RNA Detection

Chapter 12

Hybridization Chain Reaction for Direct mRNA Detection

Without Nucleic Acid Purification

Yao Xu and Zhi Zheng

Abstract

Hybridization chain reaction (HCR) provides a feasible solution for nucleic acid detection without target
amplification. By highly specific sandwich hybridization, target RNA can be directly captured onto solid
support and detected using HCR with fluorescent dyes. Here, we describe a novel method for malaria RNA
detection based on sandwich hybridization and two-dimensional HCR, without involving nucleic acid
purification or any enzymatic reaction, using ordinary oligonucleotides without labeling or modification.

Key wordsHybridization chain reaction, mRNA detection, Sandwich hybridization

1 Introduction

Hybridization chain reaction is a new class of enzyme-free fluores-

cent signal amplification method for nucleic acid detection. In this

procedure, the target DNA/RNA initiates a hybridization cascade

between two hairpin sequences through toehold mediated strand

displacement to realize signal amplification [1, 2]. The two species

of hairpin monomers of the HCR are metastable and can coexist in

the solution until triggered by the target DNA to form a nicked

DNA double helix analogous to alternating copolymers (Fig.1),

which can be seen in agarose gel electrophoresis (Fig.2). Although

HCR-based signal amplifications have been described for nucleic

acid biosensing [3, 4], there are several drawbacks significantly

impeding the practical application of these methods: (1) as a highly

sensitive signal amplification method, HCR inevitably amplifies

backgrounds, decreasing specificity. These backgrounds could be

produced by physical nonspecific adsorption of hairpin sets, or

caused by leakiness in toehold-mediated HCR (i.e., hairpin poly-

merization in the absence of initiating target DNA), especially for

real sample detection when the components are complicated. (2)

The sensitivity of toehold-mediated HCR amplification is not

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_12,©Springer Science+Business Media LLC 2018

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