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Primer Extension Analysis of HBV DNA with Strand-Specific Primers
使用链特异性引物进行HBV DNA引物延伸分析   

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Abstract

We performed primer extension assay to determine which steps of HBV DNA synthesis (i.e., minus- and plus-strand DNA synthesis and circularization of RC DNA) are affected by phosphoacceptor site mutations in C protein. In these experiments, we used several specific oligonucleotide primers. For quantitation, the level of extended DNA (ED) was normalized to the level of a single internal standard (IS) DNA.

Materials and Reagents

  1. Huh7 hepatoma cells (Japanese Collection of Research Bioresources Cell Bank, catalog number: JCRB0403 )
  2. Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco®, catalog number: 12800-017 )
  3. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
  4. Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  5. OptiMEM (Life Technologies, Gibco®, catalog number: 31985-062 )
  6. 500 μl Opti-MEM (Life Technologies, Gibco®, catalog number: 31985-062 )
  7. PEG (USB, catalog number: 19959 )
  8. NaCl (Sigma-Aldrich, catalog number: S3014 )
  9. EDTA (Sigma-Aldrich, catalog number: E5134 )
  10. Polyethylenimine (Polysciences, catalog number: 23966 )
  11. Vent Exo (-) polymerase (New England Biolabs, catalog number: M0257S )
  12. Micrococcal nuclease 1 μl (45 unit/μl) (Worthington Biochemical, I.U.B.: 3.1.31.1, catalog number: LS004798 )
  13. γ -32P-ATP (PerkinElmer Inc., catalog number: NEG035C )
  14. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201s )
  15. Internal standard (IS) DNA (from HBV WT DNA Sac II/Xho I digested fragment) 1ng/1μl
  16. 2.5 mM dNTP mixture (Takara Bio Company, catalog number: BH7901 )
  17. RNase A (Fermentas, catalog number: EN05331 )
  18. Tris-HCl (pH 8.8) (Sigma-Aldrich, catalog number: T6066 )
  19. (NH4)2SO4 (Sigma-Aldrich, catalog number: T6066)
  20. KCl (Sigma-Aldrich, catalog number: P9541 )
  21. MgSO4 (Sigma-Aldrich, catalog number: 230391 )
  22. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  23. Polyacrylamide (SERVA Electrophoresis GmbH, catalog number: 10687 )
  24. UREA (Duksan Hi-Metal, catalog number: CAS 57-13-6 )
  25. APS (Sigma-Aldrich, catalog number: A3678 )
  26. TEMED (Sigma-Aldrich, catalog number: T9281 )
  27. Boric Acid (Sigma-Aldrich, catalog number: B0394 )
  28. EDTA (Sigma-Aldrich, catalog number: E5134)
  29. 1x DNA-containing reaction buffer (see Recipes)
  30. 5% polyacrylamide gel (see Recipes)
  31. 5x TBE (see Recipes)

Equipment

  1. 10 cm dishes (Corning Incorporated, catalog number: 430167 )

Software

  1. Fujifilm Image Gauge software (version 4.0)

Procedure

  1. Huh7 hepatoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under a humidified atmosphere at 37 °C in 5% CO2.
  2. Cells were passaged every three days. 2 x 106 of Huh7 cells were seeded in 10 cm dish, one day before the transfection.
  3. Next day, cells were (co)-transfected using polyethylenimine (PEI).
    PEI transfection method:
    1. In a sterile tube, total 10 μg of plasmid DNA (5 μg of phosphoacceptor site mutant and 5 μg of P-deficient mutant) was mixed with 500 μl Opti-MEM.
    2. Add 30 μl of PEI solution (1 μg/1 μl) to DNA-Opti-MEM solution and then vortex immediately.
    3. Incubate 15 min at room temperature.
    4. Then add PEI/DNA-Opti-MEM mixture to cells.
  4. Transfection experiments were repeated at least three times.
  5. Cytoplasmic core particles were prepared as previously described (Kim et al., 2004).
    Three days after transfection, cells were used for core particle preparation.
    Cytoplasmic core particle preparations:
    1. Discard medium and wash with 10 ml PBS.
    2. Add PBS 1 ml, scrape the cells, and transfer to 1.5 ml tube.
    3. Spin down at 13,000 rpm for 10 sec and discard supernatant.
    4. Add 1 ml lysis buffer, vortex, and then incubate on ice for 10 min.
    5. Spin down at 13,500 rpm for 2 min at 4 °C.
    6. Transfer supernatant to fresh 1.5 ml tube.
    7. Add micrococcal nuclease 1 μl (45 unit/μl), 1 M MgCl2 10 μl (final 10 mM), 1 M CaCl2 8 μl (final 8 mM).
    8. Incubate 37 °C, 1 h.
    9. Add 40% PEG 250 μl (26%), 5 M NaCl 100 μl (1.4 M), 0.5 M EDTA 118 μl (40 mM).
    10. Put them in ice for 1 h.
    11. Spin down at 13,500 rpm at 4 °C for 15 min.
    12. Dissolve pellet in 20 μl nuclease free distilled water.
  6. To analyze HBV DNA synthesis by primer extension analysis, HBV DNA was extracted from isolated core particles (Kim et al., 2004).
    Prior to primer extension analysis, HBV DNA synthesis was analyzed by Southern blotting to see the levels of relaxed circular, double-stranded linear, and single-stranded DNAs (Jung et al., 2014) (Figure 1).


