(652b) Advances In DNA Sequencing And Forensic Sizing By Free-Solution Conjugate Electrophoresis In Microfluidic Devices

Recent advances have been made in the development of Free-Solution Conjugate Electrophoresis (FSCE) as a method for long-read DNA sequencing and rapid forensic sizing (?DNA fingerprinting?) in microfluidic devices. Size-based separations of DNA molecules are critically important to current methods of DNA sequencing, genotyping and forensic analyses; these separations are presently achieved by the use of a polymeric sieving matrix that sorts the DNA fragments in order from smallest to largest. Achieving size-based separations of DNA molecules on microchips in free-solution electrophoresis with no polymer matrix will eliminate the time-consuming, difficult step of loading viscous polymeric matrices into the glass or plastic devices. Additionally, there are many speed and cost advantages to performing electrophoretic separations in miniaturized ?lab-on-a-chip? devices, which promise higher throughput and potentially, a lower per-run cost for genetic analysis.

In FSCE, DNA primers are conjugated end-on to a monodisperse polyamide ?drag-tag? that subsequently enables the size-separation of DNA in free solution. The drag-tags effectively act as hydrodynamic ?parachutes,? breaking the linear scaling of the charge-to-friction ratio of DNA in electrophoresis. If each DNA molecule is modified by the addition of a single, monodisperse polyamide drag-tag, one unique peak results for each uniquely sized DNA molecule, and high-resolution separations of a mixture of different-sized DNA molecules can be accomplished by microchannel electrophoresis without a polymeric gel or sieving matrix. Both non-natural, highly repetitive, genetically engineered polypeptide drag-tags and poly-N-substituted glycine (?peptoid?) drag-tags synthesized in our lab have been used to separate DNA fragments for sequencing, genotyping and forensic analysis applications.

High-resolution FSCE separations have been previously hindered by the lack of suitable, monodisperse drag-tags. Newly developed polypeptide drag-tags, however, have been used to separate DNA sequencing fragments with substantially higher resolution and cleaner results than previously reported for this method. The full potential of rapid FSCE separations of DNA sequencing fragments is demonstrated on microfluidic chips, where sequencing fragments are resolved in approximately 110 seconds. In addition to sequencing, drag-tags have also been used to separate DNA fragments for genotyping and ?DNA fingerprinting? forensic applications. A highly multiplexed single-base extension (SBE) assay using peptoid drag-tags was developed to genoptype point mutations in exons 5-9 of the p53 gene, a gene associated with many types of human cancer; sixteen point mutations are analyzed in under 70 seconds by microchip electrophoresis in free solution. Preliminary ?DNA fingerprinting? analysis by FSCE using the ?miniSTR? primers developed by Butler et al. (J. Forensic Sci. 2003) has also been developed to size fragments up to 150 bases in length. Several challenges essential to further successful, rapid FSCE analyses on microchips are also currently under investigation, including production of significantly larger drag-tags and development of a novel, post-PCR bioconjugation method. Overall, the transition of FSCE to microfluidic devices is greatly simplified by the elimination of the need for viscous sieving matrices, and this technology promises to be easily incorporated into fully integrated microfluidic bioanalysis devices.