Analysis in forensic DNA profiling

The last step is the actual analysis of DNA. And this is a process known as capillary electrophoresis. The DNA fragments are sorted by size. And, so we have here the raw data that’s come out of the analyzer and the smaller fragments of DNA are at the left and the larger fragments of DNA are at the right. The DNA is able to be visualized because of those fluorescent dye tags. Remember from the previous slide, blue, green, yellow or red fluorescent dye tags are added to each DNA molecule. And you can see them there. There’s blue peaks, green peaks, there’s red peaks.

Now, the yellow peaks are always shown in the electropherogram as black peaks, because yellow just simply often isn’t visible enough, so the yellow dye tag is displayed as black on screen. So once that process is complete, we now have the electropherogram. But that still needs to be read and interpreted by a trained forensic scientist . Before they go through that process, of course, they actually need to make sure that the, the whole process of analysis passed all of the quality assurance steps. There will be various QA processes built into the process along the way, and all of those checks must pass.

If they don’t, if there’s any failures of quality assurance, then the results may be unusable and may have to go right back to the start and begin again. Once the DNA profile has passed quality assurance, it is examined, and the first thing we need to do is try to establish the number of contributors. So is this DNA from one person or is it actually DNA from multiple people? And we’ll be talking more about mixed DNA profiles later, but this is important. It is. The DNA doesn’t have to be from one person.

It may be a mixture from two, three or four people where it is even possible that there may be DNA from so many people that we simply cannot be certain about the number of contributors. And that is also important to know. Is there signs of DNA being degraded? We’ll see what that looks like later. Is there signs of inhibition? It might be that the sample needs to be retested, either by adding more or less DNA to try and get a better result. There’s even other forms of testing that may be more appropriate. There’s a special kind of DNA test called Y chromosome testing, and that could also be applied and for certain cases.

Are the results suitable to be put into the DNA database? And once that crime scene profile has been interpreted and perhaps the resulting profile put into the database, it’s then suitable for being compared to the DNA profiles of people who are involved in that case, whether they are the victim or perhaps a suspect. So talk for a minute about controls and standards. Controls are samples of known types, and there are both positive and negatives that are run in parallel, i.e. at the same time as your experimental samples, and these are used to demonstrate that the procedure has worked correctly. So some examples, a positive control sample that we use in the laboratory is blood from a staff member.

The staff member has volunteered some blood and that blood is then spotted onto swabs just a very small amount of blood spotted onto each swab. And then one of those positive control swabs is processed alongside each batch of swabs that are processed. Now because we know who that donor was and we know we’ve put some of their blood on the swab, we know what DNA profile should be expected at the end of the process. So once we go through that entire process of extracting, quantifying, amplifying and analyzing the DNA, we know that we should get that particular person’s DNA profile. If we don’t, something has gone wrong.

So the samples, all of those samples have failed quality assurance and may not be usable. At the same time, we’ll also include a negative control sample. Now this might be a swab that has nothing on it. Now the point here, of course, is because there’s nothing on it, we would expect to see nothing at the end of the process. The resulting DNA profile, there should be nothing there. It should be just baseline all the way along. No alleles should be detected. If we do detect alleles, then again, something has gone wrong. It could be that one of the reagents is contaminated and that contamination might be present in every sample.

Or it could be that the scientist doing the extraction work has made a mistake. Again, both the positive outcome and the negative outcome, both have to occur for results to for samples to pass quality assurance. Next important topic that should be discussed during the process of DNA profiling is the use of controls and standards. These are basically for ensuring accurate results in DNA profiling process. And here we use controls and reagents. So first of all, we should be using reagent or extraction blanks. So these are basically we are using reagent, which is used for extraction and we are not putting any sample to it.

So basically, we are like doing a mock extraction and this sample should not give any results at the last. They shouldn’t be any profile coming out of it. So that means your extraction process is clean. Second level, you use the negative controls, which are basically samples which are lacking DNA. So for PCR, you should run a negative control. So if your PCR is not giving any amplification for the negative control, that means your PCR reagents are clean. They are not having any extraneous DNA. The next level is the use of positive controls, is basically a known sample from your lab for which you know the DNA profile.

So you should be running a positive control from your lab, and you should be having the profile similar to, identical to the profile which you got in your previous experiments. Then you also have the control DNA or the standard DNA, which comes along with the kit. Like in case of Promega kits, you get 2800M standard. And similarly, in case of Applied Biosystem kits, you get 9947A standard. So this is basically the standard DNA which is provided along with the kit, and it’s the DNA from a known source. You know the profile for that. So the profile, which comes from the standard, should be identical to the profile, which has been described by the company.

So that means your entire process is running, has been run properly. The amplification is proper and even the capillary electrophoresis and data analysis also proper.