    Figure 1. HBV DNA synthesis in core particles formed by STSSSS (WT) and mutant C proteins. To examine HBV DNA synthesis in core particles formed by phosphoacceptor site mutant C proteins, phosphoacceptor site mutant and P-deficient mutant were co-transfected into Huh7 cells. HBV DNA was extracted from isolated core particles (Kim et al., 2004), and Southern blot analysis was performed (Jung et al., 2014). Replicative-intermediate DNAs, relaxed circular (RC), double-stranded linear (DL), and single-stranded (SS) DNAs, are indicated.

  7. Oligonucleotide DNA primers were 5’-end–labeled with 30 μCi γ-32P-ATP at 37 °C for 3 h using T4 polynucleotide kinase.
    Labeling method
    Primer 10 pmole/ul
    2 μl
    γ-32P-ATP
    3 μl
    D.W
    3 μl
    10x T4 TNK buffer
    1 μl
    T4 polynucleotide kinase
    1 μl
    The 5’-end–labeled primers HBV1665+ (5’-CTCTTGGACTCTCAGCAATGTCAAC-3’), HBV1744- (5’-CAGCTCCTCCCAGTCCTTAAACA-3’), and HBV1952- (5’- GAGAGTAACTCCACAGTAGCTCC -3’) were used to measure the levels of the elongated minus-strand, plus-strand, and circularized RC DNAs, respectively.
    HBV DNA (total 20 μl) extracted from core particles isolated from co-transfected Huh7 cells in 10-cm dishes was divided into four batches (5 μl): One batch for Southern blotting and three batches for primer extensions to measure minus-strand, plus-strand, and circularized RC DNAs.
    For primer extension analyses of each C protein variant, 5ul viral DNA were heated to 95 °C for 5 min, treated with 1 U RNase A at 37 °C for 1 h, ethanol precipitated [ethanol precipitation: To 5 μl extracted DNA, add 195 μl distilled water and 20 μl 3 M sodium acetate, and mix by vortexing briefly. Add 440 μl 100% ethanol (molecular grade), vortex, and keep them overnight at -20 °C. Precipitate DNA by conventional method] and resuspended in distilled water (5 μl). End-labeled primers were extended with Vent Exo (-) polymerase, yielding products that annealed to the respective complementary HBV DNA sequences (Figure 2).
    Viral DNA
    5 μl
    IS DNA (1 ng/μl)
    1 μl
    D.W
    7 μl
    DNA-containing reaction buffer
    2 μl
    2.5 mM dNTP
    2 μl
    Vent (-)
    1 μl
    Labeled primer
    2 μl
    Total
    20 μl
    Vortex and spin down
    95 °C 1 min
    95 °C 30 sec
    60 °C 30 sec 20 cycles
    72 °C 30 sec
    72 °C 1 min


    Figure 2. Schematic representation of oligonucleotides used for primer extension analysis. Minus- and plus-strand DNA elongation and circularization were detected using 32P-end-labeled HBV1665+, HBV1744-, and HBV1952-, respectively.

  8. 5 μl from 20 μl of the extended products were electrophoresed through 5% polyacrylamide gels containing 8 M urea (1,000 voltage 6 h). Gel was dried in gel dryer about 30 min at 60 °C. Dried gels were subjected to autoradiography (Figure 3), and relative levels of radioactivity were measured using the Fujifilm Image Gauge software, version 4.0.

