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Australasian Biotechnology (backfiles)
AusBiotech
ISSN: 1036-7128
Vol. 6, Num. 5, 1996
Australasian Biotechnology,
Volume 6 Number 5, September/October 1996, pp.283-284

High Speed and High Throughput Microsatellites

Simone Peric,

Corbett Research, 1/14 Hilly St., Mortlake, NSW 2137, Australia


Code Number: AU96014
Size of Files:
    Text: 6.3K
    Graphics: Photographs (jpg) - 117.3K

Described is a dedicated electrophoresis system (Corbett GS-2000) for microsatellite and other advanced DNA analysis, using laser scanning fluorescence detection for measurement.

Evenly distributed, highly polymorphic genetic markers are crucially important for modern animal and plant breeding programs, and in medical genetics. Microsatellites (short sequence repeats), typically consisting of nucleotide triplet our doublet repeats, are gaining widespread attention as the most useful marker, mainly because of their ease of detection by the PCR technique. The Corbett GS-2000 offers users an automated fragment analyser for high speed, high throughput microsatellite analysis at a fraction of the cost of similar imported products (Fig.1). In addition, it can also be used for RFLP's, SSCP, RAPD's, quantitation and sequencing.

    Figure 1. The Corbett GS-2000 Electrophoresis System

The GS-2000 is a real time gel electrophoresis system with built in solid state laser for detection of dye labelled DNA fragments. When labelled DNA fragments pass through the beam of the scanning laser, the fluorescent energy is picked up by a lense and focused onto a fibre optic cable, which directs the light to the photomultiplier. The amplified signal is then sent to the computer where together with synchronised scan information a gel image is developed.

The laser can be used to excite most of the common fluorophores used to label DNA. The GS-2000 system is capable of detecting and sizing 100 attomoles of fluorescently labelled DNA in a single peak. For applications that do not require high resolution, non-denaturing gels can be run. Ethidium bromide is used to detect as little as 500 attomoles thus eliminating the need to label primers in some applications.

The GS-2000 is fully automatic with a built-in microprocessor to control all operating parameters and functions. A 586 pentium computer captures gel images and performs all data processing. Once the data has been collected, the software automatically time aligns all the lanes and compares them with a calibration curve developed in one of the lanes where external standards have been loaded. From this calibration curve, molecular weight and concentrations can be calculated.

High speed and high throughput microsatellite analysis

Manual methods for microsatellite analysis are time consuming and prone to errors. Mobility differences across the gel can result in inaccurate size calling and allele identification.

The GS-2000 offers accurate high speed and high throughput microsatellite analysis. The data is collected in real time resulting in relatively even band spacing and more accurate allele calling. Exact sizing is obtained by use of external standards, which are loaded throughout the gel. The marker size is entered into the calibration setting, a calibration curve is calculated and then applied to all lanes. Mobility differences are also corrected in this manner, as the external standards are used to correct for any migration differences that may occur.

High speed and high throughput microsatellite analysis is achieved in a number of ways. . The GS-2000 has a 96 lane capacity and the polyacrylamide gel can be reused up to four times. This reduces time spent on pre-running and preparing and pouring gels. Microsatellites up to 200bp, can be resolved to 2 bp in less then an hour, on a 12cm well to read polyacrylamide gel run at high voltage (Fig. 2). Samples maybe time multiplexed so that 384 samples can be run in less then 4 hours, on the same gel. The high speed and throughput of this system means that not only are more samples run in a day but there is less `hands-on' time, and reuse of the gel means less money spent on reagents.

    Figure 2- Microsatellite analysis. The first panel shows the image of a complete electropherogram, with details of lanes 1 and 5 in the other panels. 17 Hex labelled 2bp repeat microsatellites, in the size range 100-140bp, were run for 40 minutes on a 0.25mm, 12cm well to read polyacrylamide gel. From left to right, a picture of the gel image, a chromatogram of the molecular weight marker and band report and a chromatogram of a two bp repeat microsatellite, 119 and 121bp.

Conclusion

The Corbett GS-2000 DNA Fragment Analyser offers precise high speed and high throughput analysis at a competitive price. The system is ideal for automated fluorescent microsatellite analysis using a single dye. An external standard is used for mobility correction and for accurate allele sizing and identification. The system can also be used for rapid analysis of RAPD's, SSCP's, quantitation and sequencing offering a fast, accurate, inexpensive system for DNA analysis.

Corbett Research

Corbett Research is a manufacturer of scientific instrumentation for Molecular Biology and Life Sciences. The company was formed in 1989, and is wholly Australian owned. The first product developed, in 1990, was a high speed capillary thermal cycler. Since then, a whole range of thermal cyclers have been developed, from 0.5mL tube units to 384 well microplate machines.

Over the last two years Corbett has developed an Automated DNA fragment analyser, applications include quantitation, microsatellite analysis and DNA sequencing. The instrument is the first of its kind to be manufactured in Australia and is very competitively priced. Internationally, Corbett has over 14 distributors and over 50% of production is exported.

Copyright 1996 Australian Biotechnology Association Ltd.


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