Continuation Part Nine: Amanda Knox/Raffaele Sollecito

[Planigale]

Once 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 SevenPart 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.

 

[Planigale]

The reason why you need a certain amount of DNA for typing is so you start with several copies of the areas of interest (STRs). From memory ideally about 15 copies, so about 15 cells worth of DNA. Too few and you run into the problem with uneven replication of areas of interest - the LCN issues. You do not want too much as you want to selectively amplify the areas of interest to stand out from the background DNA.

 

[anglolawyer]

I agree. The PG commenters typically focus on gap of about 46 days, while ignoring the lack of a sealed crime scene and the other problems you list. In addition, and perhaps most importantly, they have no good explanation for the other DNA found on the clasp. Unless one believes that 2-4 additional men took part in the assault, the presence of these donors greatly weakens the probative value of the clasp even on its own.

I don't doubt for a second the validity and usefulness of this exchange but I do mean to be irritating and to interject every now and again to say there is no evidence of anything at all on the clasp, since Stefanoni inexplicably destroyed it as evidence. The same is true of the knife but without the destruction.

Can I raise again something that none of the experts here or elsewhere really seem to go for but which leaves me baffled. Blood consists of plasma, white cells and red cells (probably other stuff too but never mind). The red cells have no DNA and they exist in a ratio upwards of 1,000 to 1 with white cells. If the knife was stuck in the victim's throat and withdrawn it will have had blood on the blade. Then it get's washed, leaving a tiny residue of blood lodged in a scratch, supposedly. Stefanoni swabs it and finds, she says, enough DNA to infer the presence of what, 3-5 white cells or so in her sample? She must have swabbed 3,000-5,000 red cells too, right? That seems like quite a lot to me. Surely that is more than enough to react with TMB?

That's the first thing. The second thing is - how did she manage to scoop up every single one of those cells in one operation? Dan O and I had a conversation about the size of these things and IIRC Dan was happy with this: if the cells were the size of tennis balls, the scratch would need to be about the size of the Titanic. So imagine a gigantic swab slurping through the hull of the Titanic and unfailingly picking up every single ball/cell leaving nothing behind at all.

It must be remembered and stressed - she performed no cytology. She did not look at her sample to see what she had. Conti-Vechiotti were able to photograph the handful of 'hexagonal structures' they sampled from the knife. How come there are no photographs of this huge number of red and white cells?

 

[Diocletus]

I don't doubt for a second the validity and usefulness of this exchange but I do mean to be irritating and to interject every now and again to say there is no evidence of anything at all on the clasp, since Stefanoni inexplicably destroyed it as evidence. The same is true of the knife but without the destruction.

Can I raise again something that none of the experts here or elsewhere really seem to go for but which leaves me baffled. Blood consists of plasma, white cells and red cells (probably other stuff too but never mind). The red cells have no DNA and they exist in a ratio upwards of 1,000 to 1 with white cells. If the knife was stuck in the victim's throat and withdrawn it will have had blood on the blade. Then it get's washed, leaving a tiny residue of blood lodged in a scratch, supposedly. Stefanoni swabs it and finds, she says, enough DNA to infer the presence of what, 3-5 white cells or so in her sample? She must have swabbed 3,000-5,000 red cells too, right? That seems like quite a lot to me. Surely that is more than enough to react with TMB?

That's the first thing. The second thing is - how did she manage to scoop up every single one of those cells in one operation? Dan O and I had a conversation about the size of these things and IIRC Dan was happy with this: if the cells were the size of tennis balls, the scratch would need to be about the size of the Titanic. So imagine a gigantic swab slurping through the hull of the Titanic and unfailingly picking up every single ball/cell leaving nothing behind at all.

It must be remembered and stressed - she performed no cytology. She did not look at her sample to see what she had. Conti-Vechiotti were able to photograph the handful of 'hexagonal structures' they sampled from the knife. How come there are no photographs of this huge number of red and white cells?

Very clever, but the dodge is that it's just skin or flesh or something that didn't get washed off when all of the blood did. Either that or it's something that got on there during repackaging/transport or Stef's lab put it there. You decide.

 

[Desert Fox]

Checked out Mitochondria DNA for Red Blood Cells and looks like that is lacking as well.

