Nonisotopic detection methods for strand displacement assays of nucleic acids.

Abstract

Using the enzymes terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) and polynucleotide phosphorylase (EC 2.7.7.8), we constructed polyriboadenylic acid tracts, approximately 8000 AMP residues long, attached to the 3'-terminus of a synthetic deoxynucleotide. The polyadenylated DNA, termed the "signal strand", was used in a displacement-type nucleic acid probe assay (see pp 1631-6, this issue). A probe-signal strand complex was made by hybridizing the signal strand to a deoxycytidylate-terminal probe DNA. The probe-signal strand complex was immobilized on an oligo (dG)-cellulose support and subsequently displaced from the immobilized hybrid complex with various amounts of analyte DNA. After the displacement procedure, the polyadenylate tracts were converted to ATP by the combined action of polynucleotide phosphorylase and pyruvate kinase. ATP was quantified by a bioluminescence assay with luciferase from Photinus pyralis. Displacement events were also quantified with biotinylated signal strand bound to avidin-conjugated horseradish peroxidase. Such enzyme-amplified assays offer considerable versatility: they may be coupled to a variety of detection systems including colorimetry, fluorimetry, and luminometry.

Hybridization kinetics between immobilized double-stranded DNA probes and targets containing embedded recognition segments

“…Early experimental efforts used chromatography to monitor the displacement of a shorter DNA strand (1.6 Kb) by a much longer strand (6.1 Kb) in oligonucleotide solutions ( 24 ). In other early studies, the duplex region of the probe was comprised of an RNA–DNA hybrid in which the displaced RNA was detected through a multistep bioluminescence approach ( 25 , 26 ). Related experimental efforts incorporated intentional single base-pair mismatches within the duplex region of the dsProbes ( 27 ).…”

“…( 49 ), for example, have particularly focused on the kinetics of these displacement events for driving DNA-based reaction schemes in oligonucleotide solutions. Even fewer kinetics studies address immobilized double-stranded DNA probe (dsProbe) systems ( 26 , 28 ). Collectively, however, these reports demonstrate the versatility of these systems for characterizing hybridization behavior, especially if colloidal substrates ( 28 , 42 ) are employed such as the microspheres used in the current kinetics study.…”

Advances in nucleic acid-based detection methods.

“…One variation of the solid-phase format is the strand displacement assay (25,96,99). In this format (Fig.…”

A homogeneous nucleic acid hybridization assay based on stand displacement

“…This practice results in lower inherent sensitivity since adsorbed nucleic acids can become partially inaccessible to hybridization (1U). To circumvent these problems, an alternative approach based on strand displacement has been described (11,12). This analytical geometry utilizes a signal strand DNA homologous to the target DNA sequence which is in turn hybridized to a larger single-stranded probe DNA.…”

“…For instance, poly-(dC) tailed probe DNAs may be captured on oligo-(dG) cellulose supports whereas displaced signal strands are not retained by the oligo-(dG) cellulose. Further simplification is possible since displacement reactions can be conducted within the solid support itself (12).…”