Winnowing DNA for Rare Sequences: Highly Specific Sequence and Methylation Based Enrichment

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue

A novel electrophoretic mechanism and separation parameter for selective nucleic acid concentration based on synchronous coefficient of drag alteration (SCODA)

Molecular manipulation and separation techniques form the building blocks for much of fundamental science, yet many separation challenges still remain, in fields as diverse as forensics and metagenomics. This thesis presents SCODA (Synchronous Coefficient of Drag Alteration), a novel and general molecular separation and concentration technique aimed at addressing such challenges. SCODA takes advantage of physical molecular properties associated with the non‐linear response of long, charged polymers to electrophoretic fields, which define a novel parameter for DNA separation. The SCODA method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is non-linear. This thesis presents the development of SCODA for extraction of DNA, and outlines the design of the instrumentation required to achieve the SCODA effect. We then demonstrate the selectivity, efficiency, and sensitivity of the technique. Contaminant rejection is also quantified for humic acids and proteins, with SCODA displaying excellent performance compared to existing technologies. Additionally, the ability of this technology to extract high molecular weight DNA is demonstrated, as is its inherent fragment length selection capability. Finally, we demonstrate two applications of this method to metagenomics projects where existing technologies performed poorly or failed altogether. The first is the extraction of high molecular weight DNA from soil, which is limited in length to fragments smaller than 50 kb with current direct extraction methods. SCODA was able to recover DNA an order of magnitude larger than this. The second application is DNA extraction from highly contaminated samples originating in the Athabasca tar sands, where existing technology had failed to recover any usable DNA. SCODA was able to recover sufficient DNA to enable the discovery of 200 putatively novel organisms.Science, Faculty ofPhysics and Astronomy, Department ofGraduat

Measurement of mobility versus temperature for methylated and unmethylated targets.

<p>Data points were fit to equation (3). The upper limit of these curves were cut off because target DNA had migrated off the end of the gel used for taking these measurements. Both sets of data points were fit to equation (2) using the non-linear least squares fitting tool in the Origin 7.5 software package (OriginLab Corporation, Northampton MA). For the unmethylated curve Mfold calculated values were used for the enthalpy and entropy terms; μ₀ and α were determined by the fit. Using ΔH = 144.4 kcal/mol and ΔS = 0.3988 kcal/mol K the unmethylated curve fit resulted in values of μ_o = 1.34e-9+/−0.05e-9 m²/Vs and α = 1.12e-6+/−0.07e-6. For the methylated curve, it was assumed that the parameters μ₀, α and ΔS were unaffected by the addition of the methyl group and the parameters obtained in the unmethylated fit were used to obtain a value for ΔH = 144.62+/−0.04 kcal/mol. Inset: Separation of methylated (6-FAM, green) and unmethylated (Cy5, red) targets by focusing with an applied DC bias at 69°C.</p

Measurement of temperature dependence of DNA target mobility through a gel containing immobilized complementary oligonucleotide probes.

<p>The upper limit of these curves were cut off because target DNA had migrated off the end of the gel used for taking these measurements. Both sets of data points were fit to equation (2) using the non-linear least squares fitting tool in the Origin 7.5 software package (OriginLab Corporation, Northampton MA). Mfold calculated values were used for the enthalpy and entropy terms; μ₀ and α were determined by the fit. For the mismatch curve μ_o = 1.34e-9+/−0.05e-9 m²/Vs and α = 1.12e-6+/−0.07e-6. For the perfect complement μ₀ = 1.28e-9+/−0.05e-9 m²/Vs and α = 2.01e-7+/−0.13e-7.</p

Rejection ratio of snMM DNA. Four different ratios of snMM:PC were injected into a gel and focused under bias to remove excess snMM.

<p>The PC DNA was tagged with 6-FAM and the snMM DNA was tagged with Cy5. Top: fluorescence signal from the final focus spot after excess snMM DNA had been washed from the gel. The fluorescence signals are normalized to the fluorescence measured on an initial calibration run where a 1∶1 ratio of PC-FAM:PC-Cy5 DNA was injected and focused to the centre of the gel. Bottom: rejection ratios calculated by dividing the initial ratio of snMM:PC by the final ratio after washing.</p

Enrichment of EZH2 Y641N mutation from a 1∶1 mixture of wild type (green) and mutant (red) amplicons.

<p>30 ng of each 470 bp target was diluted into a 250 µl solution of 0.9 mM tris, 0.9 mM boric acid and 2 mM NaCl. The sample was placed in a boiling water bath for 5 min to denature the double stranded DNA prior to injection. The targets were injected from a chamber adjacent to the lower right corner of the gel. After injection, focusing and bias fields were applied to simultaneously concentrate the mutant amplicon while washing the wild type amplicon from the gel.</p

Time series of ssSCODA focusing under bias. PC DNA is tagged with 6-FAM (green) and snMM DNA is tagged with Cy5 (red).

<p>Images were taken at 2 min intervals with the first image taken immediately following injection. The camera gain was reduced from 32 to 16 on the green channel after the first image was taken. DNA was injected from a chamber adjacent to the right side of the gel. After injection, focusing plus bias fields are applied. The PC target (green) experiences a convergent drift velocity and focuses to the centre of the gel. The more weakly focusing snMM target (red) is washed completely from the gel by the bias field.</p

Demonstration of length independent focusing.

<p>Left: Focus location under bias for 250 bp (green) and 1000 bp (red) fragments. Right: Image of the gel at the end of the run. Green and red channels have been superimposed.</p

Washing of unmethylated DNA from the gel.

<p>Top two images were taken after an initial focus but before attempts to wash. The bottom two images were taken after washing the unmethylated target from the gel. All images were taken with the same gain and shutter settings which resulted in sensor saturation and some ghost images from reflections off of lenses in the top left image. Note that the dyes have been swapped compared to the image in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031597#pone-0031597-g007" target="_blank">Figure 7</a>.</p

Time series of a demonstration of enrichment and extraction of target DNA fragments from a mixture of 20 fmol of 100 nt target (shown in red) and 1 µg of non-target 460 bp dsDNA (shown in green).

<p>The original images in the green and red channels are in gray scale; a–c. The sample mixture is electrophoretically injected from the sample chamber on the bottom left towards to top right. d–g. A focusing field and a time-multiplexed DC bias field is applied to focus the target fragments (red) while washing the background (green) back towards the bottom left. The extraction well in the center is still empty, though condensation on the walls of the well can be observed. h. The extraction well is filled with 12 µl of buffer and only the focusing field is applied. The target fragments enter the buffer in the extraction well. Once focusing is complete, the output buffer can be collected with a pipettor.</p