The hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment, has been introduced earlier for reactions in solution. Two species of DNA hairpins are present in solution without interaction, until an initiator DNA triggers a cascade of hybridization events resulting in an elongated strand of alternating opened hairpins forming a double helix. Hybridization chain reaction (HCR) was introduced as an attractive method to amplify the signal of targeted nucleic acids. We report here the results of the HCR for the amplification of DNA microarray signals. Hairpins were fluorescently labelled with Cy3 or Cy5, or with alternative couples of fluorophors. First results indicate that the HCR-induced fluorescence at the surface might be hard to detect directly. Protocols involving the removal of the amplification product from the surface evidence an amplification factor of about 5. Performing the HCR in solution before hybridizing the product to the DNA microarray, i.e. using HCR for multiple labelling the target for the detection, produces a signal amplification of a factor 3-4. The effect of buffer composition, initiator concentration and hairpin concentration was investigated. The low sensitivity that is currently attainable (approximately 0,05 µM) severely limits the practical use of the HCR.

Assessment of the hybridization chain reaction for the amplification of signal on DNA microarrays

VINELLI, ALESSANDRA;ONOFRI, MANUELE;ZUCCHERI, GIAMPAOLO
2009

Abstract

The hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment, has been introduced earlier for reactions in solution. Two species of DNA hairpins are present in solution without interaction, until an initiator DNA triggers a cascade of hybridization events resulting in an elongated strand of alternating opened hairpins forming a double helix. Hybridization chain reaction (HCR) was introduced as an attractive method to amplify the signal of targeted nucleic acids. We report here the results of the HCR for the amplification of DNA microarray signals. Hairpins were fluorescently labelled with Cy3 or Cy5, or with alternative couples of fluorophors. First results indicate that the HCR-induced fluorescence at the surface might be hard to detect directly. Protocols involving the removal of the amplification product from the surface evidence an amplification factor of about 5. Performing the HCR in solution before hybridizing the product to the DNA microarray, i.e. using HCR for multiple labelling the target for the detection, produces a signal amplification of a factor 3-4. The effect of buffer composition, initiator concentration and hairpin concentration was investigated. The low sensitivity that is currently attainable (approximately 0,05 µM) severely limits the practical use of the HCR.
11th Status Seminar Chip Technologies New approaches in chip technologies: from dynamic measurements to biomolecule synthesis
C. Del Rio; A. Vinelli; M. Onofri; C. Mittermayr ; P. Winklehner; G. Zuccheri
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/96885
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