Continuation Part Eight: Discussion of the Amanda Knox/Raffaele Sollecito case

[Diocletus]

But according to Chris, the slope of 544 is the only normal one showing a 100% efficiency (slope = -3.31). All of the others are hyper efficient.

So Stef abandons the machine whenever she "only" gets 100% efficiency?

There is apparently a change in baseline specific to these high-threshold runs, what does that mean?

 

[Strozzi]

Photo od ABI 7700 and plate or tray

I am one of the scientific illiterates (aka: idiots) on this forum. If you are like me and want to see a picture of the ABI 7700 machine, please go to the link below. The .pdf also includes photos of printouts and plate or tray. The .pdf concerns how to maintaining (clean) the machine. Even an idiot can understand that that is necessary.

http://tools.lifetechnologies.com/content/sfs/brochures/cms_042499.pdf

 

[acbytesla]

I doubt you would stick your neck out if not sure of your ground and I also know that Tom Zupacnic endorses your conclusions. I also know from your posts you are someone deserving respect. None of which is a substitute for understanding this myself. I am in the idiot's corner with Bill until I do. No big deal. It wouldn't be the first time. Would you please explain:

1 what a 'plate' is?
2 At what point in the DNA profiling operation are plates used?
3 What do they look like - something like the ice cube tray in the freezer?
4 what are these ID numbers?
5 when are they applied to an item and what type of item (a knife, a sock, a sample from a blade etc)
6 describe the process 'at a basic level' and say how the pieces of data fit together

Treat this as a request for further and better particulars. I will apply for an order in case of default

Anglo. I downloaded the manual for the ABI 7700 which is the DNA machine we are talking about. There are pictures of most everything.

The plate is essentially a plate of wells for either DNA samples or control samples. On page 46 of the manual is a picture of a 96 well plate.

The manual can be obtained at the following link. http://www.genomica.uaslp.mx/Databases/ABI7700 Users Manual.pdf

 

[acbytesla]

I am one of the scientific illiterates (aka: idiots) on this forum. If you are like me and want to see a picture of the ABI 7700 machine, please go to the link below. The .pdf also includes photos of printouts and plate or tray. The .pdf concerns how to maintaining (clean) the machine. Even an idiot can understand that that is necessary.

http://tools.lifetechnologies.com/content/sfs/brochures/cms_042499.pdf

You sort of beat me to it but I do think the link I am providing is better. But of course mine is 198 pages long.

 

[Diocletus]

Anglo. I downloaded the manual for the ABI 7700 which is the DNA machine we are talking about. There are pictures of most everything.

The plate is essentially a plate of wells for either DNA samples or control samples. On page 46 of the manual is a picture of a 96 well plate.

The manual can be obtained at the following link. http://www.genomica.uaslp.mx/Databases/ABI7700 Users Manual.pdf

But remember, the "plate" that we're talking about isn't the quantification plate. It's the "plate" referenced in the egrams.

 

[Chris_Halkides]

troubleshooting real time PCR, no-template control

Amplification of the No Template Control Due to Reagent Contamination

If you get an amplification product in your no template control (NTC), you may have one of these problems:

Contamination of your reactions by DNA
Reaction contamination can be separated into two types (select one):

Random contamination
Reagent contamination
Primer dimer formation (SYBR® Green chemistry only)
link

EDT
It looks as if random versus reagent contamination can be distinguished on the basis of how the replicates behave, relative to each other. I realize that this is not pertinent to the present discussion, but it might be helpful with respect to previous discussions of the DNA that was in a no-template control.

 

[acbytesla]

But remember, the "plate" that we're talking about isn't the quantification plate. It's the "plate" referenced in the egrams.

It seems as if the 7700 also analyzes the DNA and it provides e-grams or am I misunderstanding this? I've been reading the manual. Doesn't it do it in the same well plate? And aren't there different size well plates that can go into the machine?

 

[Diocletus]

It seems as if the 7700 also analyzes the DNA and it provides e-grams or am I misunderstanding this? I've been reading the manual. Doesn't it do it in the same well plate? And aren't there different size well plates that can go into the machine?

The egrams come from a 3130.

 

[acbytesla]

The egrams come from a 3130.

Can a well plate from a 7700 be placed in a 3130? Do they pull individual wells out and place them into a different well plate designed for the 3130?

 

[Diocletus]

Can a well plate from a 7700 be placed in a 3130? Do they pull individual wells out and place them into a different well plate designed for the 3130?

The 7700 only uses a bit of template material to quantify. Those samples are discarded. A whole new array, using the main template material, is set up to perform the STR amplification.

 

[Dan O.]

Can a well plate from a 7700 be placed in a 3130? Do they pull individual wells out and place them into a different well plate designed for the 3130?

With the right kit, it is possible to produce an gram with the same sample as used for quantification. But this is new and not normally done as the quantification typically over amplifies the DNA (50 cycles where the standard is 28). Stef is only using a single color tag for the quantifications and grams are typically produced with 4 colors.

The normal procedure is to split the sample, quantify part of it and then use that result to know how much sample is needed for amplification for the gram to get the best result.

 

[acbytesla]

With the right kit, it is possible to produce an gram with the same sample as used for quantification. But this is new and not normally done as the quantification typically over amplifies the DNA (50 cycles where the standard is 28). Stef is only using a single color tag for the quantifications and grams are typically produced with 4 colors.

The normal procedure is to split the sample, quantify part of it and then use that result to know how much sample is needed for amplification for the gram to get the best result.

