使用链特异性引物进行HBV DNA引物延伸分析

Jaesung Jung; Kyongmin Kim

doi:10.21769/BioProtoc.1552

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Peer-reviewed

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

JJ Jaesung Jung

Kyongmin Kim email

发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1552 浏览次数: 7857

评审: Smita NairVarpu MarjomakiAnonymous reviewer(s)

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Cover of Journal of Virology, featuring study using the protocol.

Aug 2014

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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

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

Equipment

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

Software

Fujifilm Image Gauge software (version 4.0)

Procedure

English

中文翻译

文章信息

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如何引用

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.