Short tandem repeat (STR) analysis is increasingly being used in forensic case analysis because of the large number of STR loci in the human genome and their highly polymorphic nature. An automated DNA sequencer using high sensitivity infrared (IR) fluorescence technology was used to detect STR allele patterns from simulated forensic samples. The amplification strategy used a 19 base pair extension on the 5′ end of one of the PCR primers. This sequence is identical to the sequence of a universal M13 Forward sequencing primer which is included in the amplification reaction. Allelic bands were detected by incorporation of the M13 primer-fluorescent dye conjugate into PCR products thus eliminating the need for direct conjugation of fluorescent dye to individual STR primers. By using an IR-based automated DNA sequencer and Tth DNA polymerase, polymorphic STR alleles were detected on-line rapidly and efficiently from bloodstains using only a high temperature incubation to extract DNA from blood cells. Five STR loci were also amplified using Chelex extracted DNA from simulated forensic samples. Multiplexing of three primer pairs in a single PCR mixture for amplification was accomplished using Taq polymerase. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for radioactivity and the gel handling required with silver staining and fluor detection systems. Real-time detection permits immediate visualization of the data and STR alleles are displayed as familiar autoradiogramlike images that can be analyzed by computer. By loading a 64 lane gel twice and multiplexing with three primer pairs, forensic scientists can type at least three loci from 120 samples in one day.

Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well-to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm wellto-read distance) produces sequence read lengths of up tol000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases! sample X 2 ten hour runs!day) to be sequenced with the advantages of infrared technology.Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany) and Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer & template combinations. The inclusion of cz-Thio-dNTP's in the labeling reaction increases signal intensity two-to three-fold.Enhanced throughput of determining DNA sequence information by increasing the number of signal channels is an overriding objective of several groups, including our own. Signal channels can be discriminated in several ways including: geometrically (increased number of parallel channels); temporally (increased speed of data collection); optically (increased number of fluorophores); by intensity (increased number of signal amplitude states); and/or by fluorescence lifetime (via timeor phase-resolved fluorescence detection).In addition, the amount of information collected per signal channel is important. Trade-offs occur between difference approaches depending on which parameters are emphasized. For example, increasing the speed of data collection may decrease the amount of useable information. On the other hand, techniques such as pulsed field electrophoresis may succeed at increasing the information collected per signal channel at the expense of time. In addition, improvements in molecular biology techniques can increase the information content per signal channel.This report reflects results of attempts to enhance throughput by: 1) improving the information content in each lane by using longer electrophoresis gels; 2) increasing the number of gel electrophoresis lanes by progressively reducing their physical size; and, 3) improving the information content for each sample by optimizing the molecular biology of internal labeling.These enhancements exploit the advantages of near-infrared fluorescence technology, such as improved sensitivity and economy of operation. 66 !SPIE Vol. 2386 O-8194-1733-5/95/$6.OO Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/25/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx

The detection of prostate-specific antigen (PSA) and visualization of spermatozoa from forensic-type samples containing semen exposed to simulated gastric juice was investigated as a support for forensic practice. Samples of simulated gastric juice mixed with semen were prepared and incubated for up to 4 h at 37 degrees C. Samples were deposited on cotton cloth and on ceramic plates and allowed to dry. The samples were examined for the presence of PSA using the Seratec PSA Semiquant immunochromatographic membrane test. Microscope slides were prepared, stained, and analyzed for spermatozoa. Spermatozoa were detected in all samples, and PSA was detected on neat samples and on samples from the ceramic plate after incubation for up to 4 h. PSA was not detected in the samples deposited on cotton cloth at incubation times greater than 15 min. This may serve as a support for examinations performed when vomit or vomit-stained evidence is submitted for analysis.

The conditions governing the intensity and stability of the pink colour given by phenolphthalein in alkaline solution have been examined and a procedure for its colorimetric determination proposed. The results of collaborative trials of this method and of its application to certain pharmaceutical preparations are presented.

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