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

The Potential and Challenges of Nanopore Sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.Molecular and Cellular BiologyPhysic

Simple, robust methods for high-throughput nanoliter-scale DNA sequencing

We have developed high-throughput DNA sequencing methods that generate high quality data from reactions as small as 400 nL, providing an approximate order of magnitude reduction in reagent use relative to standard protocols. Sequencing of clones from plasmid, fosmid, and BAC libraries yielded read lengths (PHRED20 bases) of 765 ± 172 (n = 10,272), 621 ± 201 (n = 1824), and 647 ± 189 (n = 568), respectively. Implementation of these procedures at high-throughput genome centers could have a substantial impact on the amount of data that can be generated per unit cost

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

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