DNA EXTRACTION FROM CHALLENGING SAMPLES

10 Apr

DNA EXTRACTION FROM CHALLENGING SAMPLES

Hair shafts
Hair shafts that have been pulled out often possess a root that is rich in cellular material and DNA can be extracted using any of the commonly used techniques – plucked roots have been shown to contain as much as 0.5 μg of DNA [14]. Hair that has been shed whenitisintherestingtelogenphaseoftencontainsnocellularmaterialaroundtheroot. The hair shafts are composed of keratin, trace metals, air and pigment – cell fragments, including DNA can get trapped in the matrix of the hair and provide enough DNA to produce a profile. However, hair is notoriously difficult to analyse and in many cases it is only possible successfully to profile mitochondrial DNA [14], although nuclear DNA can, in some cases, be recovered [15]. The hair shaft, like the spermatozoa acrosome, is rich in disulphide bridges and requires either mechanical grinding [16] or the addition of a reducing agent such as dithiothreitol [14, 15] that will break the disulphide bonds and allow proteinase K to digest the hair protein and release any trapped nucleic acids. Once released the DNA can be extracted using the salting-out procedure [17] or organic phenol-chloroform based extraction [14-16]. Alternative methods include digestion in a buffer containing proteinase K followed by direct PCR [18,19] or dissolving the hair shaft in sodium hydroxide and, after neutralization, the released DNA is concentrated using filter cen- trifugation [20]. Because the hair shaft contains very low levels of DNA it is prone to contamination but unlike many other types of biological evidence with low levels of DNA it is possible to clean the hair shaft prior to DNA extraction. Several methods have been used to clean hair including washing in mild detergents, water and ethanol and also using a mild lysis step in the same way as is used in the differential extraction of semen [21].

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 decom- position, 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 providearefugeforDNA.Inadditiontothephysicalbarriers,thehydroxyapatite/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].

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