Statistical analysis of fragment mobility shifts obtained from multi-color fluorescent SSCP
- Applied Biosystgems Division, Foster City, CA (United States); and others
Single strand conformational polymorphism (SSCP) analysis is a widely used method for the detection of point mutations. Recently, fluorescent SSCP analysis using the Applied Biosystems 373 DNA Sequencer along with GeneScan{sup {trademark}} 672 Software has shown several distinct advantages over standard SSCP methods. The main advantage, however is that, since up to four different dyes can be detected simultaneously, it is possible to run an internal standard in every lane. This internal lane standard can then be used to normalize data from each lane with respect to each other, thus effectively eliminating lane to lane variability. An added advantage to using the internal lane standard is that it becomes possible to obtain accurate quantitative data to characterize mobility shifts. In this regard, we have used the numbers generated to assess the reproducibility of SSCP, and to determine how large a mobility shift must be in order to be termed {open_quotes}significant{close_quotes}. The ultimate goal of determining a {open_quotes}significant{close_quotes} mobility shift is to have mutations automatically called or {open_quotes}flagged{close_quotes}. In general, we have examined a large number of CHO hprt exon 3 samples and have determined that the standard deviation between lanes is very small. For example, when samples were analyzed using normalized scan numbers (one scan every six seconds), the coefficient of variation ranged form 0.006% to 0.06%. Another measurement we found informative was to determine the distance between the two strands. In this way, it is possible to utilize information from both strands simultaneously. When the samples were analyzed in this manner, the coefficient of variation ranged from 0.23% to 2.8%. Using these standard deviation values and varying confidence values, we then applied limits to the Applied Biosystems Genotyper{sup {trademark}} software and automatically flagged any samples that did not lie within these limits as mutants.
- OSTI ID:
- 134017
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-0752
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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