 

[anglolawyer]

Very clever, but the dodge is that it's just skin or flesh or something that didn't get washed off when all of the blood did. Either that or it's something that got on there during repackaging/transport or Stef's lab put it there. You decide.

So what was the human species test? Is that (as has been said) just another blood test? Not blood, not human, no cytology, negative to quantification = nothing. Except in Italy where it's slam dunk proof of murder, of course.

ETA is there in there annals of forensic science a case in which a bladed murder weapon yielded DNA from the victim which was not and/or did not include any blood?

 

[Diocletus]

I would like to ask those who have looked at the lab data of evidence such as blood samples collected outside the downstairs apartment and inside it the following:

1. Do you believe a human being dripping blood entered the downstairs apartment after the murder?
2. Do you believe the person is male or female?
3. Do you believe that some of the blood drop patterns indicate the person may have been wearing clothes wet with water!
4. Do you believe the person lay down on a bed?
5. Do you believe the person used the downstairs shower?
6. Do you believe Stefanoni understood it was a human being and not solely a cat?
7. Do you believe Stefanoni suppressed evidence of this?

I believe that the dripping blood going down the outside stairs is watered-down blood of Meredith Kercher, dripping off of a wet item of clothing.

I am not certain whether the person who was dripping the blood (let's call him "Rudy") went inside. It does look like a cat could have gone through the window. On the other hand, there are some things about the inside blood that look wrong: it's on a lightswitch; the bloody bedding looks like it was pressed down by someone sitting on it; the splatters on the floor look too large/sporadic to be coming from a cat.

On the issue of Stefanoni suppressing evidence, I believe that the answer is "yes." First of all, given her frequent inaccuracies/lies, I disbelieve anything said by her or written in her reports unless it is backed by contemporaneous documentation, or is a statement against the prosecution's interests. There is nothing whatsoever to back up her reference to the blood as "cat" blood, so I disregard it. She has clearly suppressed egrams that she ran on some of these samples, so there is evidence of suppression right there.

 

[Diocletus]

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.

I don't think she did that, though. Her recipe for the qubit analysis was 199 uL of "mother solution" to 1 uL of sample (this extreme dilution is why the measurement is so imprecise). I would think that she just chucked the mixture when she was done "quantifying" it.

 

[Diocletus]

So what was the human species test? Is that (as has been said) just another blood test? Not blood, not human, no cytology, negative to quantification = nothing. Except in Italy where it's slam dunk proof of murder, of course.

ETA is there in there annals of forensic science a case in which a bladed murder weapon yielded DNA from the victim which was not and/or did not include any blood?

The human species test would be the successful STR amplification (human specific). All that tells us, though, is that there was some Kercher DNA in the amplification tube. Just like there was unidentified human DNA in some of the Real Time control wells.

 

[Dan O.]

To me, the problem with learning all of this is understanding the language and terminology. All the descriptions sort of assumes that the reader understands the process. That we know what PCR, quantitation, quantification,amplification and what the dyes are and what it means to bind to double DNA and what eletropherisis is and what gene sequencing is and what STR is and what is a haplotype is and what the Speed vac is or the Qubit or the Real Time etc etc. I really wish someone with all this test equipment made a You Tube video and went through the process step by step describing each machine and each step in plain English and then used the nomenclature.

But there are youtube videos of much of this equipment already out there. I've used them myself to learn what some of the kit is and how it works and especially how it can fail. Other resources to look for are the manufacturers documentation and written lab procedures. what WE should be doing is documenting everything we know about each piece of equipment and linking to the best reference material as a guide for the next person that wants to learn.

I finally feel like I'm over the hump with it although there still is some blanks to fill in. For example, why does Stefanoni use the speedvac to concentrate the sample? (The speedvac seems to use centrifuge and heat to dry up the liquid in the well while not destroying the DNA...right?) Is this really necessary?

The speed-vac is primarily a vacuum that freeze dries the sample by lowering the atmospheric pressure and therefore lowering the temperature at which the water boils. Heat is applied to keep it from freezing because freezing can damage the DNA and the water boils off faster at higher temperatures. The centrifuge pulls the heavier DNA to the bottom of the tubes so when the lighter liquids boil off the top it doesn't take the DNA with it. The speed-vac is a prime candidate for contamination transfer because it must be operated with the lids removed from the sample tubes.

