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Fluorescence-based CAPS Multiplex Genotyping on Capillary Electrophoresis Systems
基于毛细管电泳系统的荧光标记CAPS多重基因型   

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Abstract

Recent advances in next-generation sequencing techniques allow the detection of a large number of SNPs and their use in a high throughput manner. However, Cleaved Amplified Polymorphic Sequences (CAPSs) still play a significant role as complement to other high throughput methods for SNP genotyping. Therefore, new methods focusing on the acceleration of this type of markers are highly desirable. The combination of the classical CAPS technique and a M13-tailed primer multiplexing assay was used to develop an agarose gel free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected on a capillary electrophoresis system. This method allows the cost-effective genotyping of several SNPs in a multiplexed manner at an overall low cost in a short period of time. Additionally, this method could be efficiently combined with the simultaneous detection of SSRs at the same electrophoresis run resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.

Keywords: CAPS(帽子), SNP(SNP), Fluorescence-based multiplexing(基于荧光的复用), M13 tail(M13尾巴), Marker-assisted selection(分子标记辅助选择)

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 of Taq FIREPol® DNA polymerase (Solis Biodyne FIREPol®, catalog number: 01-01-01000 ; any other suppliers should be also satisfactory) with the corresponding 10x PCR buffer (supplied with FIREPol® DNA polymerase)
    2. 25 mM MgCl2 (supplied with FIREPol® 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 (1µM) (5´- CACGACGTTGTAAAACGAC-3´) (desalted) (Microsynth)
    5. Reverse specific primer (10 µM) (desalted) (Microsynth)
    6. Fluorescence (6-FAM, HEX, NED or Cy5, D2, D3) labeled primer with a complementary sequence to the M13 tail (10 µM) (Metabion, Planegg/Steinkirchen)
  3. Restriction analysis
    1. Restriction endonucleases (New England Biolabs or Fermentas)
    2. 1x Buffer restriction endonucleases (New England Biolabs or Fermentas)
  4. 1.5% agarose (Sigma-Aldrich, catalog number: A9539 )
  5. Ethidium bromide (Roche Diagnostics, catalog number: HP46.2 )
  6. DNA loading buffer 3x [6x; 30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF]
  7. Analysis on ABI PRISM 3100 Genetic Analyzer Separation Gel (Applied Biosystems, catalog number: 4363929 )
    1. ROTISOLV® HPLC gradient grade water (Roche Diagnostics, catalog number: A511.3 )
    2. HiDiTMFormamide as Sample Loading Buffer (Applied Biosystems, catalog number: 4311320 )
    3. GeneScanTMROX size standard (Applied Biosystems, catalog number: 401734 )
    4. 96 well half skirted PCR plates (Kisker, catalog number: G060/H/1E/OA-SS )
  8. Analysis on Beckman Coulter CEQTM 8000 Genetic Analysis System
    1. GenomeLabTM Separation Gel LPA I (Beckman Coulter, catalog number: 608010 )
    2. 96 well half skirted, segmented PCR plates (Kisker, catalog number: G060/H/1E-SG )
    3. Separation Buffer plates, non-sterile (Beckman Coulter, catalog number: 609844 )
    4. GenomeLabTM DNA Size Standard 600 (Beckman Coulter, catalog number: 608095 )
    5. GenomeLabTM Separation Buffer (Beckman Coulter, catalog number: 608012 )
    6. Formamide as Sample Loading Buffer (Sigma-Aldrich, catalog number: F9037 ) or original Sample Loading Buffer (SLS) (Beckman Coulter, catalog number: 608082)
  9. Gel running buffer (see Recipes)

Equipment

  1. Thermal Cycler (Applied Biosystems)
  2. Horizontal Electrophoresis system (Bio-Rad Laboratories, Sub-Cell® Model 96 Cell)
  3. ABI PRISM 3100 Genetic Analyzer or Beckman Coulter CEQTM 8000 Genetic Analysis System
  4. Heating block, bath or oven for restriction analysis
  5. PCR 96-well plate or tubes

