Nonenzymatic detection of bacterial genomic DNA using the bio bar code assay

Abstract

The detection of bacterial genomic DNA through a nonenzymatic nanomaterials-based amplification method, the bio bar code assay, is reported. The assay utilizes oligonucleotide-functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double-stranded DNA. These blockers bind to specific regions of the target DNA upon cooling and prevent the duplex DNA from rehybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide-functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (bar codes) act as amplification surrogates. The bar codes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 fM, and this is the first demonstration of a bar code-type assay for the detection of double-stranded, genomic DNA.

Figure 1. Probe Melting Analysis

A) Melting curve for the duplex formed between an oligo-AuNP probe and its fluorophore labeled complement (sequences given in Table 1) B) Melting curve for the duplex formed between the oligo-MMP probe and its fluorophore labeled complement (sequences given in Table 1). The fluorescence of the complementary strands is quenched when they are bound to the AuNP and is recovered when the duplexes melt with the fluorophore strand being released into solution.

Figure 2. Blocking Oligonucleotide Functionality

A) Scheme showing how the blocking oligonucleotides are designed to prevent genomic DNA strand rehybridization. B) This graph shows the importance of the blocking oligonucleotides to the function of the bio-barcode assay. It is clearly seen that without blockers the signal obtained in the assay is the same as that with no target, while in the presence of blockers a large signal is obtained indicating that the genomic DNA is available for hybridization to probes.

Figure 3. Genomic DNA Detection Results

A) This image is a representative slide from a single assay; showing that 2.5 fM is distinguishable from the 0 fM (no target) sample. The grey scale image from the Verigene ID is converted to color using GenePix 6.0 software (Molecular Devices). B) The data shown above is an average of 5 independent runs of the genomic DNA bio-barcode assay.

Scheme 1. Genomic Bio-Barcode Assay

The first step is to isolate the genomic DNA from the bacterial cells and cut it with a restriction enzyme. This cut prevents the DNA from super coiling during heating and gives smaller target pieces. The next step is to introduce blocking oligonucleotides designed to flank the probe binding sites and prevent strand rehybridization after thermal denaturation. The blocking oligonucleotides are used in excess to out compete the native strand during hybridization. The target region is now “propped” open and accessible for probe binding. Magnetic microparticles (oligo-MMPs) are used to capture the targets from the sample and then washed. An excess of oligonucleotide modified gold nanoparticle probes (oligo-AuNPs) is added to the assay solutions which results in the sandwiching of the target with the oligo-MMP. Unbound oligo-AuNPs are removed by washing. The barcodes are chemically released for scanometric detection and quantification.