qSanger:通过Sanger测序对细菌培养物中的遗传变异进行量化。

Q2 Agricultural and Biological Sciences 生物设计研究(英文) Pub Date : 2023-02-07 eCollection Date: 2023-01-01 DOI:10.34133/bdr.0007
Satya Prakash, Adrian Racovita, Teresa Petrucci, Roberto Galizi, Alfonso Jaramillo
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引用次数: 1

摘要

基因变异,如突变和重组,在所有培养的生物体中都会自发产生。尽管可以通过选择或反选择来识别非中性突变,但在异质群体中识别中性突变通常需要昂贵且耗时的方法,如定量或液滴聚合酶链式反应和高通量测序。在不断变化的环境条件下,中性突变甚至可能成为优势,强制进行短暂选择或反选择。我们提出了一种新的方法,我们称之为qSanger,使用混合Sanger测序读数中排列的电泳图谱峰的振幅比来量化DNA。通过定量聚合酶链反应和荧光定量,使用表达增强型绿色荧光蛋白和mCherry荧光标记的质粒在体外和共转化的大肠杆菌中验证qSanger。我们表明,qSanger允许从混合Sanger测序读数中量化遗传变异,包括单碱基天然多态性或从头突变,与经典方法相比,大大减少了人工和成本。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing.

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.

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CiteScore
3.90
自引率
0.00%
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0
审稿时长
12 weeks
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