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Tags dna , forensic evidence

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Old 24th March 2010, 08:28 AM   #1
Kestrel
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Dead skin cells don't contain DNA?

In another thread, the assertion was made that dead skin cells don't contain DNA. Based on my knowledge, I can't see any support for this idea. Yet this fact was stated in court by a prosecution expert witness and accepted by the court as true. Rather than drag all the experts into a he said, she said argument about a crime, it seemed better to open a separate thread to discuss this.

Has anyone ever heard this before and is there a scientific basis for this claim?
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Old 24th March 2010, 08:39 AM   #2
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Its exactly false. They do. It might be quite degraded, however.
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Old 24th March 2010, 08:56 AM   #3
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This is not what was asserted in the other thread. Doubtless it's possible to find quotes where people were speaking imprecisely, but this is absolutely not the thrust of the argument.

Last edited by shuttlt; 24th March 2010 at 08:57 AM.
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Old 24th March 2010, 08:58 AM   #4
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Originally Posted by BenBurch View Post
Its exactly false. They do. It might be quite degraded, however.
How fast it degrades would depend on the conditions. But considering that a complete CODISWP profiles have been obtained from crime scene items stored for over a decade, the degradation rate can't be all that fast.
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Old 24th March 2010, 09:00 AM   #5
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Actually, what Kestrel really wants to ask, is for confirmation for his contention that a complete DNA profile of an individual convicted of murder, ended up on a key piece of evidence via ambient 'dust' (the argument being, dust also contains dead skin cells along with many other things).

The evidence in question is a bra clasp that was hacked off of the bra of the murder victim. His DNA on the clasp is in the ratio of 1:6 of to that of the victim's. The volume of his DNA is 1.4 ng or 1400 picograms. The victim was murdered in the bedroom of her home in which the convicted individual was not a resident. He had visited the home on a very few previous occasions, spending no more then a total of several hours in her home. He also maintained he had never entered her bedroom on any previous occasion. The apartment was multiple occupancy (3 other residents). The individual convicted was the only other (then the vicyim's) complete profile found on the clasp.

It is Kestrel's assertion that this was via 'dust'.
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Old 24th March 2010, 09:09 AM   #6
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Originally Posted by Kestrel View Post
How fast it degrades would depend on the conditions. But considering that a complete CODISWP profiles have been obtained from crime scene items stored for over a decade, the degradation rate can't be all that fast.
Correct.
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Old 24th March 2010, 09:09 AM   #7
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Well; in human, skin cells are dead when they arrive to the surface.
Basically, they start as normal cells in the stratum germinativum and then start migrating toward the surface. In the process, they die and get emptied of most of their biological apparatus. Their nucleus is degraded; and the cells fill up with keratohyalin that dehydrate them and cross-link them with long chains of keratine molecules.
By the time they arrive to the stratum corneum they are mostly inert interlinked bricks of keratin.
They might still contain DNA, I don't know how complete the degradation process is, but it is no longer complete of functional. Might still be enough for some forensic, though, I really can't say...

Anyway; I think that is what the posters was about...
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Old 24th March 2010, 09:11 AM   #8
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Originally Posted by Fulcanelli View Post
Actually, what Kestrel really wants to ask, is for confirmation for his contention that a complete DNA profile of an individual convicted of murder, ended up on a key piece of evidence via ambient 'dust' (the argument being, dust also contains dead skin cells along with many other things).

The evidence in question is a bra clasp that was hacked off of the bra of the murder victim. His DNA on the clasp is in the ratio of 1:6 of to that of the victim's. The volume of his DNA is 1.4 ng or 1400 picograms. The victim was murdered in the bedroom of her home in which the convicted individual was not a resident. He had visited the home on a very few previous occasions, spending no more then a total of several hours in her home. He also maintained he had never entered her bedroom on any previous occasion. The apartment was multiple occupancy (3 other residents). The individual convicted was the only other (then the vicyim's) complete profile found on the clasp.

It is Kestrel's assertion that this was via 'dust'.
Extremely unlikely. Unless he had handled THAT bra. You need more than one or two cells to drive PCR.
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Old 24th March 2010, 09:18 AM   #9
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Essentially the issue is this:

The clasp of a bra was cut off during, or shortly after the Murder of Meredith Kercher. It wasn't collected but was left in the room that the murder took place in for 46 days before being collected. The entire appartment that the murder took place in was secure throughout that time.

