THESIS
2014
xvi, 65 pages : illustrations (some color) ; 30 cm
Abstract
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...[
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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.
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