One of the standard techniques for DNA detection is the fluorescent intercalator based approach as fluorescent dyes preferentially bind to double-stranded DNAs than single-stranded DNAs. The problem with this approach is that any non-specific amplification products can lead to false positive results due to that intercalators bind non-specifically to any double helix. To deal with this issue, a peptide nucleic acid (PNA) based sequence-specific intercalator method is used. PNA is a mimic of DNA and the charge-neutral backbone gives its higher binding affinity and efficiency to DNA due to the elimination of electrostatic repulsion. PNAs with certain sequences can form stable triplex structures with DNA duplex by sequence recognition and hybridization so that non-specific amplified...[ Read more ]

One of the standard techniques for DNA detection is the fluorescent intercalator based approach as fluorescent dyes preferentially bind to double-stranded DNAs than single-stranded DNAs. The problem with this approach is that any non-specific amplification products can lead to false positive results due to that intercalators bind non-specifically to any double helix. To deal with this issue, a peptide nucleic acid (PNA) based sequence-specific intercalator method is used. PNA is a mimic of DNA and the charge-neutral backbone gives its higher binding affinity and efficiency to DNA due to the elimination of electrostatic repulsion. PNAs with certain sequences can form stable triplex structures with DNA duplex by sequence recognition and hybridization so that non-specific amplified sequences cannot be intercalated. Through tagging these PNA sequences with redox species, an electrochemical sequence-specific intercalator detection scheme can be fulfilled, which can reduce false positive results and increase specificity.

Another limitation of the fluorescent approach is that it requires bulky and precise instrument, sophisticated analysis and professional operation, making it constrained within the centralized hospitals and laboratories. One way to address this problem is to develop portable devices for point-of-care analysis in decentralized areas. Microfluidic technology offers a good solution. In view of this, a continuous flow microfluidic device for real time PCR monitoring is built. By injecting the sample into the microfluidic PCR chip, amplification of the target and simultaneous electrochemical measurement can be performed on the single chip. In order to make it a more self-contained device, an electrolytic pump is fabricated on the chip, eliminating the dependence on an external syringe pump. Realization of on-chip PCR and measurement is promising for application in point-of-care scenarios.