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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00414-011-0590-5) contains supplementary material, which is available to authorized users.

Protocolo unificado de digestión y descalcificación de tres métodos de extracción de ADN de restos humanos

“…Las cantidades de ADN obtenidas mostraron fluctuaciones grandes, entre 0,02 y 0,007 ng/µl (no se muestran los datos), pero por lo general inferiores a la cantidad necesaria para alcanzar el umbral analítico validado en el Laboratorio, lo que repercutió en perfiles con pérdidas o ganancias alélicas y desequilibrios en heterocigotos. Las modificaciones propuestas del protocolo original, es decir, el aumento de la cantidad de material pulverizado y el empleo de dispositivos concentradores antes y después de la digestión, resultaron en un aumento en la cantidad de ADN recuperado, corroborando así lo sugerido en estudios anteriores (Barrio-Caballero, 2013;Jakubowska, et al, 2012;Davoren, et al, 2007). Además, se encontró que cerca de 50 % del polvo de hueso quedaba sin digerir, probablemente por no haberse usado la cantidad de solución tampón necesaria.…”

The auditory ossicles as a DNA source for genetic identification of highly putrefied cadavers

“…DNA quantity per milligram bone averaged 419 pg with mean values of 670 pg for fresh and 260 pg for putrefied samples. The DNA yield from auditory ossicles is thus higher than or comparable to the average DNA yields from bone powder [15,16]. See Table 1 for single values from the auditory ossicles.…”

“…In addition, the kit is easy to handle, fail-safe, and less toxic than the frequently preferred phenol-chloroform-based methods [17]. Especially for bones, conventional extraction protocols are mostly time-consuming and labor-intensive [15,16,18] due to an additional decalcification step and to subsequent decontamination procedures of the bone powder mills.…”

Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones

“…The main problems are environmental factors and the presence of inhibitors. A universal method is yet to be developed which allows inhibitor-free DNA sample extraction [49]. To overcome the isolation of PCR inhibitors with intact DNA, two comparative types of buffers were used, one of which was a standard protocol (lysis buffer) and the other one was a demineralization protocol (decalcification buffer).…”