Fluorescence-based mutation detection |
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Authors: | Jane S Ellison |
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Institution: | (1) National Institutes of Health, National Center for Human Genome Research, 9000 Rockville Pike, Building 49, 20892 Bethesda, MD |
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Abstract: | Conventional SSCP analysis of DNA amplified by polymerase chain reaction (PCR-SSCP) is one of the simplest and most reliable
tools for identifying point mutations, and small insertions or deletions. The sensitivity of the technique is increased by
using the Applied Biosystems (ABI) semiautomated DNA sequencer equipped with GENESCAN 672 software for F-SSCP. The four-dye
ABI system permits a red dye-labeled internal lane standard to be run in the same lanes as the DNA being examined, leaving
three dye colors for labeling DNA of interest. The internal lane standard is used to normalize gels or correct for minor differences
in apparent electrophoretic mobility between lanes. Correction for these lane-dependent differences in migration and the capability
to stack data from two different lanes on the computer screen makes it possible to detect sequence variants that produce very
small mobility shifts. Coelectrophoresis of control and unknown DNA in the same lane, using different dye labels for each,
is also helpful for detecting sequence variants that produce small mobility changes. Multiplexing multiple F-SSCP targets
in the same lane increases sample throughput. |
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Keywords: | DNA mutational analysis point mutation suppressor mutation DNA single-stranded single-strand conformation polymorphism nucleic acid conformation heterozygote detection genetic screening nonradioisotope detection polymerase chain reaction/methods |
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