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Extraction, Quantitation and Amplification

In this video, Dr Gavin Turbett deals with extraction, differential extraction, quantitation and amplification.
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There are four main stages associated with DNA profiling, and I’ll briefly step through each of those. So the first, of course, is the extraction stage, and this is where the cells that were recovered from the biological material. And it doesn’t matter if it’s from blood or semen or skin cells or it’s a piece of tissue that has been left. The cells are broken open and that releases the DNA, and that DNA can then be stored in a test tube. And you can see a test tube. They’re quite small. These can be frozen, and DNA will quite reasonably last for many years, many, many years if stored frozen. One special variation is in relation to sexual assault cases.
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The exhibit or the swab that is sent to the laboratory may well contain a mixture of cells from both the victim and the offender. So we can perform what’s known as a differential extraction, and this can be used to separate the sperm cells from the other types of cells present in the sample. So here’s a shot from a, down a microscope, and you can see the sort of those brownish items in the background are the epithelial or skin cells. And then the small oval shapes are the sperm cells because they’re both present in the one sample. If you extracted all of the DNA and tested it, you would get a mixture of DNA from both people.
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Profiles are easier to interpret if they can be separated, so there are some chemical techniques that the laboratory can apply. And this allows the sperm cells to be separated from the epithelial cells. And so in these particular cases, the one sample will actually generate two different DNA profiles, one of them being the sperm cell fraction, which hopefully, of course, is the male DNA profile and then the other being the epithelial or skin cell fraction, which would be the DNA profile of the victim. We then quantitate, we actually try to work out how much DNA was actually recovered.
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An important thing to remember with forensic exhibits is we don’t always know what it is that we’ve been given and asked to analyze. And that’s something that looks like blood may not actually be blood, or it might be blood, but it might not be human blood. So the quantitation stage is specifically to work out how much human DNA had been recovered. Was there no DNA recovered? Was there a little bit of DNA recovered, or was lots of human DNA recovered? We need to know that because that then is important for the next step. We need to know how much DNA to actually use. So this amplification stage is using a technique known as polymerase chain reaction or PCR.
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Think of it as photocopying. We basically photocopy the areas of DNA that we’re interested in, and we’ve spoken previously about the different loci that are tested and those loci are short tandem repeat sequences. So we amplify or we photocopy the short tandem repeat sequences that we’re interested in. Each locus is amplified at the same time. And at this stage, fluorescent dye tags either blue, green, yellow or red are added to each DNA fragment. So you can see here we might only have a very small amount of DNA to start with, but we photocopy. We amplify the DNA with that polymerase chain reaction, and we end up with lots of copies of that fragment of DNA, all identical.
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But they’ve also been tagged now with one of those fluorescent dye tags.

What are the different steps in forensic DNA profiling? Extraction, quantitation, amplification – you may have come across these terms while examining DNA evidence in a case or while reading about forensic DNA profiling. It is important to understand the scientific principles underlying these stages to meaningfully examine DNA evidence. Watch Dr Turbett explain these concepts in this video.

*References for images

Certain images in this video are sourced from here, here and here.

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Decoding Forensics for Legal Professionals

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