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

Xiao-Yu Li,De-Ming Kong,Yu-Peng Zhang,Yi-Chen Du

doi:10.1038/s41598-017-06594-1

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.

Highlights

Sequence-specific detection of target DNA using reliable, cost-effective and sensitive strategies often attracts broad attention due to its importance in clinical diagnosis and genetic research
Detection of target DNA embedded in DNA fragments by exponential exonucleolytic digestion-assisted rolling circle amplification (RCA)
Because the resulting short DNA fragments have much simpler self-folding structures than long genomic DNA, the linear padlock probe can access the target DNA sequence embedded in them and be ligated by T4 DNA ligase to form a circular padlock probe, which can be used as the template in subsequent RCA

Summary

Introduction

Sequence-specific detection of target DNA using reliable, cost-effective and sensitive strategies often attracts broad attention due to its importance in clinical diagnosis and genetic research. As an important isothermal DNA amplification technique, RCA has attracted more and more attention in biosensing applications and various RCA-based DNA detection strategies have been reported[19, 22,23,24,25,26,27,28,29,30,31,32] Most of these works are restricted to the detection of artificial synthetic short-stranded DNA oligonucleotides[19, 27,28,29,30,31,32]. Compared to the first two ways, the last one might provide relatively easier experimental operation, simpler reaction components and lower background signal Such a strategy has never been combined with ultrasonication to detect target DNA embedded in long-stranded genomic DNA. An easy-to-operate biosensing strategy was designed and was demonstrated to work well for the sensitive and sequence-specific detection of target DNA embedded in long-stranded genomic DNA using the human pathogen Cryptococcus neoformans (C. neoformans) as a model. Target DNA-triggered production of circular RCA template confers that the method has extraordinarily high detection specificity, and can discriminate single-nucleotide polymorphisms (SNPs)

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Conclusion