Hard tissues

Following murders, terrorist attacks, wars and fatal accidents it is desirable to group together body parts from individuals when fragmentation has occurred and ultimately to identify the deceased. If the time between death and recovery of the body is short then muscle tissues provide a rich source of DNA [22], which can be extracted using, for example, any of the Chelex®, salting-out and organic extraction methods. If, however, the soft tissues are displaying an advanced state of decomposition they will not provide any DNA suitable for analysis. When the cellular structure breaks down during decomposition, enzymes that degrade DNA are released and the DNA within the cell is rapidly digested. This process is accelerated by the action of colonizing bacteria and fungi.

Osteocytes are the most common nucleated cells in the bone matrix. In the teeth odontoblasts within the dentine and fibroblasts in the cell rich zone of the pulp cavity provide a source of nucleated cells [23]. The hard tissues of the body, bone and teeth provide a refuge for DNA. In addition to the physical barriers, the hydroxyapatite/apatite mineral, which is a major component of the hard tissues, stabilizes the DNA which becomes closely bound to the positively charged mineral - this interaction limits the action of degrading enzymes [24].

Figure 4.3 Bone and tooth material can be vigorously cleaned using: (a) abrasion to remove the outer surface and (b) washing in detergent and bleach to remove contaminating materials. (c) Exposure to strong UV light introduces thymine dimers into any contaminating exogenous DNA -preventing amplification during PCR

Figure 4.3 Bone and tooth material can be vigorously cleaned using: (a) abrasion to remove the outer surface and (b) washing in detergent and bleach to remove contaminating materials. (c) Exposure to strong UV light introduces thymine dimers into any contaminating exogenous DNA -preventing amplification during PCR

Hard tissues provide an advantage over other forms of biological material because they have a surface that can be cleaned to remove any contaminating DNA by using detergents to remove any soft tissue [25], followed by physical abrasion soaking in sodium hypochlorite (bleach) (Figure 4.3) [26], and exposure to strong ultraviolet light.

After cleaning, the bone/tooth material is normally broken down into a powder by drilling [27] or grinding under liquid nitrogen. The resulting material is decalcified using 0.5-M EDTA either before or at the same time as cell lysis [28]. The organic phenol-chloroform and the silica binding extraction methods are commonly used to extract the DNA [29-34]. The process of extracting DNA from bone samples takes much longer than with any other type of sample.

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