Amplified On-Chip Fluorescence Detection of DNA Hybridization by Surface-Initiated Enzymatic Polymerization

Tjong, Vinalia; Yu, Hua; Hucknall, Angus; Rangarajan, Srinath; Chilkoti, Ashutosh

doi:10.1021/ac200946t

Cited by 102 publications

(95 citation statements)

References 40 publications

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“…Although asymmetric PCR43 or terminal transferase reaction with deoxyribonucleotides can produce long ssDNA44 with custom modifications at the 5′-end, the ssDNA product from these methods is a mixture of different lengths or sequences. To get higher-purity ssDNA constructs, dsDNA constructs can be produced with all the required modifications using standard PCR first, followed by subsequent removal of one strand from the constructs, via either overstretching45, chemical or thermal denaturation46, or exonuclease treatment47.…”

Section: Resultsmentioning

confidence: 99%

Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

et al. 2013

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Fluorescence imaging of single-protein dynamics on DNA has been largely limited to double-stranded DNA or short single-stranded DNA. We have developed a hybrid approach for observing single proteins moving on laterally stretched kilobase-sized ssDNA. Here we probed the single-stranded DNA translocase activity of Escherichia coli UvrD by single fluorophore tracking, while monitoring DNA unwinding activity with optical tweezers to capture the entire sequence of protein binding, single-stranded DNA translocation and multiple pathways of unwinding initiation. The results directly demonstrate that the UvrD monomer is a highly processive single-stranded DNA translocase that is stopped by a double-stranded DNA, whereas two monomers are required to unwind DNA to a detectable degree. The single-stranded DNA translocation rate does not depend on the force applied and displays a remarkable homogeneity, whereas the unwinding rate shows significant heterogeneity. These findings demonstrate that UvrD assembly state regulates its DNA helicase activity with functional implications for its stepping mechanism, and also reveal a previously unappreciated complexity in the active species during unwinding.

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Section: Resultsmentioning

confidence: 99%

Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

et al. 2013

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“…This LOD corresponds to 0.16 fmol target in an 80 μL hybridization buffer, which is comparable to the previously reported LOD for unamplified samples on other array platforms for short RNA targets such as miRNA 7,8,33,34 and is similar to the LOD for DNA detection using TdT reported by us previously. 18 This result is the first demonstration of direct on-chip RNA fluorescence detection without the need for RNA manipulation or the additional capture/detection probe. The hybridized RNA target is directly quantified with sensitivity that is competitive to other more complicated techniques 19,33−35 by implementing a simple two-step, isothermal, and on-chip reaction, using commercially available reagents.…”

Section: ■ Experimental Sectionmentioning

confidence: 85%

“…17,18 To the best of our knowledge, this is the first demonstration of an on-chip fluorescence detection methodology that can directly interrogate the RNA target [short (miRNA) and long (mRNA)] without reverse transcription or other prehybridization labeling.…”

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confidence: 99%

Direct Fluorescence Detection of RNA on Microarrays by Surface-Initiated Enzymatic Polymerization

Tjong

Hucknall

et al. 2012

Anal. Chem.

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We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3'-OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3'-OH of an RNA-DNA hybrid. We found that the 3'-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection (LOD) and a 3 log-linear dynamic range for hybridization of a short 21 base-long RNA target to an immobilized peptide nucleic acid probe, while fragmented mRNA targets from three different full length mRNA transcripts yielded a ∼10 pM LOD with a similar dynamic range in a microarray format.

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“…LAMP is relatively simple and can amplify DNA with high specificity (Mori et al, 2004). With the development of gene chips, more attention has been paid to PCR or LAMP on microfluidic gene chips (Tjong et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

DNA detection of Clostridium difficile infection based on real-time resistance measurement

Liu¹,

Jiang²,

Xiang³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. We used a newly developed electrochemical method, real-time resistance measurement, based on loop-mediated isothermal amplification (LAMP), with real-time resistance monitoring and derivative analysis. DNA extracted from specimens was amplified through LAMP reaction. The 2 products of LAMP, DNA and pyrophosphate, both are negative ions; they combine with positive dye (crystal violet) and positive ions (Mg 2+ ), which leads to an increase in the resistivity of the reaction liquid. The changes of resistivity were measured in real-time with a specially designed resistance electrode, to detect Clostridium difficile DNA. We found that electrochemical detection of C. difficile could be completed in 0.5-1 h, with a detection limit of 10 2 CFU/mL, with high accuracy (95.0%), sensitivity (91.1%), and specificity (97.3%) compared to PCR methods. C. difficile is commonly associated with antibioticinduced diarrhea. Due to the difficulty in performing anaerobic culture and cytotoxicity neutralization assays, a simple, rapid, sensitive, and accurate method is preferred. We conclude that realtime resistance measurement is a rapid, sensitive, and stable method for the diagnosis of C. difficile infection that could be applied to gene chips and pocket instruments.

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Amplified On-Chip Fluorescence Detection of DNA Hybridization by Surface-Initiated Enzymatic Polymerization

Cited by 102 publications

References 40 publications

Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

Direct Fluorescence Detection of RNA on Microarrays by Surface-Initiated Enzymatic Polymerization

DNA detection of Clostridium difficile infection based on real-time resistance measurement

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