Effects of Lyse-It on endonuclease fragmentation, function and activity.

Tonya M Santaus,Shan Li,Fan Zhang,Chris D Geddes,O Colin Stine

doi:10.1371/journal.pone.0223008

Abstract

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.

Highlights

Ribonuclease A (RNase A), Ribonuclease B (RNase B), and Deoxyribonuclease I (DNase I) are three types of stable endonucleases that can contaminate RNA and DNA samples and effectively cleave a wide variety of genomic DNA and RNA prior to being detected by methods like quantitative polymerase chain reaction
Prior to studying the effects of lysing on nuclease activity, it was imperative to investigate nuclease size band intensity via SDS polyacrylamide gel electrophoresis (PAGE) to ascertain if there were any affects due to buffer at 60 ̊C or an increase in temperature. 60 ̊C was used as the conventional heating temperature because of the microwave irradiation power and times used, which generated an average upper sample temperature of 60 ̊C ± 5 ̊C
RNase B was conventionally heated at varying temperatures to determine if an increasing temperature reduced the ability of the Oriole stain to bind to the nuclease (S3B Fig)

Summary

Introduction

Ribonuclease A (RNase A), Ribonuclease B (RNase B), and Deoxyribonuclease I (DNase I) are three types of stable endonucleases that can contaminate RNA and DNA samples and effectively cleave a wide variety of genomic DNA and RNA prior to being detected by methods like quantitative polymerase chain reaction (qPCR). Ribonuclease A is a small 13.7 kDa protein with 124 amino acid residues, including eight cysteines that create four crosslinking disulfide bonds. The presence of the disulfide bonds, in addition to the protein’s ability to refold, lends to its immense stability. RNase A hydrolyzes the phosphodiester bond of a nucleoside 2’3’cyclic phosphodiester of both single and double-stranded RNA.[1, 2] Like RNase A, RNase B has an identical amino acid sequence to RNase A, but it is glycosylated by an N-linked oligosaccharide chain at Asn34.[3] The carbohydrate chain extends outwards from the protein in to the cytosol and can be hydrated. RNase B has the same catalytic ability as A, but it is slightly

Methods

Results

Conclusion