When the bra clasp was collected it was tested for DNA. The DNA of the murder victim (was wearing the bra during the murder) was present, 6-10x (more accurate figures are available) weaker was the DNA of Raphaele Sollecito (recently convicted for the murder), weaker still was the DNA of his girlfriend Amanda Knox (also convicted), there was also the DNA of 2? females that was too faint to get a profile on. The rest of the bra returned a profile for Rudy Guede (also convicted).

Raffaele had only been to the appartment a couple of times before and never into the room in which the murder took place and the bra clasp was discovered. He claimed to have tried and failed to break down the door to the victim's bedroom where the body was later found. He has offered no explanation as to how

The forensic personel who processed the crime scene changed gloves between rooms, but not I think between items. The only other source of Raffaele's DNA that was discovered was a cigarette butt in the kitchen. There are claims that the bra clasp had moved between the initial visit to the crime scene and the return visit when the bra clas was collected.

Footage of the collection of the bra clasp shows that there is a small amount of dust on the finger tips of the latex glove (photographs are available) holding the bra clasp. The claim as I understand it is that this is household dust, which presumably contains quite a lot of human skin cells. This dust is supposed to have contaminated the bra clasp with Raffaele Sollecito's DNA.

The argument that Kestrel summarizes is basically that it's kind of odd that only Raffaele Sollecito's skin cells contributed to this contaminating dust despite him hardly ever having been to the appartment and never into the murder room. It is also the understanding of the people who accept the bra clasp as evidence that household dust, while it may technically contain DNA, is not normally a contaminant to the degree implied.

The quantity of Raffaele's DNA was described by the prosecution expert as copious and the same expert claimed that it could only have come from vigorous contact.
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Old 24th March 2010, 09:20 AM   #10
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Originally Posted by Simon39759 View Post
Well; in human, skin cells are dead when they arrive to the surface.
Basically, they start as normal cells in the stratum germinativum and then start migrating toward the surface. In the process, they die and get emptied of most of their biological apparatus. Their nucleus is degraded; and the cells fill up with keratohyalin that dehydrate them and cross-link them with long chains of keratine molecules.
By the time they arrive to the stratum corneum they are mostly inert interlinked bricks of keratin.
They might still contain DNA, I don't know how complete the degradation process is, but it is no longer complete of functional. Might still be enough for some forensic, though, I really can't say...

Anyway; I think that is what the posters was about...
Thanks! Exactly the answer we've been looking for.
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Old 24th March 2010, 09:20 AM   #11
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Originally Posted by Fulcanelli View Post
Actually, what Kestrel really wants to ask, is for confirmation for his contention that a complete DNA profile of an individual convicted of murder, ended up on a key piece of evidence via ambient 'dust' (the argument being, dust also contains dead skin cells along with many other things).

The evidence in question is a bra clasp that was hacked off of the bra of the murder victim. His DNA on the clasp is in the ratio of 1:6 of to that of the victim's. The volume of his DNA is 1.4 ng or 1400 picograms. The victim was murdered in the bedroom of her home in which the convicted individual was not a resident. He had visited the home on a very few previous occasions, spending no more then a total of several hours in her home. He also maintained he had never entered her bedroom on any previous occasion. The apartment was multiple occupancy (3 other residents). The individual convicted was the only other (then the vicyim's) complete profile found on the clasp.

It is Kestrel's assertion that this was via 'dust'.
Actually, that hasn't been my assertion. Others have however made that argument.

This thread was opened to discuss a specific question of science. if you want to discuss case details, may I suggest taking it back to the Amanda Knox thread?

ETA: My contention was the evidence technicians handled the item in question with visibly dirty gloves. If these gloves had previously touched places where material containing DNA had been deposited, DNA could be transferred to the item being collected.

