Simple cDNA normalization using kamchatka crab duplex-specific nuclease
We developed a novel simple cDNA normalization method [termed duplex‐specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full‐length cDNA sequences. DSN normalization involves the denaturation–reassociation of cDNA, degradation of the double‐stranded (ds) fr...
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fthighwire:oai:open-archive.highwire.org:nar:32/3/e37 2023-05-15T16:58:52+02:00 Simple cDNA normalization using kamchatka crab duplex-specific nuclease Zhulidov, Pavel A. Bogdanova, Ekaterina A. Shcheglov, Alex S. Vagner, Laura L. Khaspekov, George L. Kozhemyako, Valery B. Matz, Mikhail V. Meleshkevitch, Ella Moroz, Leonid L. Lukyanov, Sergey A. Shagin, Dmitry A. 2004-02-18 00:00:00.0 text/html http://nar.oxfordjournals.org/cgi/content/short/32/3/e37 https://doi.org/10.1093/nar/gnh031 en eng Oxford University Press http://nar.oxfordjournals.org/cgi/content/short/32/3/e37 http://dx.doi.org/10.1093/nar/gnh031 Copyright (C) 2004, Oxford University Press NAR Methods Online TEXT 2004 fthighwire https://doi.org/10.1093/nar/gnh031 2013-05-26T15:37:46Z We developed a novel simple cDNA normalization method [termed duplex‐specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full‐length cDNA sequences. DSN normalization involves the denaturation–reassociation of cDNA, degradation of the double‐stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single‐stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA–RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first‐strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique. Text Kamchatka Kamchatka crab HighWire Press (Stanford University) Nucleic Acids Research 32 3 37e 37 |
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NAR Methods Online Zhulidov, Pavel A. Bogdanova, Ekaterina A. Shcheglov, Alex S. Vagner, Laura L. Khaspekov, George L. Kozhemyako, Valery B. Matz, Mikhail V. Meleshkevitch, Ella Moroz, Leonid L. Lukyanov, Sergey A. Shagin, Dmitry A. Simple cDNA normalization using kamchatka crab duplex-specific nuclease |
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NAR Methods Online |
description |
We developed a novel simple cDNA normalization method [termed duplex‐specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full‐length cDNA sequences. DSN normalization involves the denaturation–reassociation of cDNA, degradation of the double‐stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single‐stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA–RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first‐strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique. |
format |
Text |
author |
Zhulidov, Pavel A. Bogdanova, Ekaterina A. Shcheglov, Alex S. Vagner, Laura L. Khaspekov, George L. Kozhemyako, Valery B. Matz, Mikhail V. Meleshkevitch, Ella Moroz, Leonid L. Lukyanov, Sergey A. Shagin, Dmitry A. |
author_facet |
Zhulidov, Pavel A. Bogdanova, Ekaterina A. Shcheglov, Alex S. Vagner, Laura L. Khaspekov, George L. Kozhemyako, Valery B. Matz, Mikhail V. Meleshkevitch, Ella Moroz, Leonid L. Lukyanov, Sergey A. Shagin, Dmitry A. |
author_sort |
Zhulidov, Pavel A. |
title |
Simple cDNA normalization using kamchatka crab duplex-specific nuclease |
title_short |
Simple cDNA normalization using kamchatka crab duplex-specific nuclease |
title_full |
Simple cDNA normalization using kamchatka crab duplex-specific nuclease |
title_fullStr |
Simple cDNA normalization using kamchatka crab duplex-specific nuclease |
title_full_unstemmed |
Simple cDNA normalization using kamchatka crab duplex-specific nuclease |
title_sort |
simple cdna normalization using kamchatka crab duplex-specific nuclease |
publisher |
Oxford University Press |
publishDate |
2004 |
url |
http://nar.oxfordjournals.org/cgi/content/short/32/3/e37 https://doi.org/10.1093/nar/gnh031 |
genre |
Kamchatka Kamchatka crab |
genre_facet |
Kamchatka Kamchatka crab |
op_relation |
http://nar.oxfordjournals.org/cgi/content/short/32/3/e37 http://dx.doi.org/10.1093/nar/gnh031 |
op_rights |
Copyright (C) 2004, Oxford University Press |
op_doi |
https://doi.org/10.1093/nar/gnh031 |
container_title |
Nucleic Acids Research |
container_volume |
32 |
container_issue |
3 |
container_start_page |
37e |
op_container_end_page |
37 |
_version_ |
1766050981419679744 |