The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applications such as multiplex PCR rely on primers binding to unique regions in a genome. Competing side reactions with other primer pairs or template DNA decrease PCR efficiency: Freely available primer design software such as Primer3 screens for potential hairpin and primer-dimer interactions while selecting a single primer pair. The development of multiplex PCR assays (in the range of 5 to 20 loci) requires the screening of all primer pairs for potential cross-reactivity. However, a logistical problem results due to the number of total number of comparisons required. Comparing the primer set for a 10-plex assay (20 total primer sequences) results in 210 primer-primer combinations that must be screened. The ability to screen sets of candidate oligomers rapidly for potential cross-reactivity reduces overall assay devlelopment time. Here we report the application of a familiar sliding algorithm for comparing two strands of DNA in an overlapping fashion. The algorithm has been employed in a software package wherein the user can compare multiple sequences in a single computational run. After the screening is completed, a score is assigned to potential duplex interactions exceeding a user-defined threshold. Additional criteria of predicted melting temperature (Tm) and free energy of melting (deltaG) are included for further ranking. Sodium counterion and total stand concentrations can be adjusted for the Tm and deltaG calculations. The predicted interactions are saved in a text file for further evaluation.

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or tm of short duplex DNA oligomers (approximately 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting tm from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental tm's of short duplex DNA oligomers is assessed.

A likelihood ratio (LR) system is defined as the entire pipeline of the measurement and interpretation processes where probabilistic genotyping software (PGS) is a piece of the whole LR system. To gain understanding on how two LR systems perform, a total of 154 two-person, 147 three-person, and 127 four-person mixture profiles of varying DNA quality, DNA quantity, and mixture ratios were obtained from the filtered (.CSV) files of the GlobalFiler 29 cycles 15s PROVEDIt dataset and deconvolved in two independently developed fully continuous programs, STRmix v2.6 and EuroForMix v2.1.0. Various parameters were set in each software and LR computations obtained from the two software were based on same/fixed EPG features, same pair of propositions, number of contributors, theta, and population allele frequencies. The ability of each LR system to discriminate between contributor (H1-true) and non-contributor (H2-true) scenarios was evaluated qualitatively and quantitatively. Differences in the numeric LR values and their corresponding verbal classifications between the two LR systems were compared. The magnitude of the differences in the assigned LRs and the potential explanations for the observed differences greater than or equal to 3 on the log10 scale were described. Cases of LR < 1 for H1-true tests and LR > 1 for H2-true tests were also discussed. Our intent is to demonstrate the value of using a publicly available ground truth known mixture dataset to assess discrimination performance of any LR system and show the steps used to understand similarities and differences between different LR systems. We share our observations with the forensic community and describe how examining more than one PGS with similar discrimination power can be beneficial, help analysts compare interpretation especially with low-template profiles or minor contributor cases, and be a potential additional diagnostic check even if software in use does contain certain diagnostic statistics as part of the output.

Anonymous liquid blood samples with self-identified ethnicities were purchased from Interstate Blood Bank (Memphis, TN) and Millennium Biotech, Inc. (Ft. Lauderdale, FL) and extracted using a modified salt out procedure (1). The extracted DNA was then quantified using UV spectrophotometry at 260 nm and a PicoGreen assay (2). A 150-µL aliquot of the extracted DNA solution was directly quantified in a Cary 100 double-beam spectrophotometer (Varian Analytical Instruments, Walnut Creek, CA). Low volume micro-cuvettes allowed for accurate absorbance measurements (A = 0.2 to 0.6) without prior dilution of the stock extracted DNA. Sample concentrations were adjusted to 1 ng/µL for typing purposes using the PicoGreen assay values. Fifteen autosomal STR markers (the 13 CODIS core loci and D19S433 and D2S1338) were typed along with amelogenin using the Applied Biosystems AmpF1STR® Identifiler™ kit (3). PCR amplification was carried out on a GeneAmp® 9700 (Applied Biosystems) using 1 ng of DNA according to kit protocols (3) with the exception of reduced volume reactions (5 µL instead of 25 µL) and reduced cycles (26 instead of 28). Amplification products were diluted 1:15 in HiDi™ formamide and GS500-LIZ internal size standard (Applied Biosystems) and analyzed on the 16-capillary ABI Prism® 3100 Genetic Analyzer without prior denaturation of samples. POP™-6 (Applied Biosystems) rather than POP™-4 was utilized for higher resolution separations on a 36 cm array. Samples were injected

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