Last edited by Kestrel; 24th March 2010 at 09:37 AM.
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Old 24th March 2010, 09:21 AM   #12
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Originally Posted by BenBurch View Post
Extremely unlikely. Unless he had handled THAT bra. You need more than one or two cells to drive PCR.
I want to put my opinion on this. Single-cell PCR is not uncommon nowadays (the latest in technology and all that). Difference is that the single-cell is living when the DNA is PCR-ed. Nevertheless, you only need a bit of DNA to drive PCR (which is why it is so powerful). The bigger question in this case is "Was the ratio of suspect's DNA higher or lower than contaminating DNA?" It is hard to tell, but the general rule in PCR is that if you have a mixed sample, the proportion of DNA that is the highest will override the reaction. That is to say, extremely small ratios may be lost in the noise. You would have to have a significant ratio in order to make sense of it. I am not a trained forensic scientist, but I think even I could get a PCR out of a few dozen cells. With training, specialized equipment, and rock solid protocols, it might even be lower. The big question remains how accurate the reading will be.
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Old 24th March 2010, 09:24 AM   #13
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Originally Posted by Kestrel View Post
Actually, that hasn't been my assertion. Others have however made that argument.

This thread was opened to discuss a specific question of science. if you want to discuss case details, may I suggest taking it back to the Amanda Knox thread?
Surely this thread has been started to discuss an issue of science in order to apply it in the context of the Kercher case. Context is everything! Nobody on the other thread is seriously trying to claim that there can be no DNA in household dust as an absolute scientific fact. What people are claiming is that there is essentially no DNA in household dust for the purposes of generating contamination of the form that is being suggested.
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Old 24th March 2010, 09:29 AM   #14
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Originally Posted by BenBurch View Post
Extremely unlikely. Unless he had handled THAT bra. You need more than one or two cells to drive PCR.
I have read that roughly 160 cells contain 1 ng. of DNA. The reliable limits of LCN profiling are in the 100 pg. range, or about 16 cells. Increasing the replication count would get a reading from one or two cells, but it's not considered reliable for forensic work.
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Old 24th March 2010, 09:31 AM   #15
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Originally Posted by Kestrel View Post
How fast it degrades would depend on the conditions. But considering that a complete CODISWP profiles have been obtained from crime scene items stored for over a decade, the degradation rate can't be all that fast.
But a keratinized skin cell already has no nucleus, so extracting DNA will be very difficult, but a body cell properly stored with a nucleus will yield extractable DNA for a long time.

I guess there are two different meanings of "degrade" being used.
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Old 24th March 2010, 09:33 AM   #16
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Originally Posted by Kestrel View Post
I have read that roughly 160 cells contain 1 ng. of DNA. The reliable limits of LCN profiling are in the 100 pg. range, or about 16 cells. Increasing the replication count would get a reading from one or two cells, but it's not considered reliable for forensic work.
The CPS (Crown Prosecution Service) in the UK say the limit of LCN is 5 cells, but I guess it depends on the cell and the condition.
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Old 24th March 2010, 09:34 AM   #17
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While we're here and making such progress...

Another piece of evidence in the case is a knife from which the murder victim's profile was extracted by Low Copy Number (LCN) methods. One quarter of this sample was taken for blood testing using tetramethylbenzidine (TMB). It has been claimed that since the TMB test is more sensitive than LCN methods and, since it was negative, the DNA on the knife cannot be associated with the murder and must instead be from contamination.

The knife was recovered from the appartment of Raffaele Sollecito by a seperate team to the one that processed the crime scene. It has been claimed that the profile of the victim was either "touch DNA" that Amanda Knox walked back and then transferred to the knife, or due to lab contamination. Amanda Knox's profile did not show up on the LCN test the returned the profile of the victim, but was discovered by conventional DNA techniques on the handle of the knife.
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Old 24th March 2010, 09:45 AM   #18
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Not that it matters, but here is the photograph of the dust we're talking about:


and here's where it was found:


A quote from Science Spheres, which I believe is one of Kestrel's primary sources:

Quote:
What would happen to any random object left on the same floor and kicked about for 47 days? Especially an object with cloth attached , making it a virtual dust mop. It would be covered with dust, and the DNA that comes with that dust. Raffaele was at the apartment visiting Amanda on several occasions. The presence of his DNA there means nothing.

Control experiments to check for this would have been simple. The clasp was retrieved from a pile of debris, shown in the picture, left by the fastidious investigators in Meredith’s room. Testing a few other items from that pile to see if they, too, had picked up DNA dust from the floor would tell us whether there was anything special about the clasp. Of course, that wasn’t done.

