mardi 24 juin 2014

Continuation Part Nine: Amanda Knox/Raffaele Sollecito















Mod InfoOnce again, the thread has become quite lengthy, so here is another new continuation thread. This continues from Part Eight. For further reference, see also Part Seven Part Six , Part Five , Part Four , Part Three , Part Two , and Part One .

Posted By:Loss Leader


















The quantification of the DNA and the typing are separate processes. The advantage of the real time quantitative polymerase chain reaction (RTqPCR) is that it is specific for human DNA (it uses human specific primers), and is more sensitive (it can measure smaller amounts) than the Qubit. It does however destroy the sample tested, unlike the Qubit. The 50 cycles for quantification are nothing to do with the cycles for typing.



Having taken a sample of your extract of DNA e.g. blood you take a subsample and run it through the RTqPCR. This tells you how much human DNA was in your subsample therefore how much was in your original sample. You then know how much of the original sample you need for typing. The sample for typing is then put through PCR again usually 28 cycles, with specific primers for the areas of interest (short tandem repeat - STR), the product of this replication cycle is then analysed in this case using capillary electrophoresis to size separate the products, and laser tagging to separate out STR of a similar size. The electrophoretogram (EPR) gives the graph, on the Y axis the height of the peaks (technically AUC) give the magnitude of the number of STR copies at a particular size, the x axis is the size of the copy.



The Qubit is old technology it measures DNA in a sample directly, you reversibly label DNA with a fluorescent marker, and the amount of fluorescence then tells you how much DNA is present in the sample. You can then go on and directly do STR typing on that sample. Stefanoni may have had an argument that with a small and unique sample she did not want to waste any by putting some through a destructive test. Of course one does not know before any sample is quantitated how much DNA is present. Arguably for a kitchen knife which might be expected to have non human tissue a human specific measure would have been better. To be fair a negative test for blood would indicate any DNA detected would be low at LCN level.





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