(141a) Free-Solution Conjugate Electrophoresis Sequencing of Over 250 Bases of ssDNA

The list of genomes that have been or will be studied by sequencing continues to grow, with applications ranging from agriculture to molecular medicine and understanding cancer. Next-generation sequencing methods are enabling a majority of these ultra-high throughput projects by lowering the cost and time associated with genomic sequencing studies. However, a need remains for low cost, targeted, high-accuracy sequencing of limited medical samples for which rapid electrophoresis-based methods are ideal. Significant research has been done to translate electrophoretic separations onto microfluidic devices, resulting most recently in successful sequencing of 600 bases in 6.5 minutes by chip electrophoresis. The advantages of microfluidic separations for these targeted medical sequencing applications (faster read length, smaller sample volume, potential integration into lab-on-a-chip devices), however, are dampened by the difficulties associated with loading the viscous polymer solutions necessary to achieve size-based separations of sequencing fragments. Free-Solution Conjugate Electrophoresis (FSCE) enables size-based separations with no entangled polymer network through the specific conjugation of exactly identical mobility modifiers to each DNA molecule, which breaks the linear scaling of the charge and friction of DNA with its length. In FSCE, these completely monodisperse, mostly charge-neutral perturbing entities (?drag-tags?) are chemically conjugated to the 5'-end of ssDNA oligomers or sequencing primers. An ideal drag-tag, in addition to being completely monodisperse, must be uniquely attached to DNA and large in size in order to provide long and accurate reads; genetically engineered, highly repetitive ?protein polymers? have been developed for this use. Recent advances in FSCE drag-tags and separations have improved the sequencing length to over 250 bases of DNA separated in free-solution (using a 267-amino acid protein polymer), which is approximately a 40% increase in read-length over previously published FSCE sequencing results and is comparable to the read-length of next-generation sequencing technologies. A drag-tag with 516-amino acids has recently been produced that is almost completely monodisperse, and sequencing with this drag-tag is underway. Two examples of medical sequencing where FSCE is applicable are the donor-recipient matching process for organ transplants, where 40 HLA regions < 450 bases must be sequenced, and the detection and identification of nucleotide mutations in individual exons can affect the prognosis, diagnosis and treatment of cancer.