Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase
Autor(a) principal: | |
---|---|
Data de Publicação: | 1998 |
Outros Autores: | |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/679 http://dx.doi.org/10.1590/S0100-879X1998001000001 |
Resumo: | For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA ) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component. |
id |
UFSP_4cd9c554d868d98b213ccfd9f0cee17b |
---|---|
oai_identifier_str |
oai:repositorio.unifesp.br:11600/679 |
network_acronym_str |
UFSP |
network_name_str |
Repositório Institucional da UNIFESP |
repository_id_str |
3465 |
spelling |
Diaz, Ricardo Sobhie [UNIFESP]Sabino, Ester Cerdeira [UNIFESP]Universidade Federal de São Paulo (UNIFESP)Irwin Memorial Blood CentersFundação Pró-Sangue, Hemocentro de São Paulo2015-06-14T13:24:47Z2015-06-14T13:24:47Z1998-10-01Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 31, n. 10, p. 1239-1242, 1998.0100-879Xhttp://repositorio.unifesp.br/handle/11600/679http://dx.doi.org/10.1590/S0100-879X1998001000001S0100-879X1998001000001.pdfS0100-879X199800100000110.1590/S0100-879X1998001000001WOS:000076614900001For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA ) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.Universidade Federal de São Paulo (UNIFESP)Irwin Memorial Blood CentersFundação Pró-Sangue, Hemocentro de São PauloUNIFESP, EPM, São PauloSciELO1239-1242engAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Researchpolymerase chain reaction fidelityThermus aquaticus (Taq) DNA polymeraseThermotoga maritima DNA polymeraseAccuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymeraseinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESPORIGINALS0100-879X1998001000001.pdfapplication/pdf63014${dspace.ui.url}/bitstream/11600/679/1/S0100-879X1998001000001.pdfef5412179dd674f69800c5756228d7f3MD51open accessTEXTS0100-879X1998001000001.pdf.txtS0100-879X1998001000001.pdf.txtExtracted texttext/plain14962${dspace.ui.url}/bitstream/11600/679/2/S0100-879X1998001000001.pdf.txt61463f277a201c8c355871f76999191eMD52open access11600/6792022-09-27 09:59:57.453open accessoai:repositorio.unifesp.br:11600/679Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652023-05-25T12:23:56.967787Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase |
title |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase |
spellingShingle |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase Diaz, Ricardo Sobhie [UNIFESP] polymerase chain reaction fidelity Thermus aquaticus (Taq) DNA polymerase Thermotoga maritima DNA polymerase |
title_short |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase |
title_full |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase |
title_fullStr |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase |
title_full_unstemmed |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase |
title_sort |
Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase |
author |
Diaz, Ricardo Sobhie [UNIFESP] |
author_facet |
Diaz, Ricardo Sobhie [UNIFESP] Sabino, Ester Cerdeira [UNIFESP] |
author_role |
author |
author2 |
Sabino, Ester Cerdeira [UNIFESP] |
author2_role |
author |
dc.contributor.institution.none.fl_str_mv |
Universidade Federal de São Paulo (UNIFESP) Irwin Memorial Blood Centers Fundação Pró-Sangue, Hemocentro de São Paulo |
dc.contributor.author.fl_str_mv |
Diaz, Ricardo Sobhie [UNIFESP] Sabino, Ester Cerdeira [UNIFESP] |
dc.subject.eng.fl_str_mv |
polymerase chain reaction fidelity Thermus aquaticus (Taq) DNA polymerase Thermotoga maritima DNA polymerase |
topic |
polymerase chain reaction fidelity Thermus aquaticus (Taq) DNA polymerase Thermotoga maritima DNA polymerase |
description |
For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA ) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component. |
publishDate |
1998 |
dc.date.issued.fl_str_mv |
1998-10-01 |
dc.date.accessioned.fl_str_mv |
2015-06-14T13:24:47Z |
dc.date.available.fl_str_mv |
2015-06-14T13:24:47Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 31, n. 10, p. 1239-1242, 1998. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/679 http://dx.doi.org/10.1590/S0100-879X1998001000001 |
dc.identifier.issn.none.fl_str_mv |
0100-879X |
dc.identifier.file.none.fl_str_mv |
S0100-879X1998001000001.pdf |
dc.identifier.scielo.none.fl_str_mv |
S0100-879X1998001000001 |
dc.identifier.doi.none.fl_str_mv |
10.1590/S0100-879X1998001000001 |
dc.identifier.wos.none.fl_str_mv |
WOS:000076614900001 |
identifier_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 31, n. 10, p. 1239-1242, 1998. 0100-879X S0100-879X1998001000001.pdf S0100-879X1998001000001 10.1590/S0100-879X1998001000001 WOS:000076614900001 |
url |
http://repositorio.unifesp.br/handle/11600/679 http://dx.doi.org/10.1590/S0100-879X1998001000001 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.none.fl_str_mv |
Brazilian Journal of Medical and Biological Research |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
1239-1242 |
dc.publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica |
publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
bitstream.url.fl_str_mv |
${dspace.ui.url}/bitstream/11600/679/1/S0100-879X1998001000001.pdf ${dspace.ui.url}/bitstream/11600/679/2/S0100-879X1998001000001.pdf.txt |
bitstream.checksum.fl_str_mv |
ef5412179dd674f69800c5756228d7f3 61463f277a201c8c355871f76999191e |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
|
_version_ |
1783460285630119936 |