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Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization.

Publication ,  Journal Article
Tjong, V; Yu, H; Hucknall, A; Rangarajan, S; Chilkoti, A
Published in: Analytical chemistry
July 2011

We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer; we term this methodology surface initiated enzymatic polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the polymerization conditions to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using TdT as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a ~45 fold signal amplification compared to the incorporation of a single fluorophore. SIEP was then employed to detect hybridization of DNA, by the posthybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of target strands that hybridized to DNA probes that were printed on a surface. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection and a linear dynamic range of 2 logs.

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Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

July 2011

Volume

83

Issue

13

Start / End Page

5153 / 5159

Related Subject Headings

  • Surface Properties
  • Spectrometry, Fluorescence
  • Polymers
  • Nucleic Acid Hybridization
  • DNA Primers
  • DNA
  • Base Sequence
  • Analytical Chemistry
  • 4004 Chemical engineering
  • 3401 Analytical chemistry
 

Citation

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Tjong, V., Yu, H., Hucknall, A., Rangarajan, S., & Chilkoti, A. (2011). Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization. Analytical Chemistry, 83(13), 5153–5159. https://doi.org/10.1021/ac200946t
Tjong, Vinalia, Hua Yu, Angus Hucknall, Srinath Rangarajan, and Ashutosh Chilkoti. “Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization.Analytical Chemistry 83, no. 13 (July 2011): 5153–59. https://doi.org/10.1021/ac200946t.
Tjong V, Yu H, Hucknall A, Rangarajan S, Chilkoti A. Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization. Analytical chemistry. 2011 Jul;83(13):5153–9.
Tjong, Vinalia, et al. “Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization.Analytical Chemistry, vol. 83, no. 13, July 2011, pp. 5153–59. Epmc, doi:10.1021/ac200946t.
Tjong V, Yu H, Hucknall A, Rangarajan S, Chilkoti A. Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization. Analytical chemistry. 2011 Jul;83(13):5153–5159.
Journal cover image

Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

July 2011

Volume

83

Issue

13

Start / End Page

5153 / 5159

Related Subject Headings

  • Surface Properties
  • Spectrometry, Fluorescence
  • Polymers
  • Nucleic Acid Hybridization
  • DNA Primers
  • DNA
  • Base Sequence
  • Analytical Chemistry
  • 4004 Chemical engineering
  • 3401 Analytical chemistry