So we have “Raffaele’s DNA was found on Meredith’s bra clasp,” rather than, “Raffaele’s DNA, along with DNA from lots of other people, was found at various random locations throughout Amanda’s apartment, which he visited several times before the murder.” The first phrase sounds incriminating. The second, accurate phrase, shows how meaningless this test result is without a control experiment.
http://www.sciencespheres.com/
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Old 24th March 2010, 10:08 AM   #19
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Originally Posted by JoeTheJuggler View Post
But a keratinized skin cell already has no nucleus, so extracting DNA will be very difficult, but a body cell properly stored with a nucleus will yield extractable DNA for a long time.

I guess there are two different meanings of "degrade" being used.
In the Peggy Hettrick murder caseWP, the DNA evidence that finally freed Tim Masters was collected from places where someone merely touched the victims clothing two decades earlier. The same is true of the touch evidence processed about a decade after the JonBenét RamseyWP murder.

Extracting DNA has little to do with the cell nucleus. The extraction process simply dissolves the rest of the cell.
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Old 24th March 2010, 10:15 AM   #20
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Surely the nucleus is where most if the DNA is? If that's already had it before the skin cells even make it to the surface in order to drop off you're talking about quite small quantities of DNA.

Also, in the cases you quote, the DNA was transferred by touch. Here we are talking about household dust, not Raffaele Sollecito transferring his DNA by touching the bra clasp.
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Old 24th March 2010, 10:33 AM   #21
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Originally Posted by Kestrel
ETA: My contention was the evidence technicians handled the item in question with visibly dirty gloves. If these gloves had previously touched places where material containing DNA had been deposited, DNA could be transferred to the item being collected.
The dirt on the gloves clearly has come 'from' the clasp where he's held it. The clasp is clearly soiled...with the victim's blood for one thing.

And Kestrel please, you were maintaining on the other thread the DNA could have come from 'dust'.

The important matter, whatever you maintain in regard to the glove is essentially meaningless since Raffaele's DNA was found in only one other place in the cottage...a cigarette butt in the kitchen. According to Raffaele he'd never entered Meredith's room nor ever handled her bra. Yet you are tying to argue, when not arguing about dust, that a glove that had not been used outside of the victims room transferred the DNA onto the clasp. From where was the source of this DNA? How is it that the only profile found on the clasp was the victim's? What is the dynamic whereby DNA of only one person, from a source you cannot identify, got on that clasp? Raffaele had claimed he'd never entered her room, so whence his DNA?

In the case of contamination, we would be looking at a ratio of Raffaele's DNA to the victim's on that clasp in the region of 1:100 + . What we have is 1:6. 1.4 nanograms or 1400 picograms. As the prosecution experts maintained, this indicates 'direct and vigorous contact'. And also it must be noted, if it was not Raffaele who cut off the clasp then who was it...since there are no other complete profiles on that clasp?

To argue his profile landed on the clasp either via dust, or 3rd party transference by a gloved forensic team is not likely or even plausible.

Originally Posted by Kestrel
This thread was opened to discuss a specific question of science. if you want to discuss case details, may I suggest taking it back to the Amanda Knox thread?
Do you not believe it fair and proper that the scientists are given the context?
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Old 24th March 2010, 10:38 AM   #22
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Quote:
ETA: My contention was the evidence technicians handled the item in question with visibly dirty gloves. If these gloves had previously touched places where material containing DNA had been deposited, DNA could be transferred to the item being collected.
If this is the argument then can we drop the concern about it taking 46 days to collect the clasp?
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Old 24th March 2010, 10:53 AM   #23
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Originally Posted by Simon39759 View Post
Well; in human, skin cells are dead when they arrive to the surface.
Basically, they start as normal cells in the stratum germinativum and then start migrating toward the surface. In the process, they die and get emptied of most of their biological apparatus. Their nucleus is degraded; and the cells fill up with keratohyalin that dehydrate them and cross-link them with long chains of keratine molecules.
By the time they arrive to the stratum corneum they are mostly inert interlinked bricks of keratin.
They might still contain DNA, I don't know how complete the degradation process is, but it is no longer complete of functional. Might still be enough for some forensic, though, I really can't say...

