了其他DNA来源(例如,PCR产物),也应该使用至少50 ng。
  3. 运行以下PCR程序:94℃5分钟; 12个循环,94℃30秒,62℃30秒(初始12个循环的接触0.5℃/循环 - 剩余35个循环的56℃的最终退火),72℃30秒;和在72℃下10分钟的最终延伸步骤。在该步骤中,将生物素掺入PCR产物中,如Schuelke(2000)所述用于PCR片段的荧光标记。
  4. 运行3μl的PCR产物在用溴化乙锭染色的琼脂糖凝胶(1.5%)。
  5. 检查单个和强大的DNA片段与预期大小的存在。如果存在任何非特异性产物,如果引物设计准确地进行则这是非常不寻常的,可以将HotStart Taq 聚合酶 可以与上述相同的PCR混合物一起使用,除了MgCl 2(2mM),dNTP(0.125mM)和Taq(0.6U)。 在这种情况下的PCR程序如下:在95℃下15分钟; 12个循环,95℃30秒,62℃30秒(初始12个循环的触底为0.5℃/循环 - 剩余45个循环的56℃的最终退火),72℃30秒; 和在72℃下10分钟的最终延伸步骤
  6. 在开始焦磷酸测序测定之前,让缓冲液达到室温
  7. 向PCR样品中加入高纯度水至终体积为40μl
  8. 制备链霉亲和素Sepharose珠(每个样品3μl)和结合缓冲液(每个样品37μl)的混合物。
  9. 将40μl步骤8中制备的混合物加入到PCR板的每个孔中
  10. 密封板,并摇动PCR板恒定在ca。 在室温下以1,000rpm离心10分钟
  11. 在退火缓冲液中加入40μl0.4μM的测序引物到待使用的PSQ板的每个孔中
  12. 摇动后,将PCR板和PSQ板放在真空工作台上
  13. 用以下溶液填充真空工作站中的五个独立的槽中的每一个:110ml乙醇(70%),90ml变性溶液,110ml洗涤缓冲液,110ml高纯度水和180ml高纯度水。
  14. 将真空准备工具(VPT)放入水槽,打开真空泵(图1)。


  15. 冲洗过滤器探头20秒。
  16. 将VPT移入PCR板以捕获含有固定化模板的珠子
  17. 将VPT顺序传输到包含以下内容的槽:
    1. 70%乙醇5秒
    2. 变性溶液5秒。
    3. 洗涤缓冲液5秒。
  18. 将VPT以90°角升高5秒,以从过滤器探头中排出液体。
  19. 关闭真空泵并使VPT返回水平位置。
  20. 将VPT放在PSQ 96板中以释放珠。 轻轻摇动VPT,让它在孔底部停留几秒钟。
  21. 将VPT移动到水槽,通过搅拌10秒来清洗过滤器。
  22. 将PSQ板在80℃下加热2分钟以使模板变性。 在室温下冷却PSQ板至少5分钟,以允许测序引物退火
  23. 根据制造商的说明选择仪器参数,包括在步骤1g中获得的"分析顺序"。 将估计量的PSQ试剂装入墨盒。
  24. 将板放在PSQ 96仪器中,小心插入墨盒。 开始运行
  25. 在运行结束时,在图形显示(图2)中显示了热谱图,并且PSQ软件通过与基于"分析序列"的先前定义的理论模式相比较来为每个样品指派基因型。

    图2.焦磷酸测序分析后获得的热解图,显示在T/C SNP 两种可能的基因型中的每一种


  1. 在该方案中仅测试了有尾引物。 反向引物的尾巴应以相同的有效方式工作
  2. 如果在扩增后获得清晰且强的PCR产物,则25μl反应混合物可以减少至仅仅15μl,而对随后的焦磷酸测序分析没有任何负面影响。
  3. 检查各种引物化学计量(1:2,1:5,1:10;尾部正向引物:通用引物),以评估哪种浓度的通用引物产生最高量的生物素化PCR片段。 基于尾部正向引物和通用引物的不同比率,在热裂解图中没有观察到差异。 选择1:10比例,因为其允许降低标记引物的费用(Silvar等人,2011)。


  1. 退火缓冲区
    20 mM Tris-乙酸盐 2mM Mg-醋酸盐 pH 7.6
  2. 绑定缓冲区
    10mM Tris-HCl
    2 M NaCl
    1mM EDTA
    0.1%Tween 20
    pH 7.6
  3. 变性溶液
    0.2 M NaOH
  4. 洗涤缓冲液
    10mM Tris-乙酸盐 pH 7.6
  5. 凝胶运行缓冲液
    1 Tris-Borate-EDTA
    89 mM Tris碱
    89mM硼酸 2mM EDTA(pH8.0)
  6. 6x DNA加载缓冲液
    30%(v/v)甘油 0.25%(w/v)溴酚蓝
    0.25%(w/v)二甲苯cyanol FF


该协议改编自Silvar等人(2011)。 焦磷酸测序测定,包括退火平板制备,将PCR产物固定在链霉抗生物素珠上和制备单链焦磷酸测序模板DNA,基本上如制造商的说明书中所述进行。


  1. Schuelke,M。(2000)。 荧光标记PCR片段的经济方法。 Nat Biotechnol 18(2):233-234。
  2. Silvar,C.,Perovic,D.,Casas,A.M.,Igartua,E。和Ordon,F。(2011)。 在大麦中开发用于SNP基因分型的成本有效的焦磷酸测序方法。 植物品种 130(3):394-397
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引用:Silvar, C., Perovic, D., Casas, A. M., Igartua, E. and Ordon, F. (2015). Pyrosequencing Approach for SNP Genotyping in Plants Using a M13 Biotinylated Primer. Bio-protocol 5(10): e1473. DOI: 10.21769/BioProtoc.1473.