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Forensic DNA Laboratories currently analyze Short Tandem Repeat (STR) sequences using migration of amplified STR products through polyacrylamide gels (PAG), and/or capillary gel methods. Allele detection is accomplished using a variety of methods including staining PAG or activation of fluorescent primers by lasers. The Palm Beach County Sheriff’s Office (PBSO) Crime Laboratory and the Alabama Department of Forensic Sciences (ADFS) have validated and implemented the silver-staining and SYBR Green detection systems on casework and are presently validating fluorescently tagged STR alleles using the Hitachi FMBIO^® instrument. The Broward County Sheriff’s Office (BSO) Crime Laboratory is validating the capillary electrophoresis STR detection system (CE310) from Perkin-Elmer.

Analysis of over 10,000 individuals from population and convicted offender database samples using the CSF1PO, TPOX and TH01 loci has provided approximately CTT profiles with microheterogeneity and/or three banded patterns. PBSO, ADFS and BSO Crime Laboratories have collaborated on the verification of the "off-ladder" alleles and three-banded patterns observed during the analysis of these samples. The following multiplex systems were used to detect the CTT alleles in these 43 samples: a) Promega CTT with silver and SYBR Green staining, b) Promega CTTv and/or PowerPlexÔ fluorescently tagged primers using the Hitachi FMBIO^® for detection and c) Perkin-Elmer Green STR system using the CE310 for allele detection. In addition, individual locus-specific fluorescently tagged primers from Promega were also used to verify the microheterogeneity.

Out of the over 10,000 individuals typed for CTT, three-banded patterns were detected in 20 individuals including 18 at the TPOX locus, 1 at the CSF1PO locus and 1 at the TH01 locus. Identical three-banded patterns were observed for all 20 samples using all available allele detection systems. The locus origin of the three bands were verified using locus-specific fluorescent primers. Eleven individuals had what appeared to be "9.3, 10" alleles at the TH01 locus using silver and SYBR Green staining. All were confirmed to be "9.3, 10" alleles using the CTTv, PowerPlexÔ and Green Star multiplex systems. Three samples exhibited a homozygous "10" allele at the TH01 locus which was confirmed using all detection systems available.

Amplified STR products that migrated between and above or below the allelic ladder standards were detected in nine individuals and were designated as "off-ladder" alleles. The Hitachi or CE310 software did not assign any allele size/type on these STR products that did not correspond to any of the available allelic ladders. The Hitachi designated these alleles as "out of range", the CE310 as "off ladder". Although electrophoregrams and gel files showed similar migration location with respect to the allelic ladders (example CSF1PO allele 10.x), basepair sizing of these alleles did not always agree between the instruments. Interpretation of these samples will be presented as well as original silver and SYBR Green staining gel files, and fluorescently tagged STR primer data using the Hitachi FMBIO^® and the Perkin-Elmer CE310.