Anyway; I think that is what the posters was about...
So dandruff and cut off corns have DNA? I thought so.
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Old 24th March 2010, 11:38 AM   #24
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Originally Posted by Ixion View Post
.... The bigger question in this case is "Was the ratio of suspect's DNA higher or lower than contaminating DNA?" It is hard to tell, but the general rule in PCR is that if you have a mixed sample, the proportion of DNA that is the highest will override the reaction. That is to say, extremely small ratios may be lost in the noise .... The big question remains how accurate the reading will be.
.
Thanks Ixion for your comments. From the same case, a minute amount of DNA was encountered on a knife blade and subjected to testing.

Here's the result:


Now some commenters have described this almost perfect match as contamination of a sum of partial DNA, but I find that as hard to believe as a bunch of monkeys typing up the complete works of Shakespeare through pure chance by jumping on typewriters.

I'd like to get your opinion: do you think it's a match? Or is there some statistical explanation for those coinciding peaks?

Just so you can get a feel for the different scales (due to the minute amount of DNA in the test sample), here's an amplification of the Y-scale on the left and right graphs.

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Old 24th March 2010, 11:52 AM   #25
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Well, to my untrained eye, it does look like a match. There are several caveats one must consider though when looking at evidence like this:
1) Which markers are they using for the samples as references? I am not familiar enough with forensic biology to know if the particular markers have a high degree of variability or not.
2) Along those same lines, was a null control run (IE, DNA from the dried blood of someone other than Meredith)? That way, you could confirm whether or not some of those peaks would have formed simply by chance.
3) Was the experiment repeated? PCR can be extremely sensitive and sometimes even good labs get samples mixed up. Just a single microliter in the wrong tube, and you will get a false positive.
4) Is evidence of blood on the knife compelling enough to determine it as the murder weapon? How much blood was on the knife? How long can blood remain on the knife and still get DNA from it? (I haven't kept up with the case, so I am sure these questions have been answered)
5) Was Meredith's DNA found in the dust on the clasp of the bra? Since it was her bra, it is to be expected. How does that graph of the mixed DNA sample compare to the one from the reference above?

The data does look nice and clean. As I stated, I am not a forensic biologist. I am an immunologist, but I have done a fair amount of PCR from DNA samples.
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Old 24th March 2010, 12:07 PM   #26
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Originally Posted by Ixion View Post
Well, to my untrained eye, it does look like a match. There are several caveats one must consider though when looking at evidence like this:
1) Which markers are they using for the samples as references? I am not familiar enough with forensic biology to know if the particular markers have a high degree of variability or not.
2) Along those same lines, was a null control run (IE, DNA from the dried blood of someone other than Meredith)? That way, you could confirm whether or not some of those peaks would have formed simply by chance.
3) Was the experiment repeated? PCR can be extremely sensitive and sometimes even good labs get samples mixed up. Just a single microliter in the wrong tube, and you will get a false positive.
4) Is evidence of blood on the knife compelling enough to determine it as the murder weapon? How much blood was on the knife? How long can blood remain on the knife and still get DNA from it? (I haven't kept up with the case, so I am sure these questions have been answered)
5) Was Meredith's DNA found in the dust on the clasp of the bra? Since it was her bra, it is to be expected. How does that graph of the mixed DNA sample compare to the one from the reference above?

The data does look nice and clean. As I stated, I am not a forensic biologist. I am an immunologist, but I have done a fair amount of PCR from DNA samples.
The test was not repeated. Also, Stefanoni claimed that her lab has never experienced contamination in its entire existence---are there really labs that can legitimately claim this? I'm not a scientist so I really have no idea.
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Old 24th March 2010, 12:08 PM   #27
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Originally Posted by Fulcanelli View Post
In the case of contamination, we would be looking at a ratio of Raffaele's DNA to the victim's on that clasp in the region of 1:100 + . What we have is 1:6. 1.4 nanograms or 1400 picograms.
In the interest of giving the context, I must point out the Fulcanelli is simply wrong. The total amount of DNA was stated as 1.4 ng., with most of this being the victims DNA.

