Rapid host adaptation by extensive recombination

Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature

A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

Abstract PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications

A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications

Effects of restriction site positioning within the ds DNA hybrid, and non-complementary loop addition.

<p>The HRP signals (bars) are expressed as percentages of the fully cognate positive control (40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length, non-complementary ends, and/or loops. The <i>Bgl</i>II restriction site is indicated with thick horizontal lines. Target designations are the following: 5′ (or 3′), corresponds to the 5′ (or 3′) ends of the full length positive control; C, control (fully cognate), L, loop (addition of 5 or 10 nucleotides); rs5′ (or rs3′), the end of restriction site to which 0, 3, or 5 (+0, +3, +5) complementary nucleotides were added. For rs3′+0, two targets were prepared that had different non-complementary sequences flanking the 3′-end of the restriction site (rs3′+0-A, rs3′+0-G). The target oligonucleotide sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097826#pone-0097826-t001" target="_blank">Table 1</a>.</p

Calibration curves generated with either the purified 196<i>mecA</i> amplicon (diamonds) or the unpurified PCR mixture (containing the target amplicon) (squares).

<p>The logarithmic trendlines were calculated in Excel, and proved to be identical for the purified and non-purified amplicons.</p

The oligonucleotide probe and targets used in the current study.

<p>Capital letters show sequences that are cognate between a target oligonucleotide and the probe, with the restriction site shown in bold.</p>1<p>The total length of a target sequence that is complementary to the 40-mer probe MCA-BG.</p>2<p>Tm was calculated for a target-probe hybrid in PBS (150 mM Na⁺).</p

Detection of the non-purified amplicon <i>mecA</i> in the presence of a large excess of heterologous (mouse) genomic DNA.

<p>Circles and diamonds show replicate experiments performed using the amplicon-containing PCR mixture, closed and open for addition of 100 or 0 ng of mouse DNA, respectively. The triangles show the negative control supplemented with 100 ng of mouse DNA, specifically, dilutions of the whole PCR mixture that were not subjected to thermocycling (no amplicon formation as verified by gel electrophoresis).</p

Effect of point mutations introduced into the target sequence.

<p>(<b>A</b>) Single, double and triple mutations were introduced between the target center and the 3′ end corresponding to the surface-immobilized terminus of the target-probe duplex. (<b>B</b>) Mutations were introduced between the target center and the 5′ end corresponding to the end of the target-probe duplex that was free in solution. HRP signals (bars) are expressed as the percentages of the fully cognate positive control (dark grey bar 40, for 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a 40-mer target with 1–3 mutations shown with black ovals. The <i>Bgl</i>II restriction site is indicated with thick horizontal lines. Targets are named with ‘rs’ for mutations introduced within the restriction site, otherwise the target name contains the replacement nucleotide (mostly G) and position within the sequence, starting from the 5′ target end. The rs19+24 contained two mutations at the ends of the restriction site. Target oligonucleotide sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097826#pone-0097826-t001" target="_blank">Table 1</a>.</p

A typical calibration curve of the restriction enzyme assay generated with a 40-mer oligonucleotide target AMC-40-mer (fully complementary to the MCA-BG probe).

<p>X-axis shows concentrations (nM) of the target oligonucleotide. Y-axis shows the restriction enzyme generated HRP signal that was quantified by the blue color formation as measured by the OD₆₅₅. The signal values were background-corrected by subtracting the signal generated by the negative control with no target oligonucleotide added. The experiments were performed in triplicate to generate mean values (black circles) and standard deviations (shown with error bars).</p