NCJ Number
218819
Date Published
July 2000
Length
15 pages
Annotation
This article reports on a study that documented and developed a remedy for an artifact (not a true characteristic of a specimen) produced in the polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci when using the PE Biosystems Genotyper Version 2.5 Software Package.
Abstract
The artifact identified is called "stutter." Some researchers have concluded that stutter occurs from a slipping of the polymerase during replication of a region of repeated sequence (Schlotterer and Tautz, 1992; Wash et al., 1996). During the current analysis, peaks in stutter positions that exceeded programmed stutter-filter thresholds but were not labeled were occasionally detected. This phenomenon was also detected with the PE Biosystems preset stutter-filter threshold and was therefore not due to the incorporation of the empirically derived data. Investigation of the software macros used for filtering allele labels showed unexpected effects termed "Stutter-filter Back Talk." Apparently as each locus within a color set is analyzed in the macro, labeled peaks at stutter positions are subjected in series to the stutter filters of subsequently analyzed loci. Narrowing the stutter-filter window to prevent adjacent off-ladder allele peaks from being filtered or used as the comparison peaks for the filtering of peak labels found within a repeat unit in length is being pursued. Applying the stutter filter twice to those loci analyzed last within a color set is also being pursued. This would provide the desired filtering of labels from peaks at stutter positions from alleles separated by less than a full repeat unit. Regardless of modifications made to the Genotyper macros, the software should not be relied upon as the only source of information in making allele calls. Validation of the software combined with vigilant critical analysis must be conducted in order to ensure accurate and complete genotyping. 2 tables, 7 figures, and 6 references