Efficiency of methods was determined on the basis of DNA recovery measured by Real-Time PCR assay. The obtained
R
2 value for all reactions was at least 0.99. The average amount of DNA isolated from analysed bones with TD, PCE and EA methods was 14,926, 2803 and 22 ng, respectively, per 500 mg of bone powder (Table
1). The TD method gave remarkably better average DNA yields than PCE and EA. However, it was observed that DNA extraction from aggregates of bone crystals (EA) was the most efficient method for the 62-year-old bone (B5) exhumed from the ground. With the two other methods, no DNA was extracted (TD method) or a significantly lower amount (PCE method) was obtained. For two other exhumed relatively old bones, B1 (10 years old) and B9 (8 years old), the EA method gave higher DNA amounts than PCE, but the highest yields were observed for the TD method (Table
1). Obtained results suggest that, while the TD protocol gives higher recovery of DNA in general, the EA method can give better results in cases of old and severely degraded bones. Low levels of DNA obtained via the EA method for relatively fresh bones can be explained by the fact that this method isolates the DNA pool which is fixed within crystal aggregates and eliminates the rest of genetic material, contaminants, inhibitors and exogenous DNA by strong oxidant treatment (NaOCl). In buried, more degraded bones, extra-aggregate DNA is usually severely degraded and therefore present at low levels. In contrast, for relatively fresh bones, DNA yield was considerably higher for TD and PCE than for EA method, because, due to the EA protocol, all extra-aggregate DNA was removed (see bones 2, 7 and 8, Table
1). Poor results of the organic method of DNA extraction from exhumed bones was also observed by Davoren et al. [
15] when compared with the silica columns technique. Our results are similar to those described by Salamon et al. [
11], who obtained about 2 orders of magnitude more DNA templates when DNA was extracted from whole bone powder (similar to PCE) as compared to DNA extracted from aggregates (EA).
Table 1
DNA recovery from 500 mg of bone powder for nine bones using EA, PCE and TD protocols
1a
| 0.01 | 0.9 | 13.3 |
2 | 10,702.5 | 15.45 | 8,183.3 |
3 | 0.9 | 0.23 | 45 |
4 | 1.35 | 1.05 | 23.3 |
5a
| 0.3 | 2.1 | 0 |
6 | 7.05 | 0.75 | 8.3 |
7 | 5,326.8 | 45.75 | 23,833.3 |
8 | 9,189.9 | 115.05 | 102,114.2 |
9a
| 0 | 17.4 | 116.6 |
NC | 0 | 0.002 | 0 |
Average for exhumed bones | 0.103 | 6.53 | 43.3 |
Average for all bones | 2,803.2 | 21.99 | 14,926.37 |
Among all analysed samples, 33% of TD extracts, 17% of PCE extracts and only 4% of EA extracts partially inhibited or could inhibit amplification. For EA extracts, only B5 could cause inhibition (only two out of ten repeats of B5 DNA extractions). No B3, B4, B5 PCE extracts and no B1, B4, B5 TD extracts allowed for proper IPC template amplification (Table
2). This shows that DNA extraction from aggregates (EA) removes inhibitors much better than PCE or TD. It is possible that inhibitors do not penetrate bone crystal aggregates, and this explains why very low levels of PCR inhibition for EA method were observed. Salamon et al. [
11] estimated that the PCR inhibition level in EA extracts was 5 times lower than inhibition of DNA extracts isolated from whole bone powder.
Table 2
Possible reasons of inhibition
1 | TD | −/+ | 32.97 | 1.97 | Partial inhibition |
3 | PCE | − | 36.70 | 0.84 | Partial inhibition |
PCE | − | 33.86 | 1.83 | Partial inhibition |
4 | TD | −/+ | 32.26 | 2.12 | Partial inhibition |
PCE | − | 33.25 | 2.05 | Partial inhibition |
PCE | − | 36.44 | 0.88 | Partial inhibition |
5 | TD | − | – | −0.004 | Invalid result |
EA | − | – | – | Invalid result |
EA | − | – | – | Invalid result |
PCE | − | – | – | Invalid result |
Apart from burial conditions and effects of microorganisms and inhibitors, temperature and time also had a significant influence on DNA recovery. The bone originating from remains found in winter after 3 months from death (B2) gave a higher DNA yield than the bone of the same age found in summer (B4). As expected, the fresh bone which originated from a person who died in a car accident (B8) and which was not exposed to distractive environmental conditions and the bone from a corpse found in winter soon after death (B7) gave the highest DNA recovery.