Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA

Abstract An exonucleolytic digestion-assisted exponential rolling circle amplification (RCA) strategy was developed for sensitive and sequence-specific detection of target DNA embedded in long-stranded genomic DNA. Herein, Phi29 DNA polymerase plays two important roles as exonuclease and polymerase....

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Autores principales:	Xiao-Yu Li, Yi-Chen Du, Yu-Peng Zhang, De-Ming Kong
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fc87fd01282e448dbd6191140760ae61
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spelling	oai:doaj.org-article:fc87fd01282e448dbd6191140760ae612021-12-02T16:07:56ZDual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA10.1038/s41598-017-06594-12045-2322https://doaj.org/article/fc87fd01282e448dbd6191140760ae612017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06594-1https://doaj.org/toc/2045-2322Abstract An exonucleolytic digestion-assisted exponential rolling circle amplification (RCA) strategy was developed for sensitive and sequence-specific detection of target DNA embedded in long-stranded genomic DNA. Herein, Phi29 DNA polymerase plays two important roles as exonuclease and polymerase. Long-stranded genomic DNAs can be broken into small DNA fragments after ultrasonication. The fragments that contain target DNA, hybridize with a linear padlock probe to trigger the formation of a circular RCA template. The tails protruding from the 3′-end of the target DNA sequences are then digested by the 3′ → 5′ exonuclease activity of Phi29 DNA polymerase even if they fold into a double-stranded structure. The digested DNA fragments can then initiate subsequent RCA reaction. RCA products, which are designed to fold into G-quadruplex structures, exponentially accumulate when appropriate nicking endonuclease recognition sites are introduced rationally into the RCA template. This method is demonstrated to work well for real genomic DNA detection using human pathogen Cryptococcus neoformans as a model. In addition, this work has two other important discoveries: First, the presence of a 3′-tail can protect the RCA primer from degradation by Phi29 DNA polymerase. Second, 3′ → 5′ exonucleolytic activity of Phi29 DNA polymerase can work for both single- and double-stranded DNA.Xiao-Yu LiYi-Chen DuYu-Peng ZhangDe-Ming KongNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xiao-Yu Li Yi-Chen Du Yu-Peng Zhang De-Ming Kong Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
description	Abstract An exonucleolytic digestion-assisted exponential rolling circle amplification (RCA) strategy was developed for sensitive and sequence-specific detection of target DNA embedded in long-stranded genomic DNA. Herein, Phi29 DNA polymerase plays two important roles as exonuclease and polymerase. Long-stranded genomic DNAs can be broken into small DNA fragments after ultrasonication. The fragments that contain target DNA, hybridize with a linear padlock probe to trigger the formation of a circular RCA template. The tails protruding from the 3′-end of the target DNA sequences are then digested by the 3′ → 5′ exonuclease activity of Phi29 DNA polymerase even if they fold into a double-stranded structure. The digested DNA fragments can then initiate subsequent RCA reaction. RCA products, which are designed to fold into G-quadruplex structures, exponentially accumulate when appropriate nicking endonuclease recognition sites are introduced rationally into the RCA template. This method is demonstrated to work well for real genomic DNA detection using human pathogen Cryptococcus neoformans as a model. In addition, this work has two other important discoveries: First, the presence of a 3′-tail can protect the RCA primer from degradation by Phi29 DNA polymerase. Second, 3′ → 5′ exonucleolytic activity of Phi29 DNA polymerase can work for both single- and double-stranded DNA.
format	article
author	Xiao-Yu Li Yi-Chen Du Yu-Peng Zhang De-Ming Kong
author_facet	Xiao-Yu Li Yi-Chen Du Yu-Peng Zhang De-Ming Kong
author_sort	Xiao-Yu Li
title	Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
title_short	Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
title_full	Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
title_fullStr	Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
title_full_unstemmed	Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
title_sort	dual functional phi29 dna polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target dna embedded in long-stranded genomic dna
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/fc87fd01282e448dbd6191140760ae61
work_keys_str_mv	AT xiaoyuli dualfunctionalphi29dnapolymerasetriggeredexponentialrollingcircleamplificationforsequencespecificdetectionoftargetdnaembeddedinlongstrandedgenomicdna AT yichendu dualfunctionalphi29dnapolymerasetriggeredexponentialrollingcircleamplificationforsequencespecificdetectionoftargetdnaembeddedinlongstrandedgenomicdna AT yupengzhang dualfunctionalphi29dnapolymerasetriggeredexponentialrollingcircleamplificationforsequencespecificdetectionoftargetdnaembeddedinlongstrandedgenomicdna AT demingkong dualfunctionalphi29dnapolymerasetriggeredexponentialrollingcircleamplificationforsequencespecificdetectionoftargetdnaembeddedinlongstrandedgenomicdna
_version_	1718384694133784576

Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA

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