Comparison of three methods of DNA extraction from human bones with different degrees of degradation

There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol–chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol–chloroform protocols are more efficient for successful DNA extraction

Toward Male Individualization with Rapidly Mutating Y-Chromosomal Short Tandem Repeats

Peer reviewe

On the long term storage of forensic DNA in water

A rectrospective study was conducted on the effect of the long term storage of 122 DNA samples resuspended in water, one of the elution media still suggested by well established protocols. These DNA samples come from four different kinds of forensically relevant samples (saliva swabs, FTA card bloodstains, nails and II° World War bones) extracted in 2008–2018 and stored at – 20 °C (n = 113 of groups #1-#5) and at +4 °C (n = 9 of the group #6), respectively. At the time of the present study (2019), quantitative PCR (qPCR) was employed as tool for assessing the degradation of the samples. The employment of the Human Quantifiler Kit showed that the median loss of DNA ranged from 17.8% to 66.6% in groups #1-#5 while it was 85.0% in group #6. However, it is likely that these values represent an underestimation due to the shortness of the qPCR probe (62 bp). Noteworthy, the DNA loss was statistically significant in each of the six groups (p values ≤ 0.0167). Thus, in agreement with the data on spontaneous DNA decay, no forensic DNA sample should be stored in water for long term periods. In conclusion, the results of this technical note warn against the use of water for this purpose

Highly degraded RNA can still provide molecular information: An in vitro approach

10siThe long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical–chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r2 > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed.partially_openopenFattorini P.; Bonin S.; Marrubini G.; Bertoglio B.; Grignani P.; Recchia E.; Pitacco P.; Zupanic Pajnic I.; Sorcaburu-Ciglieri S.; Previdere C.Fattorini, P.; Bonin, S.; Marrubini, G.; Bertoglio, B.; Grignani, P.; Recchia, E.; Pitacco, P.; Zupanic Pajnic, I.; Sorcaburu-Ciglieri, S.; Previdere, C

Highly degraded RNA can still provide molecular information: An in vitro approach

The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical–chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r2 > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed