El papel de las Sentencias Constitucionales en los cambios socioculturales

Accomplishing the goals of this project has resulted in the development of a refined automated system, relying on robotics and bioinformatics, for high throughput mtDNA

sequencing [we note that infrastructure, instrumentation, and software development costs were independent of NIJ funding]. In order to identify samples matching common HV types for entire mtGenome sequencing, new databases of entire control region sequences were established for an African American sample, and four regional Hispanic databases. Significant differences in haplogroup and haplotype representation among the regional Hispanic databases suggests that additional attention should be paid to the issue of population subdivision, especially involving inherently heterogeneous population categories such as “Hispanics.”

This work represents the first comprehensive demonstration that variation in the mtDNA coding region can provide substantial levels forensic discrimination, over and above the variation present in the hypervariable regions of the control region. Admittedly, knowledge of relative rates of sequence evolution in the control region versus the coding region, and their relative size, made that a bit of a foregone conclusion. However, what was very much an open question at the outset of this project was whether the additional variation was distributed in such a manner that it could be practically accessed within the context of real-world forensic mtDNA testing. It is clearly—for the immediately foreseeable future—unrealistic to sequence the entire mtDNA genome for mtDNA casework samples. Normally, retrieving sequence from the control region leaves only a small amount of evidentiary extract left, so that only small portions of the coding region could be expected to be recovered. As a result of this project, we moreover know that the distribution of variation in the coding region is such that sequencing any practically attainable number of small fragments, using the same general approach for any case in question, will rarely

provide additional forensic discrimination (this is presented in detail in Coble et al., 2005). Using high-throughput mtGenome sequencing of common HV types, we have identified SNP sites that are highly useful for providing additional discrimination for those HV-types (and their near relatives) that will most often be involved in cases where additional discrimination is needed. This approach was particularly useful in the case of common HV types in the US Caucasian population. Smaller available sample sizes for whole mtGenome sequencing somewhat limited the ability to identify a large number of discriminatory sites in African American and Hispanic common HV types; and in some Hispanic types we simply observed low levels of variation (a limitation for any approach). Nonetheless, for all population groups studied, useful SNP panels were identified. For Caucasians, multiplex ASPE assays were designed, optimized, and demonstrated to work effectively on degraded samples.

Based on the results reported from this project, the primary use of the SNP panels is to distinguish among multiple matching samples, refining inclusion and exclusion from within a closed set of compared sequences. An alternative desirable application would be to simply increase the evidentiary significance of a single match involving one of the common types. For example, instead of an HV type that matches 7% of the population, combining SNP data might result in a SNP/HV type present in 0.5% of the population, increasing the significance of the match. However, for this purpose, large population databases that combine SNPs and HV sequences are necessary. AFDIL is presently planning to add SNP panel typing to current family reference databasing, which will quickly increase to large numbers, and will make that data available as the project is implemented. In this case, it would be only be necessary to apply the SNP panels to those population samples for which they are relevant. However, in addition to having the databases available, wide use of this approach will also require labs to have access to

population search engines that accommodate the SNP data (AFDIL’s LIMS system already incorporates this, but there are no current plans for the search function to be openly available). As the field progresses toward use of discriminatory SNPs, modifications to the publicly available searchable databases such as CODISmt and EMPOP would be highly desirable.

This project is undoubtedly only the beginning of identifying methods for obtaining data ranging over the entire mtDNA genome, to powerfully increase forensic discrimination. The ASPE assays we designed give forensic laboratories this ability, now, for many of the

circumstances where need will arise. However, the implementation of the ASPE assay, while simple in practice, carries a high burden of effort regarding primer quality control, and we suspect this will be a limitation to very widespread adoption of this technique. The field of forensic DNA analysis is aggressively developing new SNP typing methods, and maximum benefit to the field would be in commercial availability of a (fast, cheap, and easy) SNP typing kit, where primary QC issues are removed from individual laboratory. Fortunately, interest has been expressed by a number of companies in this regard. We note that, with DNA sequencing becoming ever less expensive and time consuming, that it may be feasible to sequences the entire mtGenome from reference samples, in the not-distant future. This could then direct the

investigation of degraded evidentiary samples to particular portions of the mtGenome that could be targeted for a high probability of excluding unrelated individuals. In any case, the primary mtGenome data generated for this study will be a highly useful reference for guiding the development of new approaches.