Frequently the only biological material available to identify persons or find next of kin is DNA in a very degraded state. In these cases, the kits usually employed to carry out DNA identifications do not produce very positive results, given that all the DNA is not available, and that which is available is highly fragmented. Biochemist Adrian Odriozola has developed a tool for identifying persons from these small fragments of DNA.
His thesis, presented at the University of the Basque Country (UPV/EHU), is entitled "Developing molecular tools for analysis of highly degraded DNA samples."
In order to develop this tool, Dr Odriozola used sequences known as STR (short tandem repeat). These are sequences of small fragments of DNA that are continuously repeated and turn out to be highly useful for distinguishing between persons, due to the fact that the number of repetitions of the sequence varies according to the individual. Precisely, the kits that Odriozola investigated and that are currently employed in Forensic Genetics are based on STR analysis. However, they fail when the DNA is degraded, and so the researcher drew up a new route for analysing STRs, taking these conditions into account. Dr Odriozola has published a number of articles together with research colleagues at the UPV/EHU, in publications such as the International Journal of Legal Medicine. The University has also been granted two patents.
Working with shorter sequences
To carry out identification using DNA, it is first of all essential to undertake millions of copies of the fragment to be analysed (a process called amplification); in this case, of the STRs, which are obtained using the PCR (polymerase chain reaction) technique. So that PCR can function, primers have to adhere themselves to the two ends of each of the STR sequences. In this way, on undertaking PCR, copies are obtained of both the STR sequence and of the sequential fragments of the ends that have remained trapped between the two primers. With the tool he has developed, Dr Odriozola has enhanced the design of the current primers. Thus, the sequential fragments that have remained at the extremes of the STR are shorter than those with conventional techniques (they are called miniSTR, because there is an approximation of the focus on the STR), and an identification can be obtained despite the DNA to be analysed being fragmented.
The more STRs from the same sample analysed, the greater the precision when determining to whom the sample belongs. With regard to this, Dr Odriozola has managed to develop sufficient tools (primers) to study 14 and 11 mini STRs, employing two kits that are mutually combinable. Besides developing these kits, he has shown in the process of validation that, effectively, identifications can be undertaken even with very degraded DNA samples. This fact is of great relevance given that, to date, rarely has it been possible to validate a tool of this kind, and the Forensic Genetics equipment must be totally reliable to be valid.
Methodology for looking for mutations
The primers cannot be situated in any zone whatsoever: it is fundamental that there is no mutation in the fragments that remain at the STR ends. That is, they cannot adhere to sequential fragments that vary according to the person (they would thereby lose information that could be useful for discriminating persons, because it would be trapped by the primers). To avoid this kind of situation, Dr Odriozola also drew up a methodology in his thesis for seeking mutations at these locations, using DHPLC technology. Moreover, he developed a generic methodology, useful for looking for any kind of mutation. Thus, what is involved is a tool that can not only be applied to Forensic Genetics, but to other genetic disciplines also (for example, to hereditary disorders).
Given STR analysis and the mutations-seeking methodology drawn up in the thesis, more concrete data can be obtained in order to undertake positive identifications. Thus, according to Dr Odriozola, the combination of these two tools could also be effective in complex kinship tests.
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