Software

  1. GeneMapper® 4.0 Software (Applied Biosystems) or GenomeLabTM GeXP Software (Beckman Coulter)

Procedure

  1. Prepare a PCR mix in a final volume of 15 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 fluorescence labeled (6-FAM, HEX, NED or Cy5, D2, D3) M13 primer, 0.5 U of Taq polymerase and 50 ng of DNA.
  2. 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.
  3. Check 3 µl of the PCR product in an agarose gel (1.5%) stained with ethidium bromide.
  4. Restriction assay. The presence of restriction sites should be previously assessed by comparing the sequences of target samples. The search for the suitable restriction endonuclease might be carried out with the tool SNP2CAPS (http://pgrc.ipk-gatersleben.de/snp2caps/) (Thiel et al., 2004) or using the web tool NEB cutter (http://nc2.neb.com/NEBcutter2/). The prediction of restriction analysis on each fragment with the corresponding enzyme might be performed at the TAIR website (http://www.arabidopsis.org/). Fragments larger than 600 bp should be discarded.
    1. Place 12 µl (remaining PCR mix) of the PCR product into a PCR tube.
    2. Add 1 U of the respective restriction enzyme, together with the specific buffer.
    3. Incubate in a heat block, bath or oven at the adequate temperature, according to the instructions of enzyme suppliers, for 3 h.
  5. Fragment analysis on ABI PRISM 3100 Genetic Analyzer or Beckman Coulter CEQTM8000 Genetic Analysis System.
    a. Mix equal amounts of each restricted product (6-FAM, HEX, NED or Cy5, D2, D3) for multiplexing. M13-tailed products should be usualy 1/20 diluted with HPLC gradient grade water (ROTISOLV®) in a volume of 20 µl.
    b. Make mastermix by mixing HiDi-Formamide and the Rox-Standard for each sample – 14 µl HiDi and 0.04 µl Rox-Standard.
    c. Add 14 µl of mastermixes in each well ABI or Beckman PCR-plate (P/N N8010560) and 1 µl of diluted PCR-product.
    d. Denaturate the DNA plate for 5 min at 96 °C, and after change of buffer/water in tanks of ABI PRISM Analyzer or Beckman Coulter Analysis System, place into appliance.
  6. Data analysis with the corresponding software (GeneMapper® 4.0 Software or GenomeLabTM GeXP Software). Only those fragments which retain the fluorescent label will be detected (Figure 1).


    Figure 1. Schematic illustration of the expected results after analysis on capillary electrophoresis systems using the CAPS fluorescence-based multiplexing protocol. A. Digestion of labeled PCR products from two homozygous cultivars (a/a) and (b/b) and from the corresponding heterozygous F1 generation (a/b). B. Separation of digested products on agarose gel. C. Separation of digested products on capillary electrophoresis systems; only those fragments which are detected on the genetic analyzer are represented.

Notes

  1. This protocol could be efficiently combined with the simultaneous detection of simple sequence repeat (SSR) markers in the same capillary electrophoresis run (Perovic et al., 2013).

Recipes

  1. Gel running buffer
    1x Tris-Borate-EDTA
    89 mM Tris base
    89 mM Boric acid
    2 mM EDTA (pH 8.0)

Acknowledgements

This protocol is adapted from Perovic et al. (2013).

References

  1. Perovic, J., Silvar, C., Koenig, J., Stein, N., Perovic, D. and Ordon, F. (2013). A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems. Mol Breed 32(1): 61-69.
  2. Thiel, T., Kota, R., Grosse, I., Stein, N. and Graner, A. (2004). SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development. Nucleic Acids Res 32(1): e5.

