Literature DB >> 35127994

Measuring Oligonucleotide Hydrolysis in Cellular Lysates via Viscosity Measurements.

Romel Menacho-Melgar1, Michael D Lynch1.   

Abstract

Cell lysis, a process that releases host oligonucleotides, is required in many biotechnological applications. However, intact oligonucleotides in crude cellular lysates increase the viscosity of lysates, which complicates downstream processes and routine laboratory workflows. To address this, nucleases that hydrolyze the intact oligonucleotides are commonly added, either as purified enzymes or co-expressed in genetically engineered bacterial strains. To measure oligonucleotide hydrolysis, common DNA quantification methods, such as qPCR or fluorescence-based, require expensive reagents and equipment, and cannot distinguish different-sized DNA fragments. Here, we outline a simple alternative method for measuring DNA/RNA hydrolysis in cellular lysates, by measuring their viscosity. This method only requires common laboratory supplies and a cell phone camera.
Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.

Entities:  

Keywords:  Autohydrolysis; Cell lysate; DNA content; Hydrolysis quantification; Nucleotide hydrolysis; Viscosity

Year:  2022        PMID: 35127994      PMCID: PMC8799667          DOI: 10.21769/BioProtoc.4304

Source DB:  PubMed          Journal:  Bio Protoc        ISSN: 2331-8325


  5 in total

Review 1.  Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks.

Authors:  D M Prazeres; G N Ferreira; G A Monteiro; C L Cooney; J M Cabral
Journal:  Trends Biotechnol       Date:  1999-04       Impact factor: 19.536

2.  Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease.

Authors:  Romel Menacho-Melgar; Eirik A Moreb; John P Efromson; Tian Yang; Jennifer N Hennigan; Ruixin Wang; Michael D Lynch
Journal:  Biotechnol Bioeng       Date:  2020-05-28       Impact factor: 4.530

3.  Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida.

Authors:  Z L Boynton; J J Koon; E M Brennan; J D Clouart; D M Horowitz; T U Gerngross; G W Huisman
Journal:  Appl Environ Microbiol       Date:  1999-04       Impact factor: 4.792

4.  Common artifacts and mistakes made in electrophoresis.

Authors:  Biji T Kurien; R Hal Scofield
Journal:  Methods Mol Biol       Date:  2012

5.  Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.

Authors:  Yuki Nakayama; Hiromi Yamaguchi; Naoki Einaga; Mariko Esumi
Journal:  PLoS One       Date:  2016-03-03       Impact factor: 3.240

  5 in total

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