Primer Extension Reactions for the PCR- based α- complementation Assay
引物延伸反应用于基于PCR的α-互补试验
发布: 2015年06月20日第5卷第12期 DOI: 10.21769/BioProtoc.1509 浏览次数: 10525
评审: Yu ChenChang Ho LeeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2014
Abstract
The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.
Materials and Reagents
- pBSM13+ (Stratagene)
- T3 RNA polymerase (Roche Diagnostics, catalog number: P2083 )
- 10x transcription buffer (Roche Diagnostics, catalog number: P2083 )
- High-fidelity PvuII (PvuII-HF) (New England Biolabs, catalog number: R3151L )
- High-fidelity EcoRI (EcoRI-HF) (New England Biolabs, catalog number: R3101L )
- 10x CutSmart buffer (New England Biolabs, catalog number: R3101L)
- 6x gel loading dye (New England Biolabs, catalog number: R3101L)
- NdeI (New England Biolabs, catalog number: R0111L )
- T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number: M0201L )
- 10x T4 polynucleotide kinase buffer (New England Biolabs, catalog Number: B0201S )
- RNasin (RNase inhibitor) (New England Biolabs, catalog number: M0307L )
- RNase (DNase-free) (Roche Diagnostics, catalog number: 11119915001 )
- RNase-free DNase I (Affymetrix, catalog number: 784111000 )
- Pfu DNA polymerase (Agilent Technologies, catalog number: 600353 )
- 10x Pfu buffer (Agilent Technologies, catalog number: 600353)
- Ribonucleoside triphosphate set (Roche Diagnostics, catalog number: 11277057001 )
- Deoxynucleoside triphosphate (dNTP) (Roche Diagnostics, catalog number: 11969064001 )
- Gamma [γ-32P] ATP (PerkinElmer, catalog number: Blu502A001MC )
- G-25 Macro spin columns (best suited for volumes of 75-150 μl) (Harvard Apparatus, catalog number: 74-3901 )
- RNeasy RNA purification kit (QIAGEN, catalog number: 74104 )
- Phenol: Chloroform: Isoamyl alcohol (25:24:1) (Amresco, catalog number: K169-400ML )
- Ethanol (VWR Lifesciences, catalog number: EM1.00967.4003 )
- 3M Sodium Acetate (Amresco, catalog number: E521-100ML )
- Isopropyl alcohol (J.T.Baker®, catalog number: 9037-03 )
- 40% Acrylamide-Bisacrylamide (19:1) solution (VWR International, catalog number: JT4968-0 )
- 40% Acrylamide-Bisacrylamide (29:1) solution (VWR International, catalog number: JT4968-0 )
- Urea (VWR International, catalog number: 97061-926 )
- Ammonium Persulfate (VWR International, catalog number: 97064-594 )
- HIV Reverse Transcriptase, [purified as described in Hou et al. (2004)]
- Milli-Q quality [RNase, DNase free water (dH2O)]
- DNA oligonucleotides were obtained from Integrated DNA Technologies
- Extension reaction buffer (see Recipes)
- Elution buffer (see Recipes)
- 2x SDS loading buffer (see Recipes)
Equipment
- Eppendorf tubes
- Micropipette
- Petri plates
- Table top centrifuge
- Incubator
- Gel apparatus
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Achuthan, V. and DeStefano, J. J. (2015). Primer Extension Reactions for the PCR- based α- complementation Assay. Bio-protocol 5(12): e1509. DOI: 10.21769/BioProtoc.1509.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 活性
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