简介

新一代测序技术的最新进展允许以高通量方式检测大量的SNP及其使用。然而,裂解扩增多态性序列(CAPS)仍然作为补充其他高通量方法为SNP基因分型发挥重要作用。因此,非常需要关注于加速这种类型的标记的新方法。使用经典CAPS技术和M13尾引物多重测定的组合开发用于通过限制酶消化分析SNP的无琼脂糖凝胶方案。 PCR产物用通用M13引物进行荧光标记,随后用适当的限制性内切酶消化。在混合不同标记的产物后,在毛细管电泳系统上检测它们。该方法允许在短时间内以整体低成本以多路复用的方式进行成本有效的几种SNP的基因分型。此外,该方法可以有效地与在同一电泳运行中同时检测SSR组合,导致非常适合于基于标记的选择程序,绘图群体的基因分型和遗传多样性测定的程序。

关键字:帽子, SNP, 基于荧光的复用, M13尾巴, 分子标记辅助选择

材料和试剂

  1. DNA(25ng /μl)(例如来自两周龄大麦幼苗的;或根据每个特定生物的PCR实践)
  2. PCR试剂
    1. 将0.5U Taq FIREPol DNA聚合酶(Solis Biodyne FIREPol ,目录 编号:01-01-01000; 任何其他供应商也应该满意) 与相应的10x PCR缓冲液(与FIREPol DNA一起提供) 聚合酶)
    2. 25mM MgCl 2(与FIREPol DNA聚合酶一起提供)
    3. dNTP(各10μM)(Thermo Fisher Scientific,目录号:R0182)
    4. 用通用M13在5'末端尾端特异性引物 尾(1μM)(5'-CACGACGTTGTAAAACGAC-3')(脱盐)(Microsynth)
    5. 反向特异性引物(10μM)(脱盐)(Microsynth)
    6. 荧光(6-FAM,HEX,NED或Cy5,D2,D3)标记的引物 互补序列与M13尾(10μM)(Metabion, Planegg/Steinkirchen)
  3. 限制分析
    1. 限制性内切酶(New England Biolabs或Fermentas)
    2. 1x缓冲液限制性内切酶(New England Biolabs或Fermentas)
  4. 1.5%琼脂糖(Sigma-Aldrich,目录号:A9539)
  5. 溴化乙锭(Roche Diagnostics,目录号:HP46.2)
  6. DNA加样缓冲液3x [6x; 30%(v/v)甘油,0.25%(w/v)溴酚蓝,0.25%(w/v)
  7. 在ABI PRISM 3100遗传分析仪分离凝胶(Applied Biosystems,目录号:4363929)上的分析
    1. ROTISOLV HPLC HPLC梯度级水(Roche Diagnostics,目录号:A511.3)
    2. 作为样品上样缓冲液的HiDi TM TM甲酰胺(Applied Biosystems,目录号:4311320)
    3. GeneScan TM ROX大小标准(Applied Biosystems,目录号:401734)
    4. 96孔半裙板PCR板(Kisker,目录号:G060/H/1E/OA-SS)
  8. 对Beckman Coulter CEQ TM 8000遗传分析系统的分析
    1. GenomeLab TM 分离凝胶LPA I(Beckman Coulter,目录号:608010)
    2. 96孔半裙,分段PCR板(Kisker,目录号:G060/H/1E-SG)
    3. 分离缓冲板,未灭菌(Beckman Coulter,目录号:609844)
    4. GenomeLab TM DNA Size Standard 600(Beckman Coulter,目录号:608095)
    5. GenomeLab TM 分离缓冲液(Beckman Coulter,目录号:608012)
    6. 甲酰胺作为样品上样缓冲液(Sigma-Aldrich,目录号: F9037)或原始样品上样缓冲液(SLS)(Beckman Coulter,目录   编号:608082)
  9. 凝胶运行缓冲液(参见配方)

设备

  1. 热循环仪(Applied Biosystems)
  2. 水平电泳系统(Bio-Rad Laboratories,Sub-Cell Model 96 Cell)
  3. ABI PRISM 3100遗传分析仪或Beckman Coulter CEQ TM 8000遗传分析系统
  4. 加热块,浴缸或烤箱进行限制分析
  5. PCR 96孔板或管

