Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A.
Ribonuclease A (RNase A) is widely used in molecular biology research both for analytical assays and for nucleic acid preparation. The catalytic mechanism of RNase A is well understood and absolutely precludes activity on DNA; however anecdotal reports of DNA degradation by RNase A are not uncommon....
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0115008 |
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| author | Federico Donà Jonathan Houseley |
| author_facet | Federico Donà Jonathan Houseley |
| author_sort | Federico Donà |
| collection | DOAJ |
| description | Ribonuclease A (RNase A) is widely used in molecular biology research both for analytical assays and for nucleic acid preparation. The catalytic mechanism of RNase A is well understood and absolutely precludes activity on DNA; however anecdotal reports of DNA degradation by RNase A are not uncommon. Here we describe a mechanism by which RNase A treatment can lead to apparent DNA degradation. This results from the surprising finding that RNase A remains functional in a phenol:chloroform mixture, to our knowledge the only enzyme that survives this highly denaturing solvent environment. Although RNase A does not cleave the DNA backbone it is capable of binding to DNA, forming stable RNase A-DNA complexes that partition to the interphase or organic phase during phenol:chloroform purification. The unexpected survival of the RNase A DNA-binding activity in phenol means that these complexes are not dissolved and a substantial amount of RNase A-bound DNA is permanently removed from the aqueous phase and lost on phase separation. This effect will impact DNA recovery from multiple procedures and is likely to represent a source of sequence bias in genome-wide studies. Our results also indicate that the results of analytical studies performed using RNase A must be considered with care. |
| format | Article |
| id | doaj-art-9f24e3cb01184c2d9556cb6ab5d3acd4 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-9f24e3cb01184c2d9556cb6ab5d3acd42025-08-20T02:22:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01912e11500810.1371/journal.pone.0115008Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A.Federico DonàJonathan HouseleyRibonuclease A (RNase A) is widely used in molecular biology research both for analytical assays and for nucleic acid preparation. The catalytic mechanism of RNase A is well understood and absolutely precludes activity on DNA; however anecdotal reports of DNA degradation by RNase A are not uncommon. Here we describe a mechanism by which RNase A treatment can lead to apparent DNA degradation. This results from the surprising finding that RNase A remains functional in a phenol:chloroform mixture, to our knowledge the only enzyme that survives this highly denaturing solvent environment. Although RNase A does not cleave the DNA backbone it is capable of binding to DNA, forming stable RNase A-DNA complexes that partition to the interphase or organic phase during phenol:chloroform purification. The unexpected survival of the RNase A DNA-binding activity in phenol means that these complexes are not dissolved and a substantial amount of RNase A-bound DNA is permanently removed from the aqueous phase and lost on phase separation. This effect will impact DNA recovery from multiple procedures and is likely to represent a source of sequence bias in genome-wide studies. Our results also indicate that the results of analytical studies performed using RNase A must be considered with care.https://doi.org/10.1371/journal.pone.0115008 |
| spellingShingle | Federico Donà Jonathan Houseley Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A. PLoS ONE |
| title | Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A. |
| title_full | Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A. |
| title_fullStr | Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A. |
| title_full_unstemmed | Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A. |
| title_short | Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A. |
| title_sort | unexpected dna loss mediated by the dna binding activity of ribonuclease a |
| url | https://doi.org/10.1371/journal.pone.0115008 |
| work_keys_str_mv | AT federicodona unexpecteddnalossmediatedbythednabindingactivityofribonucleasea AT jonathanhouseley unexpecteddnalossmediatedbythednabindingactivityofribonucleasea |