相关实验视频

Updated: Jun 23, 2025

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

Published on: June 14, 2021

用牛津纳米孔测序扩大DNA异质性评估应用于细胞自由表达模板

Sepehr Hejazi¹, Afrin Ahsan¹, Mohammad Kashani²

¹Chemical and Biological Engineering - Iowa State University, 618 Bissell Rd, Ames, IA 50011.

bioRxiv : the preprint server for biology

|June 19, 2024

概括

牛津纳米孔测序准确量化了来自各种方法的DNA放大错误,如聚合酶链反应 (PCR) 和滚动圆放大 (RCA). 这种方法有助于提高同质蛋白质表达的基因放大精度.

科学领域:

分子生物学分子生物学
基因组学就是基因组学.
生物技术是生物技术.

背景情况:

在各种生物技术应用中,DNA放大是至关重要的.
精确量化放大DNA错误对于可靠的下游应用是必不可少的.
现有的误差量化方法可能耗时或缺乏全面分析.

研究的目的:

评估牛津纳米孔测序作为一种可访问的工具,用于量化放大DNA中的异质性.
为了比较不同DNA放大技术的错误率和类型.
建立一种方法来评估基因放大对无细胞蛋白质表达的准确性.

主要方法:

牛津纳米孔测序被用来分析通过常规聚合酶链反应 (PCR) 增强的DNA,具有不同的聚合酶忠实度 (OneTaq,Phusion,Q5) 和使用Phi29聚合酶的滚动圆放大 (RCA).
还评估了细菌等离子体放大.
在大型序列数据集上进行了错误分布分析,以确定突变类型,概率和位置.
二次验证涉及分析从放大DNA合成的sfGFP光蛋白的辐射光谱.

主要成果:

牛津纳米孔测序使得放大DNA中删除,插入和替代的快速量化成为可能.
在经过测试的PCR方法中,Q5和Phusion聚合酶的错误率是最低的.
易发生错误的PCR与更广泛的发射谱相关,在记者蛋白中达到峰值,证实了DNA错误对蛋白质功能的影响.
该研究确定了每个放大技术的特定错误模式和异质性.

结论:

牛津纳米孔测序为量化DNA放大精度提供了一种强大且易于使用的方法.
这些发现突出了Q5和聚合酶作为DNA放大高保真性选择.
这种技术可以作为评估新型基因放大方法和优化无细胞表达系统的路线图,以实现更均的蛋白质生产.

关键词:

纳米孔测序的测序细胞可以自由表达.基因模板的基因模板突变是一种突变.

更多相关视频

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Published on: October 25, 2018

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Published on: August 24, 2017

相关概念视频

RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

Sanger Sequencing

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...