A DNA machine for sensitive and homogeneous DNA detection via lambda exonuclease assisted amplification.
This work designs a DNA machine with three assistant DNAs and lambda exonuclease (Exo-λ) for sensitive and homogeneous fluorescent detection of DNA. The selective digestion of Exo-λ to blunt or recessed 5'-phosphorylated strand of probe 1-probe 2 duplex results in the release of target DNA and probe 2 to produce the fluorescence restoring of fluorophore labeled to probe 1. The released target DNA could hybridize with another probe 1-probe 2 duplex to trigger the target recycling for signal amplification, while the released probe 2 hybridized with molecular beacon to restore its fluorescence for signal enhancement. This DNA machine showed a fast response to target DNA with a linear concentration range from 0.4 pM to 4 nM. The limit of detection was 68 fM at a signal-to-noise ratio of 3. The high selectivity of the method may result from the Exo-λ's specific recognition-site of double-stranded DNA and the specific hybridization of target DNA with probe 1-probe 2 duplex. This DNA machine with the homogenous detection, rapid response as well as simplicity provides a new approach for sensitive detection of DNA.
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China.
Lin
L
Liu
Jianping
J
Lei
Fenglei
F
Gao
Huangxian
H
Ju
D000818:Animals; D001419:Bacteria; D010582:Bacteriophage lambda / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D020137:Base Pair Mismatch; D020029:Base Pairing; D001681:Biological Assay; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques; D059629:Signal-To-Noise Ratio; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
D015342:DNA Probes; D005456:Fluorescent Dyes; D014764:Viral Proteins; D004247:DNA; D005090:Exodeoxyribonucleases
DNA machine; Fluorescence; Homogeneous; Lambda exonuclease; Signal amplification