I suspect you might have gotten more than you asked for by this response, I'm sorry I wasn't more concise but I got to thinking about it and one point (and link!) led to another and I probably wrote and linked more than your questions required. If you're not interested in the details and tangents I went through perhaps someone else is.
Could somebody expand on this a bit?
1. What is the smallest amount of DNA that can be used to get a reliable DNA test result?
It kinda depends on what you mean by 'reliable.' As for what can be amplified and profiled, that can be as low as 10 pg or even less, though there's a number of technical considerations regarding the analysis of such minute material and what that might
mean compared to what we normally think of when DNA evidence is evaluated.
Anything below 100-200 picograms (the definition varies) is considered
'low template DNA' and requires special handling and testing to preclude contamination. This is a much greater concern with low template samples as that level material wouldn't even register most of the time doing high template analysis, it wouldn't produce peaks of the appropriate height to register on the machine, in fact odds are they'd not even be labeled by the analysis program as it was set to preclude them. So a sample could very well have contamination from many minor contributors either from the handling or testing but they would never be discovered. This is one big reason why contamination is considered relatively rare in
high template DNA testing, though they still take the necessary precautions, but someone's saliva, blood, semen or identifiable chunks of flesh or skin are less likely to be transferred willy-nilly which is
not the case with (
really) microscopic particles that
do adhere and transfer easily as Halides1 has documented extensively in this thread and
elsewhere from a variety of sources.
Another reason is that DNA analysis is an
identifier, not a time stamp or GPS device. By that I mean when the police or prosecution say they have 'DNA evidence' in the United States or Britain (almost always) and most of the time in Italy they have
an incriminating substance they found near or around the murder that by its very nature suggests involvement in the crime, such as blood, saliva or semen on or near the victim or perhaps skin cells underneath the fingernails. They then use DNA analysis to
identify who that belongs to, it's not just that some unestablished substance DNA was found and that somehow 'proves' that the person touched something or was even somewhere at a particular time, it's the nature of the
substance which helps establish that. That's why if you look at the flow chart for FBI DNA analysis on page 8 of
this report (page 22 of the PDF) you'll see the first step is to identify the biological substance and if that is impossible they just return it to the submitter. If you're really interested in getting in-depth on this subject pages 4-15 (18-29 of the PDF) of that report includes an excellent overview of the DNA analysis process.
In this case <Dr. Stefanoni failed to identify the substance on the blade, we only know it failed several highly sensitive blood tests. She didn't even
bother with the bra clasp which really surprised the independent court experts judging from their bolding it in
their report which Komponisto (the translator) retained from the original:
Conti-Vecchiotti said:
The SAL shows that the generic test for blood using tetramethylbenzine (TMB) was not performed on Exhibit 165B, and nor was any laboratory test performed to show the presence of biological material other than blood.
We maintain that it would have been necessary to proceed to morphological analysis in search of any cells that may have been present by means of coloration with any of the reagents commonly employed in histology (hematoxylin). Such a simple and quick test would have required a minimal amount of material that would not have in any way compromised the subsequent laboratory tests but would have been able to clarify the nature of the material taken from the item under examination.
Notwithstanding the omitted test for cells (and thus the failure to identify the material taken from Exhibit 165B), the Technical Consultant hypothesized the presence of “presumed flaking cells” on the aforementioned item.
The Technical Consultant’s hypothesis regarding the nature of the material analyzed (which was confirmed at the preliminary hearing and at the trial), is wholly arbitrary in that it is not supported by any scientifically objective finding.
2. Is amplification always used or just for situations where there is very little?
Polymerase Chain Reaction amplification is standard in DNA testing, 28 cycles (exponential) is currently the most common, though more amplification such as to 31 or 34 is common with low template samples. In the Massei Report starting on page 183 (of the PMF copy) there's an overview of this process as well drawn from Stefanoni's testimony where it says the
polizia scientifica use 28 cycles but I saw in passing where Diocletus said something about her going to 50 cycles on the knife but I defer to him on that subject as I never came across that information. It did puzzle me that she would have used 28 cycles on a sample she couldn't quantify (therefore she would
know it must be low template and it would be necessary). Incidentally that section of Massei is basically the same thing as pages 4-15 of the FBI report I linked above, but I recommend the FBI one as it is not translated thus is easier to follow--and it has pretty pictures!

))
The other type of testing which does not rely on amplification is called RFLP (restriction fragment length polymorphism) testing and requires thousands of cells and has been replaced for the most part by PCR analysis.
This page explains the basics of the RFLP process and includes another (shorter and perhaps easier going) description of the PCR process as well.
3. What is the mass figure a measurement of:
A. The total mass of DNA that is analyzed including all sources for the DNA in the sample including plant DNA if it is present?
B. The total mass of human DNA?
C. Something else?
Once the original sample (which may be of any size and composition) finishes the extraction process whatever was quantified has generally undergone human-specific quantification. Most kits are designed for (human) DNA samples of ~0.5 ng (500 picograms) to 2.0 ng, generally considering around 1 ng to be an optimum sample size (too much and the peaks will be off the chart, too little and they may not register enough RFUs). However there's also '
total DNA quantification' which may have been done before that step and is used on other procedures.
My uninformed view of the DNA on the bra clasp has been that there was a very small amount that was amplified a great deal. The test results were probably equivocal because the raw data wasn't made available to the defense. The nature of the equivocal data probably was such that matching anybody's DNA to the test results would have been equivocal because there wasn't a strong signal from any individual's DNA so that there was a large subjective element to the "matching" of the test results to RS DNA.