软件

  1. GeneMapper <4.0软件(Applied Biosystems)或GenomeLab GeXP Software(Beckman Coulter)

程序

  1. 准备PCR混合物,最终体积为15μl:1×PCR缓冲液,2.5mM MgCl 2,0.2mM dNTP,0.02μM的尾部正向引物,0.2μM的反向引物   引物,0.18μM荧光标记的(6-FAM,HEX,NED或Cy5,D2, D3)M13引物,0.5U Taq聚合酶和50ng DNA
  2. 运行   PCR程序:94℃5分钟; 12个循环,94℃30秒, 在62℃下30秒(最初12个循环的触底为0.5℃/循环 - 最后退火56℃,剩余35个循环),72℃30秒; 和a   最后延伸步骤在72℃下10分钟
  3. 检查3μl的PCR产物在用溴化乙锭染色的琼脂糖凝胶(1.5%)中。
  4. 限制性测定。 限制性位点的存在应该是 之前通过比较目标样品的序列来评估。的 可以进行合适的限制性内切核酸酶的搜索 使用工具SNP2CAPS( http://pgrc.ipk-gatersleben.de/snp2caps/) (Thiel等人,2004)或使用网络工具NEB刀具 ( http://nc2.neb.com/NEBcutter2/)。限制性分析的预测  可以在每个片段上用相应的酶进行 TAIR网站( http://www.arabidopsis.org/)。片段大于 应舍弃600 bp。
    1. 将12μl(剩余的PCR混合物)的PCR产物放入PCR管中
    2. 加入1U相应的限制酶,连同特定缓冲液
    3. 在加热块,浴或烘箱中在适当温度下孵育, 根据酶供应商的说明书,3小时。
  5. 在ABI PRISM 3100遗传分析仪或Beckman Coulter CEQ TM 8000遗传分析系统上的片段分析。
    一个。混合等量的每种限制产品(6-FAM,HEX,NED或 Cy5,D2,D3)。 M13尾产品应该是1/20  用HPLC梯度级水(ROTISOLV)稀释,体积为20μl μl。
    b。通过混合HiDi-Formamide和Rox-Standard为每个样品制备主混合物 - 14μlHiDi和0.04μlRox-Standard。
    C。在每个孔ABI或Beckman PCR板(P/N N8010560)和1μl稀释的PCR产物中加入14μl主干混合物。
    d。在96℃下将DNA板变性5分钟,然后更换 缓冲液/水在ABI PRISM分析仪或Beckman Coulter分析的罐中 系统,放入设备。
  6. 数据分析与 相应的软件(GeneMapper 4.0 Software或GenomeLab TM GeXP 软件)。只有那些保留荧光标记的片段 (图1)。


    图1.原理图说明 毛细管电泳系统分析后的预期结果 使用CAPS基于荧光的多重协议。 A.消化   来自两个纯合品种( a/a )和 b/b 从相应的杂合F1代( a/b )。 B.分离 的消化产物在琼脂糖凝胶上。 C.消化产物的分离 毛细管电泳系统; 只有那些片段 表示在遗传分析仪上检测的。

笔记

  1. 该方案可以与在同一毛细管电泳运行中同时检测简单序列重复(SSR)标记物有效地组合(Perovic等人,2013)。

食谱

  1. 凝胶运行缓冲液
    1 Tris-Borate-EDTA
    89 mM Tris碱
    89mM硼酸 2mM EDTA(pH8.0)

致谢

该协议改编自Perovic等人(2013)。

参考文献

  1. Perovic,J.,Silvar,C.,Koenig,J.,Stein,N.,Perovic,D。和Ordon,F。 用于毛细管电泳系统上的CAPS基因分型的多功能基于荧光的多重测定法。 a> Mol breed 32(1):61-69。
  2. Thiel,T.,Kota,R.,Grosse,I.,Stein,N.and Graner,A。(2004)。 SNP2CAPS:用于CAPS标记开发的SNP和INDEL分析工具 核 Acids Res 32(1):e5。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Perovic, J., Silvar, C., Perovic, D., Stein, N. and Ordon, F. (2015). Fluorescence-based CAPS Multiplex Genotyping on Capillary Electrophoresis Systems. Bio-protocol 5(10): e1472. DOI: 10.21769/BioProtoc.1472.
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