Representative data



Figure 3. Minus-DNA elongation was detected using 32P-end-labeled HBV1665+, showing that T162A exhibited the reduced minus-strand DNA elongation

Recipes

  1. Lysis buffer
    10 mM Tris (pH 8.0)
    1 mM EDTA
    0.2% NP40
    50 mM NaCl
  2. 1x DNA-containing reaction buffer
    20 mM Tris-HCl (pH 8.8)
    10 mM (NH4)2SO4
    10 mM KCl
    2 mM MgSO4
    0.1% Triton X-100
  3. 5% polyacrylamide gel
    30% polyacrylamide
    5.2 ml
    5x TBE
    6 ml
    UREA
    14.4 g
    Add distilled water up to total 30 ml
    10% AP
    120 μl
    TEMED
    30 μl
  4. 5x TBE
    Tris-Hcl
    54 g
    Boric acid
    28.5 g
    0.5 M EDTA
    20 ml
    Add distilled water up 1 L
    Total
    1,000 ml

Acknowledgments

This work was supported by National Research Foundation Grants funded by the Korean Government (NRF-2012-R1A2A2A01015370). We performed primer extension analysis by a previously described method (Lewellyn and Loeb, 2011), with minor modifications.

References

  1. Kim, H. Y., Park, G. S., Kim, E. G., Kang, S. H., Shin, H. J., Park, S. and Kim, K. H. (2004). Oligomer synthesis by priming deficient polymerase in hepatitis B virus core particle. Virology 322(1): 22-30.
  2. Jung, J., Hwang, S. G., Chwae, Y. J., Park, S., Shin, H. J. and Kim, K. (2014). Phosphoacceptors threonine 162 and serines 170 and 178 within the carboxyl-terminal RRRS/T motif of the hepatitis B virus core protein make multiple contributions to hepatitis B virus replication. J Virol 88(16): 8754-8767.
  3. Lewellyn, E. B. and Loeb, D. D. (2011). The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication. J Virol 85(3): 1298-1309.

简介

我们进行引物延伸测定以确定HBV DNA合成的哪些步骤(即,负链和正链DNA合成和RC DNA的环化)受C蛋白中的磷酸受体位点突变的影响。 在这些实验中,我们使用几种特异性寡核苷酸引物。 为了定量,将延伸DNA(ED)的水平标准化为单个内标(IS)DNA的水平。

材料和试剂

  1. Huh7肝癌细胞(Japanese Collection of Research Bioresources Cell Bank,目录号:JCRB0403)
  2. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:12800-017)
  3. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:16000-044)
  4. 青霉素/链霉素(Life Technologies,Gibco ,目录号:15140-122)
  5. OptiMEM(Life Technologies,Gibco ,目录号:31985-062)
  6. 500μlOpti-MEM(Life Technologies,Gibco ,目录号:31985-062)
  7. PEG(USB,目录号:19959)
  8. NaCl(Sigma-Aldrich,目录号:S3014)
  9. EDTA(Sigma-Aldrich,目录号:E5134)
  10. 聚乙烯亚胺(Polysciences,目录号:23966)
  11. Vent Exo( - )聚合酶(New England Biolabs,目录号:M0257S)
  12. 微球菌核酸酶1μl(45单位/μl)(Worthington Biochemical,I.U.B .: 3.1.31.1,目录号:LS004798)
  13. γ-32 P-ATP(PerkinElmer Inc.,目录号:NEG035C)
  14. T4多核苷酸激酶(New England Biolabs,目录号:M0201s)
  15. 内标(IS)DNA(来自HBV WT DNA Sac II/Xho I消化的片段)1ng /1μl
  16. 2.5mM dNTP混合物(Takara Bio公司,目录号:BH7901)
  17. RNase A(Fermentas,目录号:EN05331)
  18. Tris-HCl(pH8.8)(Sigma-Aldrich,目录号:T6066)
  19. (NH 4)2 SO 4(Sigma-Aldrich,目录号:T6066)
  20. KCl(Sigma-Aldrich,目录号:P9541)
  21. MgSO 4(Sigma-Aldrich,目录号:230391)
  22. Triton X-100(Sigma-Aldrich,目录号:T8787)
  23. 聚丙烯酰胺(SERVA Electrophoresis GmbH,目录号:10687)
  24. UREA(Duksan Hi-Metal,目录号:CAS 57-13-6)
  25. APS(Sigma-Aldrich,目录号:A3678)
  26. TEMED(Sigma-Aldrich,目录号:T9281)
  27. 硼酸(Sigma-Aldrich,目录号:B0394)
  28. EDTA(Sigma-Aldrich,目录号:E5134)
  29. 1×含DNA反应缓冲液(见配方)
  30. 5%聚丙烯酰胺凝胶(见配方)
  31. 5x TBE(请参阅配方)

