ABSTRACT
We propose a novel method for electrokinetic injection of DNA samples into capillaries from nanoliter gel micropads, deposited on glass slides, which are coated with electroconducting film. Theoretical and experimental proof is presented for the proposed method. The method allows efficient and highly precise injection without physical contact between the gel pad and the capillary. Read length of more than 700 bp at Q20 has been reproducibly demonstrated in fused-silica capillaries using the proposed injection technique. Based on the obtained results we discuss a novel DNA sequencing system which combines DNA amplification and cycle sequencing in arrays of subnanoliter gel micropads and high-throughput electrophoretic separation in monolith multicapillary arrays.
Subject(s)
DNA/isolation & purification , Electrophoresis, Microchip/methods , Models, Chemical , Nanotechnology/methods , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Base Sequence , Electrophoresis, Microchip/instrumentation , Equipment Design , Gels/chemistry , Indicators and Reagents , Polymerase Chain Reaction/instrumentation , Reproducibility of Results , Sensitivity and Specificity , Sequence Analysis, DNA/instrumentation , Spectrophotometry, UltravioletABSTRACT
Recently, we developed a family of high-performance automated capillary DNA sequencing instruments based on a single-photon detection of fluorescently labeled DNA fragments. Our machines employ digital and broadband techniques, essential for achieving superior instrument sensitivity and dynamic range. In the present paper, we discuss limitations of the instrument's performance caused by the nonlinearity of single-photon detectors as well as methods for nonlinearity compensation which increase the detection dynamic range and base-calling accuracy.
