Quantification of DNA

10 Apr

DNA EXTRACTION AND QUANTIFICATION

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.

Quantification of DNA
After extracting DNA an accurate measurement of the amount of DNA and also the quality of the DNA extract is desirable. Adding the correct amount of DNA to a PCR will produce the best quality results in the shortest time; adding too much or not enough DNA will result in a profile that is difficult or even impossible to interpret. This is especially important when profiling forensic samples, when it is very difficult to know the state of preservation of the biological material and also, in many cases, it is difficult to estimate how much cellular material has been collected. It is less important to quantify DNA when using some reference samples – where similar amounts of DNA can be expected to be extracted each time as there are not very many variables. Even so, many laboratories will still quantify the DNA from reference samples as part of their standard analysis. In response to the importance of quantification of samples recovered from the scene of crime, the DNA Advisory Board in the USA adopted rules that made quantification of human DNA mandatory [35]. The quantity of DNA that can be extracted from a sample depends very much on the type of material. Each nucleated cell contains approximately 6 pg of DNA: liquid blood contains 5000-10000 nucleated blood cells per millilitre; semen contains on average 66 million spermatozoa per millilitre (the average ejaculation produces 2.75 ml of semen) [36]. Biological samples recovered from the scene of crime are not usually in pristine condition and can often consist of a very small number of shed epithelial cells; consequently, the amount of DNA that can be recovered can be extremely low and difficult to quantify.

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