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Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization.

Publication ,  Journal Article
Tjong, V; Yu, H; Hucknall, A; Chilkoti, A
Published in: Analytical chemistry
January 2013

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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Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

January 2013

Volume

85

Issue

1

Start / End Page

426 / 433

Related Subject Headings

  • Surface Properties
  • Saccharomyces cerevisiae
  • RNA
  • Polymerization
  • Oligonucleotide Array Sequence Analysis
  • Nucleic Acid Hybridization
  • Nucleic Acid Amplification Techniques
  • Fluorescent Dyes
  • Exoribonucleases
  • Carbocyanines
 

Citation

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Tjong, V., Yu, H., Hucknall, A., & Chilkoti, A. (2013). Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization. Analytical Chemistry, 85(1), 426–433. https://doi.org/10.1021/ac303132j
Tjong, Vinalia, Hua Yu, Angus Hucknall, and Ashutosh Chilkoti. “Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization.Analytical Chemistry 85, no. 1 (January 2013): 426–33. https://doi.org/10.1021/ac303132j.
Tjong V, Yu H, Hucknall A, Chilkoti A. Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization. Analytical chemistry. 2013 Jan;85(1):426–33.
Tjong, Vinalia, et al. “Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization.Analytical Chemistry, vol. 85, no. 1, Jan. 2013, pp. 426–33. Epmc, doi:10.1021/ac303132j.
Tjong V, Yu H, Hucknall A, Chilkoti A. Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization. Analytical chemistry. 2013 Jan;85(1):426–433.
Journal cover image

Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

January 2013

Volume

85

Issue

1

Start / End Page

426 / 433

Related Subject Headings

  • Surface Properties
  • Saccharomyces cerevisiae
  • RNA
  • Polymerization
  • Oligonucleotide Array Sequence Analysis
  • Nucleic Acid Hybridization
  • Nucleic Acid Amplification Techniques
  • Fluorescent Dyes
  • Exoribonucleases
  • Carbocyanines