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

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.