Abstract
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.
Highlights
Many challenges in clinical diagnostics are rooted in the difficulties of detecting rare molecules
Sequence Specific Concentration and Enrichment With the temperature dependent mobility shown in Figure 1, sequence specific enrichment of target DNA can be achieved by superimposing a small direct current (DC) bias over the timevarying electric field required for Synchronous Coefficient Of Drag Alteration (SCODA) concentration [17,19]
The total velocity of a target DNA molecule in the gel is the sum of the SCODA velocity, resulting from the time varying electric field that drives molecules toward the center of the gel, and the velocity due to the applied DC bias
Summary
Many challenges in clinical diagnostics are rooted in the difficulties of detecting rare molecules. Unlike traditional hybridization-based enrichment methods which rely on a single hybridization event per molecule, this enrichment technique ensures that each target molecule undergoes multiple hybridizations to the immobilized probes before reaching the centre of the gel, where each period of the sequence specific SCODA (ssSCODA) cycle is effectively a hybridization-wash cycle. These repeated hybridizations impart this technique with a specificity that, to our knowledge, exceeds all other hybridization based enrichment techniques, and is capable of resolving targets identical in sequence and differing only by the methylation status of a single cytosine residue
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