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DNA PCR Assays for Igh Rearrangement
基于DNA的PCR法分析免疫球蛋白重链(Igh)的重排   

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Abstract

This protocol is used for the detection of immunoglobulin heavy (H) chain rearrangements. This PCR-based assay enables detection of DH-JH recombination in cultured hematopoietic cells (Schlissel et al., 1991; Satoh et al., 2013) [e.g. ES-derived cells (Satoh et al., 2013)].

Materials and Reagents

  1. Mouse spleen cells or ES-derived hematopoietic cells
  2. DNA extraction Kit: PerfectPure DNA Cultured Cell Kit (5 PRIME, catalog number: 2302000 )
  3. 10x PCR Buffer with KCl (Life Technologies, Applied Biosystems®, catalog number: 4338856 )
  4. MgCl2 (Life Technologies, Applied Biosystems®, catalog number: 4338856)
  5. Taq DNA polymerase (Life Technologies, Applied Biosystems®, catalog number: 4338856)
  6. dNTPs (Life Technologies, Applied Biosystems®, catalog number: 4338856)
  7. Primers (FASMAC)
    The sequence of primers are as follows.
    a. DHL(5’): GGAATTCG(AorC)TTTTTGT(CorG)AAGGGATCTACTACTGTG
    b. Mu0(5’): CCGCATGCCAAGGCTAGCCTGAAAGATTACC
    c. J3(3’): GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
  8. Agarose (UltraPureTM Agarose) (Life Technologies, InvitrogenTM, catalog number: 16500-100 )
  9. Ethidium bromide (Wako Pure Chemical Industries, catalog number: 315-90051 )

Equipment

  1. PCR Thermal Cycler (Veriti Thermal Cycler) (Life Technologies, Applied Biosystems®)
  2. Centrifuges (TOMY SEIKO, model: MX-150 )
  3. Electrophoresis apparatus (ADVANCE, Mupid-exU)

Procedure

  1. Genomic DNA was prepared for PCR by lysing mouse spleen cells (1-4 x 104) or ES-derived hematopoietic cells (3-5 x 105) in 75 µl elution solution. See the manufacturer's protocol (http://www.5prime.com/media/3415/perfectpure dna cultured cell manual_5prime_1064553_122010.pdf).
  2. 20 µl PCR reactions contained 5.5 µl template (82.5 ng or less), 10 mM Tris-HCl, 50 mM KCl, 2.0 mM MgCl2, 1 µM primers (25 mers), 200 µM dNTPs and 1 U Taq DNA polymerase.
  3. PCR program
    a. 94 °C – 2 min
    b. 94 °C – 1 min
    c. 60 °C – 1 min
    d. 72 °C – 1.75 min
    e. Repeat steps b-d, 35x
    f. 72 °C – 10 min
  4. Half of each PCR products were electrophoresed on 1% agarose gels, and their amounts were evaluated by staining with ethidium bromide.
  5. DH-JH recombination was detected as amplified fragments of 1,033 bp, 716 bp and 333 bp using a primer DHL(5’) and J3(3’). Germline alleles were detected as an amplified fragment of 1,259 bp using a primer Mu0(5’) and J3(3’).

Results


Figure 1.DNA PCR assays of germline (GL) or DH-JH rearranged Igh chain (DJ) genes were performed with mouse splenocytes. A PCR experiment using a primer DHL(5’) and J3(3’) can detect three types of DH-JH rearrangement (J1, J2, and J3) (Schlissel et al., 1991). All of three bands are present with successful DH-JH rearrangement. However, a J1 band is sometimes undetected in the ethidium bromide-based DNA-band visualization when the amount of template DNA is very small. The size marker was loaded in the left lane.

Acknowledgments

This protocol was adapted from a previously published paper by Schlissel et al. (1991). The representative data shown in the protocol was adapted from Satoh et al. (2013).

References

  1. Satoh, Y., Yokota, T., Sudo, T., Kondo, M., Lai, A., Kincade, P. W., Kouro, T., Iida, R., Kokame, K., Miyata, T., Habuchi, Y., Matsui, K., Tanaka, H., Matsumura, I., Oritani, K., Kohwi-Shigematsu, T. and Kanakura, Y. (2013). The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages. Immunity 38(6): 1105-1115. 
  2. Schlissel, M. S., Corcoran, L. M. and Baltimore, D. (1991). Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J Exp Med 173(3): 711-720. 

