Investigation of the DNA target design parameters for effective hybridization-induced aggregation of particles for the sequence-specific detection of DNA

Abstract

In a recent publication, we presented a label-free method for the detection of specific DNA sequences through the hybridization-induced aggregation (HIA) of a pair of oligonucleotide-adducted magnetic particles. Here we show, through the use of modified hardware, that we are able to simultaneously analyze multiple (4) samples, and detect a 26-mer ssDNA sequence at femtomolar concentrations in minutes. As such, this work represents an improvement in throughput and a 100-fold improvement in sensitivity, compared to that reported previously. Here, we also investigate the design parameters of the target sequence, in an effort to maximize the sensitivity of HIA and to use as a guide in future applications of this work. Modifications were made to the original 26-mer oligonucleotide sequence to evaluate the effects of: (1) non-complementary flanking bases, (2) target sequence length, and (3) single base mismatches on aggregation response. The aggregation response decreased as the number of the non-complementary flanking bases increased, with only a five base addition lowering the LOD by four orders of magnitude. Low sensitivity was observed with short sequences of 6 and 10 complementary bases, which were only detectable at micromolar concentrations. Target sequences with 20, 26 or 32 complementary bases provided the greatest sensitivity and were detectable at femtomolar concentrations. Additionally, HIA could effectively differentiate sequences that were fully complementary from those containing 1, 2 or 3 single base mismatches at micromolar concentrations. The robustness of the HIA system to other buffer components was explored with nine potential assay interferents that could affect hybridization (aggregation) or falsely induce aggregation. Of these, purified BSA and lysed whole blood induced a false aggregation. None of the interferents inhibited aggregation when the hybridizing target was added. Having delineated the fundamental parameters affecting HIA-target hybridization, and demonstrating that HIA had the selectivity to detect single base mismatches, this fluor-free end-point detection has the potential to become a powerful tool for microfluidic DNA detection.

Hybridization-Induced Aggregation Technology for Practical Clinical Testing

“…22 The method ( Figure 1) is an extension of hybridization-induced aggregation (HIA) technology, whereby the hybridization of a specific DNA target to a pair of oligonucleotide probes immobilized on the surface of microbeads, tethers the microbeads together and induces aggregation. 23,24 A digital image of the hybridization microwell is used for quantifying the extent of aggregation in terms of image saturation, which allows for a quantitative representation of hybridization efficiency. We exploit this phenomenon to differentiate target DNA sequences that differ by only a single nucleotide in a method we refer to as HIA for mutation detection (HIA MD ).…”

Nucleic Acid Analysis via Hybridization-Induced Microbead Aggregation: Opportunities for Genetic Testing

“…Previously, the feasibility of applying the HIA method for single base mismatch detection was demonstrated in short, 26-base synthetic oligonucleotides (32). While this was a promising piece of preliminary data, it was not sufficient to establish whether HIA would be viable for point mutation detection in biological samples, using PCR-generated target sequences.…”

“…Fluorescence-based detection can take on many forms, including fluorescence polarization (32) and fluorescence resonance energy transfer (33).…”

Advancing Forensic DNA Processing with Optical Detection and Microfluidic Technology

“…SERATEC ® is more sensitive than the ABAcard ® , however, it is susceptible to false positives with condom lubricants. Although PSA provides the sensitivity needed to detect semen, PSA can also be found in other body fluids including blood, vaginal fluid, and urine 36,37 , and PSA tests are susceptible to false positives when condom lubricants are present and on anal swabs 38,39 .…”

“…Rotating magnetic field mixing of magnetic silica particles and DNA is not new and has been exploited over the last several years 32,33,[39][40][41] . The strong affinity of DNA for silica surfaces in a chaotropic salt is attributed to shielded intermolecular electrostatic forces, dehydration of DNA and silica surfaces, and a hydrogen bond formation between DNA and silica particles 32,40 .…”