Assuming the 6:1 ratio is correct, the suspects DNA would be in the 200 pg. range.
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Old 24th March 2010, 12:11 PM   #28
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Originally Posted by HumanityBlues View Post
The test was not repeated. Also, Stefanoni claimed that her lab has never experienced contamination in its entire existence---are there really labs that can legitimately claim this? I'm not a scientist so I really have no idea.
Well, they can claim it all they want. Who is going to audit them? If it is true that they have never had a contamination, then someone will be footing the bill. If it is false, then loss of lots of credibility. When you are dealing with sensitive samples, reputation is very important. Maybe they have never had a contamination, but that doesn't mean that it couldn't happen (skeptic in me). This is why other controls are vital. As I said, a single microliter could change the results.
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Old 24th March 2010, 12:18 PM   #29
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Originally Posted by Ixion View Post
Well, they can claim it all they want. Who is going to audit them? If it is true that they have never had a contamination, then someone will be footing the bill. If it is false, then loss of lots of credibility. When you are dealing with sensitive samples, reputation is very important. Maybe they have never had a contamination, but that doesn't mean that it couldn't happen (skeptic in me). This is why other controls are vital. As I said, a single microliter could change the results.
When dealing with LCN samples in the 100 pg. range, could simply touching a contaminated object with a gloved hand transfer enough DNA to contaminate the sample? An example would be adjusting a light used to examine the sample, then touching the sample again.
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Old 24th March 2010, 12:33 PM   #30
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Originally Posted by Kestrel View Post
When dealing with LCN samples in the 100 pg. range, could simply touching a contaminated object with a gloved hand transfer enough DNA to contaminate the sample? An example would be adjusting a light used to examine the sample, then touching the sample again.
It is possible; depends on how careful they were when gathering evidence. A typical human cell has about 6 pg of DNA in it (that is a living cell, don't know about keratinized skin cells). So less than 20 cells would need to be transfered to get a 1:1 increase in the amount of contaminating DNA. However, the chance of contamination does drop off significantly in the lab. LCN PCR is usually performed in a sealed glove box, to prevent environmental contamination. The source of contamination in the lab would occur when the technician performing the assay forgets to change pipet tips and transfers liquid from one container to another accidentally.
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Old 24th March 2010, 01:28 PM   #31
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Originally Posted by Cainkane1 View Post
So dandruff and cut off corns have DNA? I thought so.
Actually, dandruff are similar keratinized dead skin flakes so, their DNA would be similarly degraded (as mentioned, I do not know to what extend this degradation is complete, my wild guess would be quite incomplete (leaving several sequences of hundreds of nucleotides long) and quite variable between cells).

But, if blood is involved, this is all a moot point. If blood got on the piece of tissue, it would contain plenty of intact DNA (one drop of blood is supposed to contains between 7,000 to 25,000 white Blood cells. Each of these cells should contain an average of 6pg of genomic DNA.

Low numbers of template DNA requires additional PCR cycles which in turn are well known to decrease the specificity of the reactions. However, it must be kept in mind, these errors will be non-specific.
Bob's DNA sequences will look like junk; it will not, all the sudden, looks like Jeff's... So, if you are trying to identify something, and it matches, you can be confident that the match was present in the original samples.


Now, the whole question is, how did the DNA get into the samples. Is it because the suspect handle the sample or is it due to some contamination at some point?
On that subject, I can't say, I have not followed the case and am not an expert.
However, to answer Ixion's concern, I know that forensic lab have a very rigid protocol and are regularly inspected and tested by the law agencies that employ them (just imagine the scandal, and the numbers of cases appealed and dumped, if news ever got out of a lab not being reliable). So, if contamination happened, it is pretty certain it did before the samples arrived to the lab...
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Old 24th March 2010, 01:30 PM   #32
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Ixion,

I wondered if you'd seen my post on the LCN. The object (a knife) was collected from a different location that the victim (whose dna was found on the knife) had never been to. It was collected by a seperate team. I should add there was too little material to replicate the test.
Originally Posted by shuttlt View Post

Another piece of evidence in the case is a knife from which the murder victim's profile was extracted by Low Copy Number (LCN) methods. One quarter of this sample was taken for blood testing using tetramethylbenzidine (TMB). It has been claimed that since the TMB test is more sensitive than LCN methods and, since it was negative, the DNA on the knife cannot be associated with the murder and must instead be from contamination.