简介

该方案用于检测免疫球蛋白重链(H)链重排。 这种基于PCR的测定使得能够在培养的造血细胞中检测D H H -J H亚基重组(Schlissel等人,1991; Satoh et al。,2013)[例如 ES衍生细胞(Satoh et al。,2013)]。

材料和试剂

  1. 小鼠脾细胞或ES衍生的造血细胞
  2. DNA提取试剂盒:PerfectPure DNA Cultured Cell Kit(5 PRIME,目录号:2302000)
  3. 用KCl的10x PCR缓冲液(Life Technologies,Applied Biosystems ,目录号:4338856)
  4. MgCl 2(Life Technologies,Applied Biosystems ,目录号:4338856)
  5. Taq DNA聚合酶(Life Technologies,Applied Biosystems ,目录号:4338856)
  6. dNTP(Life Technologies,Applied Biosystems ,目录号:4338856)
  7. 引物(FASMAC)
    引物的序列如下:
    一个。 D H(5'):GGAATTCG(AorC)TTTTTGT(CorG)AAGGGATCTACTACTGTG b。 Mu0(5'):CCGCATGCCAAGGCTAGCCTGAAAGATTACC
    c。  J3(3'):GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
  8. 琼脂糖(Ultra Technologies)(Life Technologies,Invitrogen TM ,目录号:16500-100)
  9. 溴化乙锭(和光纯药工业,目录号:315-90051)

设备

  1. PCR热循环仪(Veriti Thermal Cycler)(Life Technologies,Applied Biosystems )。
  2. 离心机(TOMY SEIKO,型号:MX-150)
  3. 电泳装置(ADVANCE,Mupid-exU)

程序

  1. 通过在75μl中裂解小鼠脾细胞(1-4×10 4个)或ES衍生的造血细胞(3-5×10 5个)来制备基因组DNA用于PCR 洗脱溶液。 请参阅制造商协议( http://www.5prime.com/media/3415/perfectpure dna culture cell manual_5prime_1064553_122010.pdf )。
  2. 20μlPCR反应物含有5.5μl模板(82.5ng或更少),10mM Tris-HCl,50mM KCl,2.0mM MgCl 2,1μM引物(25mer),200μMdNTP和1μl U Taq DNA聚合酶
  3. PCR程序
    一个。 94℃-2分钟
    b。 94°C - 1分钟
    C。 60°C - 1分钟
    d。 72℃ - 1.75分钟
    e。 重复步骤b-d,35x
    F。 72°C - 10分钟
  4. 将每个PCR产物的一半在1%琼脂糖凝胶上电泳,并通过用溴化乙锭染色评价其量。
  5. 使用引物D H L(5')检测到1033bp,716bp和333bp的扩增片段,其结果显示,使用引物D H )和J3(3')。 使用引物Mu0(5')和J3(3'),将种系等位基因检测为1,259bp的扩增片段。

结果


图1.进行种系(GL)或D H H -J H重排的lgh链(DJ)基因的DNA PCR测定与小鼠脾细胞的比较。使用引物D H(5')和J3(3')的PCR实验可以检测三种类型的D H H H重排(J1,J2和J3)(Schlissel等人,1991)。所有三个条带都具有成功的D H H -J H重排。然而,当模板DNA的量非常小时,在基于溴化乙锭的DNA条带可视化中有时未检测到J1条带。将大小标记物装载在左泳道中。

致谢

该方案改编自Schlissel等人先前发表的论文(1991)。方案中所示的代表性数据来自Satoh et al。(2013)。

参考文献

  1. Satoh,Y.,Yokota,T.,Sudo,T.,Kondo,M.,Lai,A.,Kincade,PW,Kouro,T.,Iida,R.,Kokame,K.,Miyata,T.,Habuchi ,Y.,Matsui,K.,Tanaka,H.,Matsumura,I.,Oritani,K.,Kohwi-Shigematsu,T.and Kanakura,Y。 Satb1蛋白指导造血干细胞向淋巴谱系分化。 免疫 38(6):1105-1115。 
  2. Schlissel,M.S.,Corcoran,L.M。和Baltimore,D。(1991)。 病毒转化的前B细胞显示有序活化,但不会失活免疫球蛋白基因重排和转录 。 J Exp Med 173(3):711-720。 
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Satoh, Y., Sudo, T. and Yokota, T. (2014). DNA PCR Assays for Igh Rearrangement. Bio-protocol 4(4): e1046. DOI: 10.21769/BioProtoc.1046.
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