Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.

Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG wer...

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Autores principales:	Xi-Peng Liu, Jian-Hua Liu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/106c9a11192d41ff980582234ebfd92b
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spelling	oai:doaj.org-article:106c9a11192d41ff980582234ebfd92b2021-11-18T07:34:45ZCharacterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.1932-620310.1371/journal.pone.0027248https://doaj.org/article/106c9a11192d41ff980582234ebfd92b2011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22087273/?tool=EBIhttps://doaj.org/toc/1932-6203Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.Xi-Peng LiuJian-Hua LiuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 11, p e27248 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xi-Peng Liu Jian-Hua Liu Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.
description	Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.
format	article
author	Xi-Peng Liu Jian-Hua Liu
author_facet	Xi-Peng Liu Jian-Hua Liu
author_sort	Xi-Peng Liu
title	Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.
title_short	Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.
title_full	Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.
title_fullStr	Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.
title_full_unstemmed	Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.
title_sort	characterization of family iv udg from aeropyrum pernix and its application in hot-start pcr by family b dna polymerase.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/106c9a11192d41ff980582234ebfd92b
work_keys_str_mv	AT xipengliu characterizationoffamilyivudgfromaeropyrumpernixanditsapplicationinhotstartpcrbyfamilybdnapolymerase AT jianhualiu characterizationoffamilyivudgfromaeropyrumpernixanditsapplicationinhotstartpcrbyfamilybdnapolymerase
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Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.

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