And why is the quantification so important? In other words, I have a good profile, why is it so important to know precisely how much DNA is in the sample? That seems superfluous.

I thought this was already answered. If you have too much DNA you get uneven amplification in the PCR and it can gum up the capillary tubes in the electrophoresis. If you have too little DNA you get stochastic effects that interfere with interpretation of the results.

 

[Bill Williams]

Barbie Nadeau's latest..... you have to give her something - that she's been by far the most successful in monetarizing this horrible tragedy.

She's done it through complete, one-sided reporting, all based on her sluttly book, and the notion that Perugian college life runs on threesomes....

Now she is chiming in on the "separation" issue of the two defence appeals.

Lo and behold! She now claims something that does not put PLE in good light!

"...Sollecito was called in alone to clear up a few fine points, including whether or not he really believed his girlfriend was telling the truth. According to the transcripts of that unrecorded interrogation, he hedged when he was asked to specify just what time Knox joined him in his Perugia apartment the night Kercher’s throat was fatally slit. Sollecito’s leap of faith in Knox sent alarm bells ringing."​

There are transcripts! And her use of "According to...." might suggest that she has read them?

Or is she just lying?

 

[Anode]

Barbie Nadeau's latest..... you have to give her something - that she's been by far the most successful in monetarizing this horrible tragedy.

She's done it through complete, one-sided reporting, all based on her sluttly book, and the notion that Perugian college life runs on threesomes....

Now she is chiming in on the "separation" issue of the two defence appeals.

Lo and behold! She now claims something that does not put PLE in good light!

​

There are transcripts! And her use of "According to...." might suggest that she has read them?

Or is she just lying?

Is she talking court testifying transcripts and not interrogation transcripts?

 

[carbonjam72]

Could it be a feint to Follain?

Is she talking court testifying transcripts and not interrogation transcripts?

Quote:
"...Sollecito was called in alone to clear up a few fine points, including whether or not he really believed his girlfriend was telling the truth. According to the transcripts of that unrecorded interrogation , he hedged when he was asked to specify just what time Knox joined him in his Perugia apartment the night Kercher’s throat was fatally slit. Sollecito’s leap of faith in Knox sent alarm bells ringing."

Is she possibly referring to something in Follain's book? (I haven't read it but I heard he includes a transcript of some sort regarding the interrogation).

 

[acbytesla]

But there are youtube videos of much of this equipment already out there. I've used them myself to learn what some of the kit is and how it works and especially how it can fail. Other resources to look for are the manufacturers documentation and written lab procedures. what WE should be doing is documenting everything we know about each piece of equipment and linking to the best reference material as a guide for the next person that wants to learn.

The speed-vac is primarily a vacuum that freeze dries the sample by lowering the atmospheric pressure and therefore lowering the temperature at which the water boils. Heat is applied to keep it from freezing because freezing can damage the DNA and the water boils off faster at higher temperatures. The centrifuge pulls the heavier DNA to the bottom of the tubes so when the lighter liquids boil off the top it doesn't take the DNA with it. The speed-vac is a prime candidate for contamination transfer because it must be operated with the lids removed from the sample tubes.

I thought this was already answered. If you have too much DNA you get uneven amplification in the PCR and it can gum up the capillary tubes in the electrophoresis. If you have too little DNA you get stochastic effects that interfere with interpretation of the results.

Thanks Dan, I never knew that too much DNA was a problem. I knew the too little was though. But in a way that doesn't answer the question I had which is sort of a backward question. Imagine I'm a guilter, a judge and the question is, We have a good profile that matches the defendant so why should we care about how much DNA was in the sample?

Your explanation is pretty good and I have looked at a few of the youtube videos. But I haven't watched any that I thought made it a lot easier. For example, Planigale just tried to explain much of the process and I understood it, but I think that is because I'm over that hump. Most new to the process would say "huh"?

What I really think what is needed is a good glossary that explains each term in plain English not the nomenclature or better yet, plain English and the nomenclature.

The manuals and documentation for the machines have been the best sources for me, but even they are intimidating and a bit overwhelming to start with.

I know asking for the youtube videos is kind of lazy. But what I've been thinking is someone to produce the youtube video to show what Stefaanoni DID explaining the machines and the process and why what she did seems very strange.