Thanks to both you and Diocletus. I just downloaded the 3130 manual. It seems as if they both use the same 96 well plates. The 3130 manual does mention a 384 well plate also.

Any way the image of the well plate should help anglo understand what a plate is.

 

[Dan O.]

Here is a comparison of Slopes and Intercepts for the 7700 runs and the 7500 runs. Nothing stands out as abnormal.



There is also the point that these are not the final result. The quantification has minimal impact on the egram results. The smoking gun is still the clear demonstration that contamination was present in this lab and we shouldn't loose sight of that.

 

[acbytesla]

Here is a comparison of Slopes and Intercepts for the 7700 runs and the 7500 runs. Nothing stands out as abnormal.

[qimg]http://www.internationalskeptics.com/forums/imagehosting/thum_151445395fcafc54c8.png[/qimg]

There is also the point that these are not the final result. The quantification has minimal impact on the egram results. The smoking gun is still the clear demonstration that contamination was present in this lab and we shouldn't loose sight of that.

I'm still trying to understand precisely what Stefanoni testified to as how much DNA was in the sample and how much we now believe was actually in the sample. Does anyone know? Dan has said that this shows that in fact there is 16 times less DNA because of this evidence. So? can we be a little more clear? I'm still confused how we come to that determination as we are stating that instead of finding 23pg we are finding 108pg how that means that it is 4ct times less instead of more. ????

 

[Diocletus]

Here is a comparison of Slopes and Intercepts for the 7700 runs and the 7500 runs. Nothing stands out as abnormal.

[qimg]http://www.internationalskeptics.com/forums/imagehosting/thum_151445395fcafc54c8.png[/qimg]

There is also the point that these are not the final result. The quantification has minimal impact on the egram results. The smoking gun is still the clear demonstration that contamination was present in this lab and we shouldn't loose sight of that.

Dan: I stared hard at that for 30 seconds and then looked at a blank wall, and I could a perfect likeness of Howard Stern.

 

[Planigale]

I don't mean to put a downer on things. But we are only talking about amplification here. (We take a sample containing a small amount of DNA and put it through multiple replication cycles.) A slight inaccuracy in measuring the amount of DNA in the initial sample is not very important. The important issue is the negative controls and in particular the results of the substrate negative controls put through the Ambiprism 3130 for STR typing.

On the other hand this is a great example of why the EDF are needed since these will document the setting of the 7700 in particular the setting of the threshold which needs to be adjusted depending on the calibration curve.

 

[Planigale]

I don't mean to put a downer on things. But we are only talking about amplification here. (We take a sample containing a small amount of DNA and put it through multiple replication cycles.) A slight inaccuracy in measuring the amount of DNA in the initial sample is not very important. The important issue is the negative controls and in particular the results of the substrate negative controls put through the Ambiprism 3130 for STR typing.

On the other hand this is a great example of why the EDF are needed since these will document the setting of the 7700 in particular the setting of the threshold which needs to be adjusted depending on the calibration curve.

 

[acbytesla]

I don't mean to put a downer on things. But we are only talking about amplification here. (We take a sample containing a small amount of DNA and put it through multiple replication cycles.) A slight inaccuracy in measuring the amount of DNA in the initial sample is not very important. The important issue is the negative controls and in particular the results of the substrate negative controls put through the Ambiprism 3130 for STR typing.

On the other hand this is a great example of why the EDF are needed since these will document the setting of the 7700 in particular the setting of the threshold which needs to be adjusted depending on the calibration curve.

That is the question Planigale. That is what I'm trying to understand. 4Ct higher or lower? Are we saying that instead of 100 picograms of material that there is 5 or 6 picograms?

 

[Diocletus]

I don't mean to put a downer on things. But we are only talking about amplification here. (We take a sample containing a small amount of DNA and put it through multiple replication cycles.).

To be more specific, we're talking about quantification.

A slight inaccuracy in measuring the amount of DNA in the initial sample is not very important.

Well, wait a minute. The validity of this piece of evidence is based in part on testimony that there was "abundant" DNA on the bra clasp hooks, and the fact of the matter is that: (i) that amount of DNA is commensurate with contamination that we see elsewhere in the quantification run, and (ii) the entire run is unreliable due to the contamination and what might also be an irregularity in the behavior of the machine.

On the other hand this is a great example of why the EDF are needed since these will document the setting of the 7700 in particular the setting of the threshold which needs to be adjusted depending on the calibration curve.

The threshold was adjusted from .10 to .13. This suggests either a high level of background noise or early flourescence activity. Why? And, why did Stefanoni take the machine off line afterwards?

 

[Planigale]

That is the question Planigale. That is what I'm trying to understand. 4Ct higher or lower? Are we saying that instead of 100 picograms of material that there is 5 or 6 picograms?

So how precise do we need to be. Classic STR typing takes 500 - 2000pg. 200 - 500pg is iffy and less than 200 is LCN DNA (or was at the time). So broadly we could say more than 400pg good more than 200 iffy but probably OK less than 100pg seriously dubious LCN and less than 50pg...! So in someways we would be best actually talking about ng with 0.1 as the lower limit anything less being zero. We probably only need to consider 1 decimal point of precision in ng.

Primarily the amplification cycle consists of a machine; the thermal cycler, that is just a sophisticated cooker, joined with the laser quantifier. The reagents are bought in and go in the plate wells. I think it is unlikely the hardware was bust. Usually the software crashes, or the lease is up and you get new kit from the manufacturer. The calibration suggests a reagent issue.