The Visible Genetics Clipper sequencer is a new platform for automated DNA sequencing which employs disposable MicroCel cassettes and 50 μm thick polyacrylamide gels. Two DNA ladders can be analyzed simultaneously in each of 16 lanes on a gel, after labeling with far‐red absorbing dyes such as Cy5 and Cy5.5. This allows a simultaneous bidirectional sequencing of four templates. We have evaluated the Clipper sequencer, by cycle‐sequencing of an M13 single‐stranded DNA standard, and by coupled amplification and sequencing (CLIP) of reverse‐transcribed human immunodeficiency virus (HIV‐1) RNA standards and clinical patient samples. (i) Limitations of instrument. We have examined basic instrument parameters such as detector stability, background, digital sampling rate, and gain. With proper usage, the optical and electronic subsystems of the Clipper sequencer do not limit the data collection or sequence‐determination processes. (ii) Limitations of gel performance. We have also examined the physics of DNA band separation on 50 μm thick MicroCel gels. We routinely obtain well‐resolved sequence which can be base‐called with 98.5% accuracy to position ˜450 on an 11 cm gel, and to position ˜900 on a 25 cm gel. Resolution on 5 and 11 cm gels ultimately is limited by a sharp decrease in spacing between adjacent bands, in the biased reptation separation regime. Fick's (thermal) diffusion appears to be of minor importance on 6 cm or 11 cm gels, but becomes an additional resolution‐limiting factor on 25 cm gels. (iii) Limitations of enzymology. Template quality, primer nesting, choice of DNA polymerase, and choice between dye primers and dye terminators are key determinants of the ability to detect mutations and polymorphisms on the Clipper sequencer, as on other DNA sequencers. When CLIP is used with dye‐labeled primers and a DNA polymerase of the F667Y, Δ(5′ → 3′ exo) class, we can routinely detect single‐nucleotide mutations and polymorphisms over the 0.35—0.65 heterozygosity range. We present an example of detecting therapeutically relevant mutations in a clinical HIV‐1 RNA isolate.

We present a new method for the linearization and alignment of data traces generated by multilane automated DNA sequencing instruments. Application of this method to data generated with the Visible Genetics Open Gene DNA sequencing system (using MicroCel 700 gel cassettes, with a 25 cm separation distance) allows read lengths of > 1,000 nucleotides to be routinely obtained with high confidence and > 97% accuracy. This represents an increase of 10-15% in average read length, relative to data from this system that have not been processed in the fashion described herein. Most importantly, the linearization and alignment method allows usable sequence to be obtained from a fraction of 10-15% of data sets which, because of original trace misalignment problems, would otherwise have to be discarded. Our method involves adding electrophoretic calibration standards to the DNA sequencing fragments. The calibration standards are labeled with a dye that differs spectrally from the dye attached to the sequencing fragments. The calibration standards are identical in all the lanes. Analysis of the mobilities of the calibration standards allows correction for both systematic and random variation of electrophoretic properties between gel lanes. We have successfully used this method with two-dye and three-dye DNA sequencing instruments.

One of the most significant advances in light detection methods in recent years is the development of CCD technology. There is a great difference, however, in merely detecting light versus providing accurate photometric measurements of a field of view. Many factors must be taken into account in controlling CCD and camera characteristics, data acquisition and subsequent data processing. Calibration techniques are particularly significant if we are to ensure proper photometric analysis.This presentation will cover the important variables and controls needed, and provide illustrations of the value of videobased measurements in practical applications.The measurement of luminance distributions became a topic of research in the 1960's, when film cameras were used to perform absolute photometry.1 The procedures were complex, time-consuming and fraught with problems.Nevertheless, groundwork was laid which suggested that photographic photometry would be a viable and powerful technique if the chief source of the problem, the film itself, could be replaced by a more stable and reliable means of collecting the image. With the advent of the Charge Coupled Device, (CCD), most of the problems of the film cameras are removed. The CCD array, however, brings with it a new series of problems. To produce an accurate system of photometry, it is essential to understand the problems inherent to CCD camera systems. Techniques must be developed to overcome their idiosyncrasies, calibration methods must be developed, and controls must be exercised to ensure accuracy. Achieving these goals is not a trivial pursuit. This paper will address the many factors involved in the development of a high accuracy CCD measurement system to provide NIST-traceable reliable photometry. FORMS OF CCD SYSTEMMany types of CCD cameras exist. These range from inexpensive mass-produced systems for camcorders and low cost television cameras, to the highest quality systems used in scientific applications such as astronomy.Low cost cameras typically are used in non-broadcast television for surveillance and other non-critical applications. These normally use the NTS signal convention, and their purpose is to collect and transmit images rapidly. Photonic quality suffers due to high noise and low sensitivity, and such cameras can introduce insurmountable problems if it is attempted to use them to perform high accuracy photometry. At the other end of the range are cameras of very high sophistication, incredible sensitivity and stability. These are designed for critical applications in astronomical observations, but their cost is prohibitive for normal photometric applications.Between the two extremes is the "Slow Scan" camera, which offers technology quite different from the television systems. These offer a wide range of user controls, as is essential for high accuracy photometric applications. Such 28 / SPIE Vol. 2550 0-8194-1909-5/95/$6.00 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/23/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOf...

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de Haas -van Alphen (dHvA) experiments of a slowly cooled, stage-2 SbC15-graphite compound show splitting of the dHvA frequencies of the graphitic energy bands. The splitting is caused by the interaction between states separated by intercalate layers. The values of the interactions for the two graphitic bands are estimated to be 0.6 and 0.7 meV. X-ray diffraction shows a doubling of the caxis lattice vector which modifies the energy bands. With use of the estimated values of the c-axis interaction, it is shown that a band-conduction mechanism can account for the large anisotropy of the conductivity of stage-2 SbC1&-intercalated graphite.