I doubt anyone will ever do it. (Way too much work..and few people would actually watch it) If you want to learn it you just have to dive in with both feet and not let this foreign language defeat you....(It beat me more than a few times and I quit after learning a little) Finally though, I feel much less intimidated by the nomenclature and the language. So thanks to you Dan, Chris, Planigale, Diocletus and anyone else who had the patience to answer my questions.

I agree, that what is needed is links to the best sources. So far the best sources have been the mfr's manuals.

 

[carbonjam72]

DR Gill's Book?

Thanks Dan, I never knew that too much DNA was a problem. I knew the too little was though. But in a way that doesn't answer the question I had which is sort of a backward question. Imagine I'm a guilter, a judge and the question is, We have a good profile that matches the defendant so why should we care about how much DNA was in the sample?

Your explanation is pretty good and I have looked at a few of the youtube videos. But I haven't watched any that I thought made it a lot easier. For example, Planigale just tried to explain much of the process and I understood it, but I think that is because I'm over that hump. Most new to the process would say "huh"?

What I really think what is needed is a good glossary that explains each term in plain English not the nomenclature or better yet, plain English and the nomenclature.

The manuals and documentation for the machines have been the best sources for me, but even they are intimidating and a bit overwhelming to start with.

I know asking for the youtube videos is kind of lazy. But what I've been thinking is someone to produce the youtube video to show what Stefaanoni DID explaining the machines and the process and why what she did seems very strange.

I doubt anyone will ever do it. (Way too much work..and few people would actually watch it) If you want to learn it you just have to dive in with both feet and not let this foreign language defeat you....(It beat me more than a few times and I quit after learning a little) Finally though, I feel much less intimidated by the nomenclature and the language. So thanks to you Dan, Chris, Planigale, Diocletus and anyone else who had the patience to answer my questions.

I agree, that what is needed is links to the best sources. So far the best sources have been the mfr's manuals.

Has anyone read Dr Gill's Book that just came out? Is this type of review more or less what he was trying to provide, a guide for lawyers and judges on DNA. (And, with an analysis of the DNA evidence in the Kercher case.)

As others have alluded to, the timing of publication seems strangely fortuitous.

 

[acbytesla]

Has anyone read Dr Gill's Book that just came out? Is this type of review more or less what he was trying to provide, a guide for lawyers and judges on DNA. (And, with an analysis of the DNA evidence in the Kercher case.)

As others have alluded to, the timing of publication seems strangely fortuitous.

You just have to pony up the $25 for the 100 page E-Book. I'm too cheap and it might be wasted on me. I'm waiting to see what the experts on the forum have to say about it.

 

[acbytesla]

Barbie Nadeau's latest..... you have to give her something - that she's been by far the most successful in monetarizing this horrible tragedy.

She's done it through complete, one-sided reporting, all based on her sluttly book, and the notion that Perugian college life runs on threesomes....

It doesn't?

Now she is chiming in on the "separation" issue of the two defence appeals.

Lo and behold! She now claims something that does not put PLE in good light!

​

There are transcripts! And her use of "According to...." might suggest that she has read them?

Or is she just lying?

hmmmmmm let me think about that...She's lying.

 

[Anode]

hmmmmmm let me think about that...She's lying.

Well it seems either she or the PLE is.

 

[acbytesla]

Well it seems either she or the PLE is.

No, she's lying. It has never mattered to Barbie to tell the truth. She has fed more slutty nonsense into this story than anyone else. What matters most to her is to write an interesting story that people will read. And she's been very good at that.

Barbie has never been that great of source for the facts. If there are "transcripts" to the interrogations, she's never seen them.

 

[Strozzi]

No, she's lying. It has never mattered to Barbie to tell the truth. She has fed more slutty nonsense into this story than anyone else. What matters most to her is to write an interesting story that people will read. And she's been very good at that.

Barbie has never been that great of source for the facts. If there are "transcripts" to the interrogations, she's never seen them.

If interrogation transcripts exist, they would have great monetary value. Barbie doesn't have a copy because if she did she would have written and sold articles based on them. After all that has transpired in this case, transcripts would cause an earthquake at police headquarters in Perugia. Which is why they don't exist.

Remember the Perugia police cars that raced to Rome to try to seize documentation from C & V? There would be a repeat of that from Perugia to Barbie's house if she even just had notes made from interrogation transcripts.