The knife was recovered from the appartment of Raffaele Sollecito by a seperate team to the one that processed the crime scene. It has been claimed that the profile of the victim was either "touch DNA" that Amanda Knox walked back and then transferred to the knife, or due to lab contamination. Amanda Knox's profile did not show up on the LCN test the returned the profile of the victim, but was discovered by conventional DNA techniques on the handle of the knife.
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Old 24th March 2010, 01:33 PM   #33
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Originally Posted by Ixion View Post
It is possible; depends on how careful they were when gathering evidence. A typical human cell has about 6 pg of DNA in it (that is a living cell, don't know about keratinized skin cells). So less than 20 cells would need to be transfered to get a 1:1 increase in the amount of contaminating DNA. However, the chance of contamination does drop off significantly in the lab. LCN PCR is usually performed in a sealed glove box, to prevent environmental contamination. The source of contamination in the lab would occur when the technician performing the assay forgets to change pipet tips and transfers liquid from one container to another accidentally.

Ok, our message cross-posted.
Also, keep in mind, that it is standard procedure to also process blind controls (samples of known positive or negative results) randomly added to the samples proceeded so the mistake would need to occur, precisely to the samples tested.
Also, there is no reason to process the sample's DNA at the same time than the suspect's. In fact, it would be a silly (and risky) thing to do...
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Old 24th March 2010, 01:58 PM   #34
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Originally Posted by Simon39759 View Post
Ok, our message cross-posted.
Also, keep in mind, that it is standard procedure to also process blind controls (samples of known positive or negative results) randomly added to the samples proceeded so the mistake would need to occur, precisely to the samples tested.
Also, there is no reason to process the sample's DNA at the same time than the suspect's. In fact, it would be a silly (and risky) thing to do...
Actually, one think that it is, but it isn't. According to this article in NATURE only two forensic labs who perform LCN testing in the USA use blind controls as part of their protocol. Blind controls may be considered best practice, the ideal, but they certainly are not the norm.

As I understand it, Dr Stefanoni didn't process the victim's DNA on that machine, rather referred to her charts/data. Indeed, that machine had never hitherto been used to test any item or individual connected with the case.
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Old 24th March 2010, 02:02 PM   #35
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Originally Posted by Kestrel View Post
In the interest of giving the context, I must point out the Fulcanelli is simply wrong. The total amount of DNA was stated as 1.4 ng., with most of this being the victims DNA.

Assuming the 6:1 ratio is correct, the suspects DNA would be in the 200 pg. range.
I've no idea where you heard that. There was 1.4 ng of Raffaele's DNA on the clasp. This was offered in the trial during cross examination. That figure was never given to indicate the total amount of DNA on the clasp, but one individual's.
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Old 24th March 2010, 02:11 PM   #36
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Originally Posted by Simon39759 View Post
Now, the whole question is, how did the DNA get into the samples. Is it because the suspect handle the sample or is it due to some contamination at some point?
On that subject, I can't say, I have not followed the case and am not an expert.
However, to answer Ixion's concern, I know that forensic lab have a very rigid protocol and are regularly inspected and tested by the law agencies that employ them (just imagine the scandal, and the numbers of cases appealed and dumped, if news ever got out of a lab not being reliable). So, if contamination happened, it is pretty certain it did before the samples arrived to the lab...
As I expected.

Originally Posted by shuttlt View Post
Ixion,

I wondered if you'd seen my post on the LCN. The object (a knife) was collected from a different location that the victim (whose dna was found on the knife) had never been to. It was collected by a seperate team. I should add there was too little material to replicate the test.
The TMB test is used to detect the presence of blood (I don't know how sensitive it is, but it is a simple procedure; I have all the necessary reagents in my lab). It is a subjective test. TMB + Blood + Hydrogen Peroxide = blue/green. TMB + Hydrogen Peroxide (no blood) = no color change

Since there was not blood detected, then the DNA found on the knife came from some other source. How it got there seems to be a matter of contention.
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Old 24th March 2010, 02:20 PM   #37
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Originally Posted by Fulcanelli View Post
I've no idea where you heard that. There was 1.4 ng of Raffaele's DNA on the clasp. This was offered in the trial during cross examination. That figure was never given to indicate the total amount of DNA on the clasp, but one individual's.
How do you separate the DNA belonging to two or more individuals from a mixed sample in order to measure it?
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Old 24th March 2010, 02:32 PM   #38
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Originally Posted by Ixion View Post
Well, to my untrained eye, it does look like a match. There are several caveats one must consider though when looking at evidence like this:
1) Which markers are they using for the samples as references? I am not familiar enough with forensic biology to know if the particular markers have a high degree of variability or not.
2) Along those same lines, was a null control run (IE, DNA from the dried blood of someone other than Meredith)? That way, you could confirm whether or not some of those peaks would have formed simply by chance.
3) Was the experiment repeated? PCR can be extremely sensitive and sometimes even good labs get samples mixed up. Just a single microliter in the wrong tube, and you will get a false positive.
4) Is evidence of blood on the knife compelling enough to determine it as the murder weapon? How much blood was on the knife? How long can blood remain on the knife and still get DNA from it? (I haven't kept up with the case, so I am sure these questions have been answered)
5) Was Meredith's DNA found in the dust on the clasp of the bra? Since it was her bra, it is to be expected. How does that graph of the mixed DNA sample compare to the one from the reference above?