While it is true that there were so many alleles assembled by the numerous contributors to the clasp (minimum of 4) that one could find a 'matching profile' for roughly a quarter of Italian men, odds are that Raffaele's DNA contributed to the sample that generated that autosomal EDF due to the fact his Y-haplotype was present in that test as well (testing different parts of the DNA 'string').
Is this view a reasonable one? If the sample size wasn't actually small, does that make it more likely that the alleged RS DNA match was accurate? Do we know how the amplitude of the DNA signal claimed to be from RS compares with the amplitude of the DNA signal from the unknown males?
The larger the sample size the easier it is to amplify, profile and analyze. The more contributors to a sample the easier it is to 'match' people who didn't even contribute to the sample at all, however as stated above I don't think that is the case in this instance. Raffaele's is the largest of the low template contributors, however that may simply be because he had hundreds of items from his apartment analyzed in that lab that was not the case with others and/or he was at the discovery and exerted himself in attempting to break down the door, probably leaving a fair number of minute skin cells on that door, handle and floor. Meredith's contribution to the sample accounts for roughly 80%, Raffaele probably around 12% and one contributor at about 4% and one or more others of about 4% as well. Actually it's probably closer to 85% Meredith, 9% Raffaele, 3% some other male (who at one locus provided an allele that exceeded Raffaele's) and 3% at least one more male and perhaps other males and/or females as that aligns with Tagliabracci's testimony better (he had Meredith's as ten times that of Raffaele's) but Massei chose to accept Stefanoni's testimony which stated that going by peak heights Raffaele's were one seventh or so. I found looking into it that it's not directly linear with the smaller contributors who will generally produce peaks disproportionally higher than their contribution would suggest perhaps due to the nature of the PCR amplification process which starts geometric and then goes linear at the end and there's a higher percent chance the larger samples will do more reproducing at a linear rate than the smaller ones. However it's not really worth arguing!
You can also profile contamination, and it looks just the same on the EDF as anything actually deposited in the commission of a crime. However contamination does (generally) have certain identifying characteristics: it probably won't be the primary contributor to a sample, it may not even have any biological material outside the DNA (PCR amplifies DNA not cells and stuff) if it was contaminated in the lab, it will generally be found in small amounts and there may well be a number of contributors for items more likely to be subjected to contamination.
Which describes the bra clasp
very well indeed! As
C&V sum it up:
Conti-Vecchiotti Report said:
We find that the Technical Consultant arrived at this restrictive conclusion (presence of only two individuals: victim and Raffaele Sollecito) following an incorrect interpretation of the autosomic STRs as a result of having disregarded the recommendations of the ISFG concerning the correct interpretation of mixtures, recommendations which, had they been followed, would have allowed one to conclude that several minor contributors were present in the trace besides the victim’s profile (major contributor). Hence we agree with Dr. Stefanoni’s statement regarding “the extrapolation of a genetic profile deriving from a mixture of biological substances belonging to at least two individuals, at least one of male sex” but we cannot accept the conclusion stating that “the genetic profile is compatible with the hypothesis of a mixture of biological substances (presumably flaking cells) belonging” only ”to Raffaele Sollecito and to Meredith Susanna Cara Kercher”[/b] insofar as, from what has been explained above, a mixture is present in which several contributors of male sex are present (a circumstance supported by the electropherogram relative to the Y chromosome, where several alleles are clearly present which, despite being particularly evident, were not taken into consideration by the Technical Consultant);
- The item was recovered 46 days after the crime, in a context highly suggestive of environmental contamination. The risk of incorrectly interpreting such environmental contaminants from dust could have been minimized only by taking the care [avendo l'accortezza] to institute extremely stringent control procedures, including the analysis of extracts from sterile cotton swabs soaked with sterile buffer passed on ambient surfaces to take samples of dust, a procedure which was not carried out;
- Taking into account what has been explained relative to the inspection methods, having seen the documentation in the record, and in particular the DVD of the filmed investigation of the scene [indagini di sopraluogo], the official photos of the Scientific Police, and the statements made in court, we find that the universally noted inspection procedures and correct protocols of collection and sampling of items were not applied on Via della Pergola, even [those designed] to minimize environmental contamination and contamination from handling. From this it follows that it cannot be ruled out that the results obtained from Exhibit 165B derive from contamination phenomena in any phase of the collection and/or handling of the item.
Italics and bolding for emphasis and Italian retained from original; highlighting mine
If that clasp, which wasn't collected and processed under protocols required for high template work, let alone low template, isn't considered highly likely to be contaminated I don't know why they even
bother with the rituals and the bunny suits. Consider what must (and/or might) have occurred for that from the moment they collected the bra. Somehow the clasp must either have been detached and coincidentally ended up in the same spot when Rudy (probably) ripped the bra off from the front and threw it to where the bra was collected. If it wasn't already detached it must have been hanging by a string or something and fell off when the bra was packaged. So either they missed it and left it on the floor when collecting the bra or it fell off and they didn't notice it.
They then proceeded to finish collecting the other items, removed the body and then the mattress the whole time not noticing (or caring) the clasp was still on the floor. With that many people in that relatively small little room moving about and moving things, entering and exiting the door which just hours before Raffaele had tried to break down mere feet from where the clasp was eventually found, not in the same place it was originally captured on video six weeks before. That's six weeks it must have laid on the floor there
somewhere in a room with a broken lock and in a house with a broken window, that cottage looks kinda drafty even without the window broken and the lock on the door busted, I wonder would it would have been like to have been a dust bunny those six weeks.
Maybe if the bra clasp could talk it could tell you!
