ISSN:
0173-0835
Keywords:
Infrared DNA sequencer
;
Automated DNA sequencer
;
Dynamic range
;
Fluorescence
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
We are currently developing genotyping software and protocols for use on the Li-Cor model 4000(L) infrared fluorescence DNA sequencers. During the development of the genotyping software it became apparent that the potential dynamic range of the instrument was not being realized when the data collection parameters were optimized to produce a high-contrast image on the computer screen. In particular, we were unable to obtain peaks with signal to noise (S/N) greater than about 30:1 and the stronger peaks often saturated the detector. Because the numerical dynamic range available in 16-bit data collection mode exceeds 65000, our limited dynamic range of about 30 in actual data was somewhat puzzling. Hence, we undertook a study to explore the dynamic range and linearity of the Li-Cor DNA sequencer in order to minimize background fluorescence and noise as well as maximize the available S/N. Data is presented on the background and noise using different polyacrylamide gel mixes with various signal gain and offset values, and the relative contribution of the glass, gel and instrumentation to the background fluorescence is discussed. Based on these results, optimum gain and offset values were determined that maintain the background fluorescence at approximately 1-2% of the maximum dynamic range with a minimum amount of noise. Using these optimized values, we determined the detection system is linear over three orders of magnitude by titrating known quantities of an infrared fluorophore-labeled primer. In addition, we were able to detect approximately 15.2 amoles of labeled primer. The results provided by this study establish a set of guidelines for evaluating how to set the signal gain and offset in order to minimize the background and maximize the S/N and dynamic range of the Li-Cor sequencer.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150170405
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