At the National Forensic Laboratory, the most common forensic examinations involve DNA traces collected at crime scenes. For comparison with these samples, reference samples are collected from individuals of known identity, typically in the form of a buccal swab (saliva sample). From both evidentiary and reference samples, DNA is extracted in the Department of Biological Examinations, and genetic profiles are determined based on short tandem repeats (STRs).
The purpose of this master's thesis was to transfer the existing, outdated method for processing reference saliva samples to a new robotic system, the easyPunch STARlet, and to validate the new method. At the same time, the method of saliva sample collection was changed by replacing the sample carriers. Two types of new sample carriers were compared, and a protocol for working with the new robotic system was established.
Sample carriers containing saliva from selected individuals were processed using the new robotic system, and genetic profiles were subsequently obtained. In the initial analysis, a full genetic profile was successfully determined in 91.4% and 85.7% of cases, depending on the type of carrier used. These success rates are not acceptable for routine forensic work; therefore, during the validation process, a protocol was developed for further processing of samples in cases where the full profile could not be obtained in the first analysis (inconclusive results).
Samples with inconclusive results were reprocessed using the robotic system, either with the original protocol or with a modified protocol involving an increased number of amplification cycles. This approach resulted in successful full-profile determination in 33.3% and 100% of the respective cases. Samples that still did not yield a full profile after reprocessing were subjected to a manual method, in which part of the sample was manually cut from the carrier. This method resulted in complete profiles for all samples, achieving a 100% success rate for both types of new sample carriers.
As part of the validation process, the accuracy, robustness, reproducibility, and repeatability of the method were also evaluated. All genetic profiles were correctly determined when compared with known profiles, thus meeting the criterion for reproducibility. The quality of the obtained profiles was comparable between both types of carriers, indicating sufficient robustness of the method. The method’s repeatability was 93.3%, which is considered acceptable, as the profiles obtained from the same sample were consistent. The method's reproducibility in cases with initially inconclusive results was 100%, provided the protocol for such samples was followed.
In conclusion, using the protocol developed during the validation process, a successful genetic profile can be obtained from all reference saliva samples. The new robotic system is suitable for this purpose, as are both types of sample carriers. The quality of the results is appropriate and equivalent to those obtained with the previous system and sample carrier. Laboratory work will be more efficient, and the likelihood of human error will be reduced.
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