错配引物延伸法

Vasudevan Achuthan; Jeffrey J. DeStefano

doi:10.21769/BioProtoc.1508

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

Mismatched Primer Extension Assays

错配引物延伸法

Vasudevan Achuthan

JD Jeffrey J. DeStefano email

发布: 2015年06月20日第5卷第12期 DOI: 10.21769/BioProtoc.1508 浏览次数: 8271

评审: Yu ChenChang Ho LeeAnonymous reviewer(s)

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

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

Aug 2014

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

引物延伸反应用于基于PCR的α-互补试验

Vasudevan Achuthan and Jeffrey J. DeStefano

2015年06月20日 11468 阅读

基于Intein的生物传感器用于体内蛋白稳定性监测

John S. Smetana [...] Christopher W. Lennon

2025年04月20日 1781 阅读

基于Azo-Xylan及其变体的Endo-1,4-β-D-木聚糖酶活性检测方法

Luca Bombardi [...] Salvatore Fusco

2025年04月20日 2153 阅读

Abstract

Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg²⁺ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg²⁺. The relative fidelity of the polymerase can then be estimated using the standard extension efficiency. The assay described here is based on the method published in Mendelman et al. (1990).

Materials and Reagents

Deoxynucleoside triphosphate (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 )
40% Acrylamide-Bisacrylamide (19:1) solution (VWR International, catalog number: JT4969-0 )
T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number: M0201L )
10X T4 polynucleotide kinase buffer (New England Biolabs, catalog number: B0201S )
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)
DNA oligonucleotides from Integrated DNA Technologies
1. Template:
  5'-GGGCGAATTTAG[G/C]TTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTG G-3’. The underlined nucleotides in brackets indicate that templates with either a G or C at this position can be used depending on the type of mismatch examined.
2. Primer:
  5ʹ-TAACCCTCACTAAAGGGAACAAAAX-3ʹ. “X” at the 3ʹ end of the primer denotes A, T, or C depending on the mismatch examined. “X” in the case of a matched primer is G.
1 M MgCl₂
Extension reaction buffer (see Recipes)
2x loading dye (see Recipes)