设备

  1. 10cm培养皿(Corning Incorporated,目录号:430167)

软件

  1. Fujifilm Image Gauge软件(4.0版)

程序

  1. Huh7肝癌细胞在补充有10%胎牛血清和1%青霉素/链霉素的Dulbecco改良的Eagle's培养基(DMEM)中,在潮湿气氛下,在37℃,5%CO 2中生长。
  2. 细胞每三天传代。 在转染前一天,将2×10 6个 Huh7细胞接种在10cm皿中。
  3. 第二天,使用聚乙烯亚胺(PEI)(共)转染细胞。
    PEI转染方法:
    1. 在无菌试管中,总共10μg质粒DNA(5μg磷酸受体 位点突变体和5μgP缺陷突变体)与500μl混合 Opti-MEM。
    2. 加入30微升PEI溶液(1微克/1微升)到DNA-Opti-MEM溶液,然后立即涡旋。
    3. 在室温下孵育15分钟。
    4. 然后向细胞中加入PEI/DNA-Opti-MEM混合物。
  4. 将转染实验重复至少三次。
  5. 如先前所述(Kim等人,2004)制备细胞质核心颗粒。
    转染后三天,将细胞用于核心颗粒制备 细胞质核颗粒制剂:
    1. 弃去培养基并用10ml PBS洗涤。
    2. 加入PBS 1ml,刮细胞,并转移到1.5ml管
    3. 在13,000rpm下旋转10秒并弃去上清液
    4. 加入1ml裂解缓冲液,涡旋,然后在冰上孵育10分钟
    5. 在4℃下以13,500rpm旋转2分钟。
    6. 将上清液转移到新鲜的1.5ml管中
    7. 加入微球菌核酸酶1μl(45单位/μl),1M MgCl 210μl(最终10mM),1M CaCl 28μl(最终8mM)。
    8. 孵育37℃,1小时
    9. 加入40%PEG250μl(26%),5M NaCl100μl(1.4M),0.5M EDTA118μl(40mM)。
    10. 将它们放在冰上1小时。
    11. 在4℃下以13,500rpm旋转15分钟
    12. 将沉淀溶于20μl无核酸酶的蒸馏水中。
  6. 为了通过引物延伸分析分析HBV DNA合成,从分离的核心颗粒中提取HBV DNA(Kim等人,2004)。
    在引物延伸分析之前,通过Southern印迹分析HBV DNA合成,以观察松弛的环状,双链线性和单链DNA的水平(Jung等人,2014)(图1 )。


    图1.由STSSSS(WT)和突变体C蛋白形成的核心颗粒中的HBV DNA合成为了检查由磷酸受体位点突变体C蛋白形成的核心颗粒中的HBV DNA合成,磷酸受体位点突变体和P缺陷型突变体共转染Huh7细胞。从分离的核心颗粒提取HBV DNA(Kim等人,2004),并进行Southern印迹分析(Jung等人,2014)。指示了复制中间体DNA,松弛环(RC),双链线性(DL)和单链(SS)DNA。

