Dead skin cells don't contain DNA?

[Fulcanelli]

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.

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?

 

[shuttlt]

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?

 

[Cainkane1]

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.

 

[Kermit]

.... 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.

 

[Ixion]

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.

 

[HumanityBlues]

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.

 

[Kestrel]

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.

 

[Ixion]

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.

 

[Kestrel]

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.

 

[Ixion]

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.

 

[Simon39759]

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...

 

[shuttlt]

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.

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.

 

[Simon39759]

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...

 

[Fulcanelli]

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.

 

[Fulcanelli]

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.

 

[Ixion]

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.

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.

 

[Kestrel]

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?

 

[Fulcanelli]

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.

 

[Fulcanelli]

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.

 

[JoeTheJuggler]

In the Peggy Hettrick murder case^WP, 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 Ramsey^WP 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.