Enhancing the Linear Dynamic Range in Multi-Channel Single Photon Detector beyond 7OD.

We present design, implementation, and characterization of a single photon detector based on 32-channel PMT sensor [model H7260-20, Hamamatsu]. The developed high speed electronics enables the photon counting with linear dynamic range (LDR) up to 10count/s per detector's channel. The experimental characterization and Monte-Carlo simulations showed that in the single photon counting mode the LDR of the PMT sensor is limited by (i) "photon" pulse width (current pulse) of 900ps and (ii) substantial decrease of amplitudes of current pulses for count rates exceeding 10 count/s.

Detection of multi-color fluorescent objects with single photon spectrometer.

Single photon counting is the most sensitive and accurate method for detection of very weak fluorescent signals obtained in many applications such as DNA sequencing, detection of biological reporters on micro-beads, detection of droplets in micro-fluidic systems, etc. In this paper we describe the use of single photon spectrometer for detection and characterization of very weak multicolor fluorescence produced by mixtures of various fluorescent dyes and quantum dots.

Electrokinetic injection of DNA from gel micropads: basis for coupling polony technology with CE separation.

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.

Experimental study of the formation of high-resistivity zones at the gel/buffer interface in CE.

A novel, nondamaging method for experimental characterization of the formation and propagation of high-resistivity zones in CE, based on the measurement of time-dependent Joule heating on the outer capillary surface is proposed. The method detects propagation of resistive regions in capillaries in real time and allows the estimation of their velocity and resistance. The presented experimental data are in agreement with the results of the computer simulation as well as with previous data on the subject.

Novel design of multicapillary arrays for high-throughput DNA sequencing.

A novel approach to design and optimize linear multicapillary arrays (LMCAs) for high-throughput DNA sequencing is proposed. A significant increase in the number of capillary lanes is obtained due to the use of composite insertions alternately placed between working capillaries of the array and a specific combination of refractive indices of the DNA separation matrix, capillary glass, the insertions and a medium which surrounds the capillary array. Theoretical and experimental studies showed that in conjunction with a dual-side laser illumination scheme, the proposed LMCA design allows a simultaneous uniform irradiation of as many as 550 working capillaries.

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection.

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR.

Electrophoresis in capillary cells with detection gap.

A novel design of the detection zone in multicapillary arrays used for electrophoretic separation is presented. The use of a detection gap (DG), in which the reflective surfaces separating the channels of the array are eliminated, is proposed to improve the illumination and detection of the separated DNA fragments. The electric field compression in the DG is achieved by optimization of the gap geometry.

Dynamic range of fluorescence detection and base-calling accuracy in DNA sequencer based on single-photon counting.

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.

Formation of a resistive region at the anode end in DNA capillary electrophoresis.

We have studied the formation of a resistive region in the capillary during DNA separation. This effect is caused by an unequal change in the mobilities of cations and anions at the interface between the running buffer solution and the capillary. We studied the motion of the resistive region boundary by sequential removal of portions of the affected capillary end.

A family of novel DNA sequencing instruments based on single-photon detection.

We have developed a family of high-performance capillary DNA sequencing instruments based on a novel multicolor fluorescent detection technology. This technology is based on two technical innovations: the multilaser excitation of fluorescence of labeled DNA fragments and the "color-blind" single-photon detection of modulated fluorescence. Our machines employ modern digital and broadband techniques that are essential for achieving superior instrument performance.