The data does look nice and clean. As I stated, I am not a forensic biologist. I am an immunologist, but I have done a fair amount of PCR from DNA samples.
These are all very good questions and I'll do my best to answer them as accurately as I can. First of all, it must be noted that the knife is one of the most controversial and contested elements in the case, or at least that relating to the 'blade' (all accept the findings from the handle).

It's a 23 cm long blade, a standard cheap kitchen knife, that is a match for the fatal wound to the victim's neck. It was collected by a forensic team from Raffaele Sollecito's apartment (not from the cottage that was the scene of the murder) by the forensic team. The team that collected the knife did so on a different day to examinations at the cottage.

For a better view of the charts, I would suggest you view HERE

This is the documentation we have to hand, along with some of the testimony.

Was there a negative control? This we don't have the answer to. However, view my previous post in regard to negative controls.

The fact is, chance is virtually ruled out by the noise to peak ratio of the sample. As you can see, the sample is very clean. This is not what we'd expect to see on an item either contaminated, or containing the DNA of multiple persons (which may then for up by 'chance' to match the peaks of the victim) and this same fact counters the argument that it's a low rfu. There is a string argument that a sample shouldn't be judged (or limited to) by the rfu, but actually the noise to peak ratio.

The experiment was not repeated since there was not enough material to retest. But, the judge's report informs us there were 50 passes.

Blood - this matter is complex. The blood test proved negative. However, due to the small amount of material it being blood cannot be ruled out due to the fact the blood tests lack that sensitivity. In short, if the substance was blood, there was not enough to register a positive. However, it is not in doubt the DNA is from the victim and even the defence experts accept this fact. Their contention is instead limited to arguments of potential contamination. And, as we know, the body contains a whole range of biological material that a blade may come into contact with when entering a body. So, some of it may be blood and some may be other biological material from the victim. But, LCN testing does not provide the answers as to which cells are which and at that low level, blood tests are useless.

As for 'dust' on the clasp, there was no dust found on the clasp. The whole dusty argument is simply something thrown out by the defence simply on the basis that 'dust exists', not that the clasp was dusty. This has since been taken up by followers of the defence. Their logic is: Dust exists, therefore it was. That does not sound like a compelling argument to me.

The graph from the clasp has not been released. But, it was noisy. The complete profiles of two individuals were found (Raffaele and the victim Meredith Kercher), a low partial profile matching Amanda Knox and two partial trace profiles matching two unknown females. However, this is to be expected in a household where the victim shared washing and drying facilities with three other women.
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Old 24th March 2010, 02:39 PM   #39
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Originally Posted by Kestrel View Post
How do you separate the DNA belonging to two or more individuals from a mixed sample in order to measure it?
One would assume by counting, rather then weighing. There'd be an equasion. For example, if someone gets peaks from a certain profile averaging 50 rfu, they can equate the volume of DNA corresponding to that particular range, along with the mean number of cells.
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Old 24th March 2010, 02:41 PM   #40
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Originally Posted by Kestrel View Post
In the Peggy Hettrick murder caseWP, the DNA evidence that finally freed Tim Masters was collected from places where someone merely touched the victims clothing two decades earlier. The same is true of the touch evidence processed about a decade after the JonBenét RamseyWP murder.

Extracting DNA has little to do with the cell nucleus. The extraction process simply dissolves the rest of the cell.
I agree. My point was that "degradation" of DNA isn't merely a factor of time. It is probably more difficult to extract DNA from fresh dust (keratinized skin cells) than from a preserved blood sample stored for years.
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