The Y-Plex 6 kit is a Y-STR identification kit marketed by Reliagene Technologies that allows for the identification of the male component from evidentiary samples, and the identification of paternally related individuals. This kit utilizes primers designed to amplify the DYS393, DYS19, DYS389II, DYS390, DYS391, DYS 385 loci located in the NRY region of the Y chromosome. The objective of the experiments and results described below was the validation of the Y-Plex 6 kit on the ABI PRISM377 DNA Sequencer for implementation into casework at the Armed Forces DNA Identification Laboratory (AFDIL). The validation process included an intra-gel and inter gel precision study, sensitivity study, the successful typing of male DNA from; chelex extracted blood samples, organically extracted blood and tissue samples and Qiagen extracted blood samples, reproducibility, the ability to identify paternally related individuals, specificity, and the ability to distinguish between male: male and male: female mixtures. All experiments were performed according to the protocol set forth in the Y-Plex 6 Instruction Manual v3.0.

There is a general trend in the forensic field for laboratories to move toward high sample throughput with out losing data quality, therefore, the experiments described below were aimed at validating the ABI 3100 Genetic Analyzer and BigDye version 1.1 (ABI, Foster City CA) for use in processing mtDNA case samples in a more efficient manner. Prior to implementing DNA technologies into a Forensic Laboratory, the Scientific Working Group on DNA Analysis and Methods (SWGDAM) requires all laboratories perform an internal validation on those methods. An essential part of the validation process is identifying the limitations of the reagents, kits or machines being validated, thus this validation included defining the upper and lower detection thresholds for the ABI 3100 Genetic Analyzer and BigDye version 1.1, standardization of the amount of product to be sequenced, purification techniques, and evaluation nonprobative case samples.

The human cytomegalovirus (HCMV) UL70, UL102, and UL105 genes are predicted to encode essential proteins that assemble the replicative helicase–primase complex based on sequence and genome position similarities to putative herpes simplex virus type 1 (HSV-1) counterparts. Consistent with this prediction, they are required for transient complementation of DNA synthesis. However, little is known about their physical interactions and biochemical activities, primarily because of their restricted expression in HCMV-infected cells. To look for assembly of the predicted complexes, we prepared rabbit polyclonal antisera and used Semliki Forest Virus (SFV) vectors to express untagged and glutathione-S-transferase (GST)-tagged UL70, UL102 and UL105 proteins. The UL70 and UL105 proteins co-purified with the GST-tagged UL102 protein from triply-infected baby hamster kidney cells (BHK-21), and pUL70, but not pUL105, co-purified with pGST–UL102 from dually infected BHK-21 cells. In immunoprecipitation experiments with untagged SFV-expressed proteins, pUL70 or pUL105 coprecipitated with pUL102, pUL102 or pUL70 co-precipitated with pUL105; and pUL102 or pUL105 coprecipitated with pUL70. Comparison of the GST-pull down and immunoprecipitation experiments suggested that the amino-terminal GST-tag interfered with certain pairwise interactions. These results support the prediction that the HCMV helicase–primase proteins assemble a three-protein heteromeric complex, and show that each protein contacts both partners.

The primary mission of the mtDNA section of the Armed Forces DNA Identification Laboratory (AFDIL) is to aid the Central Identification Laboratory Hawaii (CILHI), in the identification of human skeletal remains recovered from World War II, the Korean War, and the Vietnam Conflict. For blood specimens, AFDIL currently sequences mtDNA control region (nt15971-nt599) amplicons with ¼ reaction (in relation to the manufacturer's recommended quantity) Big Dye v. 1.0 (ABI, Foster City, CA) and seven primers in individual reactions. Sequencing products are analyzed on the ABI 3100 Genetic Analyzer using POP6 and a 36cm capillary array. However, since the ABI 3100 Genetic Analyzers and quarter BigDye sequencing reactions were validated, several problems have arisen in generating full control region sequences. These include the necessity of eight or more primers to cover the entire control region (CR), the use of alternative primers to cover difficult HV1 and HV2 C-stretches, and the loss of peak definition due to shouldering caused by a high degree of secondary structure within parts of the control region. The experiments to validate the use of full strength or half strength BigDye reactions and 50cm capillary arrays for analyzing mtDNA family references were conducted in an effort to standardize sequencing reactions and to reduce the amount of resequencing needed to confirm control regions, thus saving time and money. First, a sensitivity study in which 300pg control region (nt15971-599) amplicons were sequenced with F15, F29, F34, R16410 and F15971 at either full or half reaction BigDye v. 1.0 and analyzed using a 50 cm array was performed. The sequence generated from these primers produced 500 or more well- defined bases, with half sequencing reactions having to be cut 20-50 base pairs sooner than full reactions. However, this did not affect the ability to confirm sequencing regions since an overlapping primer scheme was used. Further studies involved using only ½ BigDye reactions and demonstrated that the sequences obtained from a 50cm array could accurately generate full control region data using four to five sequencing primers. Next, eight case samples previously typed and reported using ¼ BigDye reactions on the 36cm array were amplified, sequenced and typed on the 3100 using a 50cm array to demonstrate accurate