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Hybridization chain reaction


A new technique, based on a chain reaction of recognition and hybridization events between two sets of DNA hairpin molecules, offers an enzyme-free alternative for the rapid detection of specific DNA sequences.


The processes that mediate target recognition and signal transduction in biosensors generally operate through distinct mechanisms. In the polymerase chain reaction (PCR), for example, recognition is provided by sequence-specific hybridization of short oligonucleotides to a larger target, whereas signal amplification requires the use of a polymerase coupled with externally driven melting and rehybridization. However, in the October 26th issue of the Proceedings of the National Academy of Sciences, Niles Pierce and graduate student Robert Dirks demonstrate how binding of DNA to a substrate can accomplish the roles of recognition and signal amplification without any external inputs. This is accomplished by the triggered self-assembly of DNA nanostructures in a novel process they term hybridization chain reaction (HCR).

According to Pierce, HCR came about not from a desire to compete with PCR but as a byproduct of engineering DNA hairpins that could be used as fuel packets to power DNA mechanical machines. By eliminating the machines from the design scenario, they found that the hairpins could be triggered to undergo a chain reaction. “The realization that got us excited was the idea that this could be used as an amplifying signal transducer,” says Pierce.

The key to HCR in its simplest form is the storage of potential energy in two hairpin species. When a single-stranded DNA initiator is added to this previously stable mixture, it opens a hairpin of one species, exposing a new single-stranded region that opens a hairpin of the other species (Fig. 1). This process, in turn, exposes a single-stranded region identical to the original initiator. The resulting chain reaction leads to the formation of a nicked double helix that grows until the hairpin supply is exhausted. Detection of the resulting products does not require any specialized detection equipment. As Pierce remarks, “All you need is a gel apparatus, which can be found in any wet lab. We are also trying to make a nanogold-based colorimetric assay that will enable detection by eye alone.”

Figure 1: Schematic of the basic hybridization chain reaction.
figure 1

Addition of an initiator strand of DNA to the stable mixture of two hairpin species triggers a chain reaction of hybridization events between the hairpins.

For more diverse biosensing applications, DNA and RNA aptamers selected to bind specific molecules hold promise for the development of HCR triggers that will initiate the chain reaction only in the presence of the target molecule. The authors have used this aptamer trigger concept to specifically discriminate ATP from GTP. Pierce remarks, “If we succeed in developing a general aptamer triggering mechanism, then HCR amplification could be incorporated in sensors for a wide range of small molecules.”

Unlike PCR, which provides exponential amplification, the current form of HCR provides linear amplification. “We are now developing nonlinear versions of HCR that provide quadratic, cubic or exponential growth after being triggered by the initiator,” says Pierce. “However, false positives become a much bigger problem, as spurious initiation events are also amplified nonlinearly.” Successful development of an exponential HCR amplification system would increase the sensitivity to target molecules at very low concentrations. This would open up the possibility of attaining a PCR-like level of sensitivity for a variety of small molecules without the need for any expensive equipment or reagents.


  1. Dirks, R.M. & Pierce, N.A. Triggered amplification by hybridization chain reaction. Proc. Natl. Acad. Sci. USA 101, 15275–15278 (2004).

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Evanko, D. Hybridization chain reaction. Nat Methods 1, 186 (2004).

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