  7. 使用T4多核苷酸激酶,寡核苷酸DNA引物在37℃用30μCiγ-32 P-ATP进行5'-末端标记3小时。
    标签方法
    引物10pmole/ul
    2微升
    γ- 32 P-ATP
    3微升
    D.W
    3微升
    10x T4 TNK缓冲区
    1微升
    T4多核苷酸激酶
    1微升
    使用5'末端标记的引物HBV1665 +(5'-CTCTTGGACTCTCAGCAATGTCAAC-3'),HBV1744-(5'-CAGCTCCTCCCAGTCCTTAAACA-3')和HBV1952-(5'-GAGAGTAACTCCACAGTAGCTCC-3'细长的负链,正链和环化的RC DNA。
    将从在10cm皿中从共转染的Huh7细胞分离的核心颗粒提取的HBV DNA(总共20μl)分成四批(5μl):一批用于Southern印迹,三批用于引物延伸以测量负链,正链和环化RC DNA 对于每个C蛋白变体的引物延伸分析,将5ul病毒DNA加热至95℃5分钟,用1U RNA酶A在37℃处理1小时,乙醇沉淀[乙醇沉淀:向5μl提取的DNA中加入195μl蒸馏水和20μl3M乙酸钠,并通过短暂涡旋混合。加入440μl100%乙醇(分子级),涡旋,并保持在-20℃过夜。通过常规方法沉淀DNA]并重悬于蒸馏水(5μl)中。末端标记的引物用Vent Exo( - )聚合酶延伸,产生退火到相应的互补HBV DNA序列的产物(图2)。
    病毒DNA
    5微升
    IS DNA(1ng /μl)
    1微升
    D.W
    7微升
    含DNA的反应缓冲液
    2微升
    2.5mM dNTP 2微升
    通风口( - )
    1微升
    标签引物
    2微升
    总计
    20微升
    旋转和旋转
    95°C 1分钟
    95°C 30秒
    60℃30秒20个循环
    72°C 30秒
    72℃1分钟


    图2.用于引物延伸分析的寡核苷酸的示意图。 使用 P端标记的HBV1665 +,HBV1744-和HBV1952-检测小核和正链DNA延伸和环化。

  8. 将5μl来自20μl延伸产物的电泳通过含有8M尿素的5%聚丙烯酰胺凝胶(1,000电压6小时)。 凝胶在凝胶干燥器中在60℃下干燥约30分钟。 干凝胶进行放射自显影(图3),并使用Fujifilm Image Gauge软件4.0版测量相对的放射性水平。

代表数据



图3.使用pAd末端标记的HBV1665 +检测到了减数DNA延伸,显示T162A显示减少的负链DNA伸长

食谱

  1. 裂解缓冲液
    10mM Tris(pH8.0) 1mM EDTA
    0.2%NP40
    50mM NaCl
  2. 1×含DNA反应缓冲液
    20mM Tris-HCl(pH8.8)
    10mM(NH 4)2 SO 4 4
    10 mM KCl
    2mM MgSO 4 0.1%Triton X-100
  3. 5%聚丙烯酰胺凝胶
    30%聚丙烯酰胺 5.2 ml
    5x TBE
    6毫升
    UREA
    14.4 g
    加蒸馏水至总量30 ml
    10%AP
    120微升
    TEMED
    30微升
  4. 5x TBE
    Tris-Hcl
    54克
    硼酸
    28.5克
    0.5 M EDTA
    20ml
    加入蒸馏水1 L
    总计
    1000 ml

致谢

这项工作得到了由韩国政府资助的国家研究基金会资助(NRF-2012-R1A2A2A01015370)的支持。 我们通过先前描述的方法(Lewellyn和Loeb,2011)进行引物延伸分析,具有微小的修改。

参考文献

  1. Kim,H.Y.,Park,G.S.,Kim,E.G.,Kang,S.H.,Shin,H.J.,Park,S。和Kim,K.H。(2004)。 通过在乙型肝炎病毒核心颗粒中引发缺陷型聚合酶来合成寡聚体。 病毒学 322(1):22-30。
  2. Jung,J.,Hwang,S.G.,Chwae,Y.J.,Park,S.,Shin,H.J。和Kim,K。(2014)。 磷酸受体苏氨酸162和丝氨酸170和178在乙型肝炎的羧基末端RRRS/T基序内病毒核心蛋白对乙型肝炎病毒复制有多重贡献。病毒核心蛋白对乙型肝炎病毒复制有多重贡献。 88(16):8754-8767。
  3. Lewellyn,E.B。和Loeb,D.D。(2011)。 乙型肝炎病毒核心蛋白的羧基末端结构域的精氨酸簇使多方向的贡献基因组复制。 J Virol 85(3):1298-1309
  • English
  • 中文翻译
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jung, J. and Kim, K. (2015). Primer Extension Analysis of HBV DNA with Strand-Specific Primers. Bio-protocol 5(15): e1552. DOI: 10.21769/BioProtoc.1552.
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