Sequence selective capture, release and analysis of DNA using a magnetic microbead-assisted toehold-mediated DNA strand displacement reaction

This paper reports on the modification of magnetic beads with oligonucleotide capture probes with a specially designed pendant toehold (overhang) aimed specifically to capture double-stranded PCR products. After capture, the PCR products were selectively released from the magnetic beads by means of a toehold-mediated strand displacement reaction using short artificial oligonucleotide triggers and analysed using capillary electrophoresis. The approach was successfully shown on two genes widely used in human DNA genotyping, namely human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) and amelogenin

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.The authors kindly acknowledge the Australian Research Council Future Fellowship Fund (Grant No. FT130100211) for funding

Toehold-Mediated Nonenzymatic DNA Strand Displacement As a Platform for DNA Genotyping

Toehold-mediated DNA strand displacement provides unique advantages in the construction and manipulation of multidimensional DNA nanostructures as well as nucleic acid sequence analysis. We demonstrate a step change in the use of toehold-mediated DNA strand displacement reactions, where a double-stranded DNA duplex, containing a single-stranded toehold domain, enzymatically generated and then treated as a molecular target for analysis. The approach was successfully implemented for human DNA genotyping, such as gender identification where the amelogenin gene was used as a model target system, and detecting single nucleotide polymorphisms of human mitochondrial DNA. Kinetics of the strand displacement was monitored by the quenched Förster resonance energy transfer effect

Random Whole Metagenomic Sequencing for Forensic Discrimination of Soils

<div><p>Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.</p></div

General characteristics of full sequencing data.

<p>Statistical data represented as mean ± Standard Deviation (SD). Percentage of sequences matching to the M5NR, M5RNA and SEED Subsystems databases was determined with an E-value cut-off of E<1×10⁻⁵. QC = quality control.</p

Comparison of the taxonomic soil profiles generated on full datasets at the phylum (A, B, C) and species (D, E, F) resolution levels.

<p>Bray-Curtis distance similarity matrix was calculated from the square-root transformed abundance of DNA fragments matching taxa in the M5NR database (E-value <1×10⁻⁵). The Bray-Curtis matrix was used for generating CLUSTER dendrogram, NMDS and CAP ordination plots. <b>CLUSTER analysis (A and D).</b> Red dotted branches on the CLUSTER dendrogram indicate no significant difference between metagenomic profiles (supported by the SIMPROF analysis, p<0.05). <b>NMDS unconstrained ordination (B and E).</b> The NMDS plot displays distances between samples. Data points that are closer to each other represent samples with highly similar metagenomic profiles. <b>CAP constrained ordination (C and F).</b> CAP analysis tests for differences among the groups in multivariate space. The significance of group separation along the canonical axis is indicated by the value of the squared canonical correlation (δ₁²) and P-value. A contour line on the NMDS and CAP ordinations drawn round each of the cluster defines the superimposition of clusters from CLUSTER dendrogram at the selected level of similarity.</p

Results of CAP model cross-validation of soil metabolic profiles discrimination generated from full sequencing datasets.

<p>Results of CAP model cross-validation of soil metabolic profiles discrimination generated from full sequencing datasets.</p

RELATE comparison of Bray-Curtis similarity matrices.

<p>The Bray-Curtis similarity matrices calculated from square root transformed abundance of DNA fragments generated based on full datasets and sub-sampled datasets.</p

Comparison of the metabolic soil profiles generated on full datasets at the subsystems level 1 (A, B, C) and subsystems function (D, E, F) resolution levels.

<p>Bray-Curtis distance similarity matrix was calculated from the square-root transformed abundance of DNA fragments matching taxa in the SEED database (E-value <1×10⁻⁵). The Bray-Curtis matrix was used for generating CLUSTER dendrogram, NMDS and CAP ordination plots. <b>CLUSTER analysis (A and D).</b> Red dotted branches on the CLUSTER dendrogram indicate no significant difference between metagenomic profiles (supported by the SIMPROF analysis, p<0.05). <b>NMDS unconstrained ordination (B and E).</b> The NMDS plot displays distances between samples. Data points that are closer to each other represent samples with highly similar metagenomic profiles. <b>CAP constrained ordination (C and F).</b> CAP analysis tests for differences among the groups in multivariate space. The significance of group separation along the canonical axis is indicated by the value of the squared canonical correlation (δ₁²) and P-value. A contour line on the NMDS and CAP ordinations drawn round each of the cluster defines the superimposition of clusters from CLUSTER dendrogram at the selected level of similarity.</p