DNA EXTRACTION AND QUANTIFICATION

10 Apr

DNA EXTRACTION AND QUANTIFICATION

the FTA® paper it is stable at room temperature for several years. Cellular material lyses on contact with the FTA® paper and the DNA becomes bound to the paper. To analyse the DNA sample, the first step is to take a small region of the card, normally a 2-mm diameter circle, place it into a 1.5-ml tube and the non-DNA components are simply washed off, leaving only DNA on the card. The small circle of FTA® paper is then added directly to a PCR. The FTA paper extractions are very simple to perform and do not require multiple tube changes, thus reducing the possibility of sample mixing.

DNA extraction from challenging samples

The extraction of the many samples encountered in the forensic laboratory, including blood and shed epithelial cells, can be carried out routinely using any of the above techniques. There are however some sample types that require variations on the basic techniques.

Semen
Semen is one of the most commonly encountered types of biological evidence. The extraction of DNA from the spermatozoa is complicated by the structure of the sper- matozoa (Figure 4.2). DNA is found within the head of the spermatozoa that is capped by the protective acrosome, which is rich in the amino acid cysteine – a large num- ber of disulphide bridges form between the cysteines in the acrosome. Proteinase K, which is a general proteinase, cannot break the disulphide bonds and this reduces the efficiency of the extraction. The addition of dithiothreitol (DTT), a reducing agent that will break the disulphide bonds, greatly increases the release of spermatozoa DNA [12].

Another complication with semen is that it is often recovered as a mixture of sper-matozoa and epithelial cells. The acrosome can be an advantage in these cases as it is possible to perform differential lysis: the epithelial cells are broken down by mild lysis conditions and the spermatozoa can be effectively separated from the lysed epithelial cells [12,13].

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