Wednesday, 17 March 2010

A Baseline for LCN DNA Analysis

This came up in a discussion about JonBenét Ramsey. The main point in the discussion was that a baseline was needed for touch DNA analysis, which became synonymous with Low Copy Number (LCN) analysis. There is quite a bit of background here that needs to be covered as well, because this case will is also an example for this post. If you want to read the short answer, just scroll all the way down to the last line.

Background and Evidence

In 1996 she was found dead in the basement of her parents' home in Boulder, Colorado and the case is still unsolved. There has been much discussion of who actually committed the crime, some arguing the parents did it, others an intruder, but there is no resolution in sight. In a way it echoes the Azaria Chamberlain disappearance here in Australia in that there are people who think that Lindy Chamberlain killed her daughter and blamed the dingoes.

Back in 2008 the Washington Times reported that the Boulder DA had cleared the Ramseys (although Patsy Ramsey had died in 2006). Forensic Scientists had analysed touch DNA on the leggings that she was wearing on the night she died. This DNA matched DNA found on her panties.

Now it gets a little tricky here. DNA was also found under her fingernails the belonged to an unknown person. Now according to CBS News 48 Hours this DNA also matched that on her panties. I'm having trouble finding corroborating evidence for this, but assuming that it's all true we've got DNA from under her fingernails, DNA on her panties, and touch DNA from her leggings. Apparently these all match.

It's the last one that caused this discussion, but there is still a little more to cover.

What is "touch DNA"?

"Touch DNA" is a term that describes DNA that is left by touching an object.

The concept relates to Locard's exchange principle, which essentially explains the idea behind trace evidence. Whenever you do something you will leave a trace behind. It may not necessarily be DNA but it will be something from you. This includes hair samples, fibres, footprints and fingerprints. The opposite is also true, you will take something from the place you are at. This includes dirt in your shoes, other fibres or hairs and the like.

Touch DNA is an extension of this idea. Since some of the things that make up fingerprints includes skin cells it is possible to extract DNA from a fingerprint. However such DNA does not have to come from a fingerprint, it may come from other body parts as well, so your clothes will contain extractable and examinable DNA as well.

This article from the FBI analysed DNA gathered from clothing. They gathered DNA from both the inside and outside of the clothes and analysed it for DNA. Interestingly they found samples of DNA from other people including spouses and in one case from someone who nobody knew at all. The fact that this was from a newly opened package of hosiery made things all the more interesting.

This article was cited as evidence as to why a baseline was needed.

But before we look at that we will need to look at what LCN DNA analysis is.

What is LCN DNA Analysis? How does it relate in this case?

Low Copy Number Analysis, also called/considered a subset of Low Template Analysis, is the analysis of small amounts of DNA. Normally this analysis is used when regular Polymerase Chain Reaction (PCR) analysis doesn't provide a testable amount of DNA. The sample sizes that we are looking at here is less then 200 pg (about 2x10-13kg). Normally samples contain about 0.5 to 2ng (5x10-13kg to 2x10-12kg) of DNA usually because you amplify it with PCR. The University of Leicester has a page on the subject.

PCR is best describes as a DNA photocopier. It uses the same proteins that are used in DNA copying, although the taq polymerase comes from Thermus Aquaticus, a thermophilic bacteria, so the polymerase is able to withstand higher temperatures, like those used to separate the DNA strands. It should be noted that it copies the DNA of everything that's in the sample, so contamination is a very real problem and great care is taken to prevent it.

Normal PCR amplification uses about 28 cycles. LCN adds an extra 6 cycles, so the amplification process uses 34 cycles.

As far as I'm aware there is, as yet, no data available as to how much touch DNA was gathered. It was assumed that it was a very small amount, so naturally it was assumed that LCN analysis was used.

Has LCN DNA Analysis been used in other cases?

In Australia such analysis was used in the murder of Peter Falconio. DNA was found on cable ties that were used to bind Joanne Lees. The DNA was found to belong to Bradley John Murdoch, who was later convicted of the murder. The court was told at the time that the odds of it belonging to someone else were 1 in 150 quadrillion.

In 2007 it was used in the Omagh bombing case in the UK. After concerns from the justice in the case the CPS suspended the use of LCN, and ordered a study. In 2008 the CPS announced that there was no inherent unreliability in the analysis.

It has also been used in cases like the murder of Anna Lindh, a Swedish politician, where the DNA came from a knife handle, and in the murder of Marion Crofts. This pdf from the Forensic Science Service has a few more examples.

So why a baseline?

The logic behind the baseline is that with the small amounts of DNA involved there might be an increase in the number of people falsely accused by DNA evidence.

Therefore a baseline of some sort should be found to see if the DNA is coincidental or not. The idea being once you have the baseline you will be able to discriminate between "coincidental" and "relevant" samples.

In my opinion though there is no way to statistically determine whether a sample is coincidental. The DNA needs to be processed and looked at in light of the other evidence. In the Ramsey case it's that this DNA appeared on the leggings, the panties, and under her fingernails. It was found to be the same DNA. But how did it get there?

DNA Transfer

LCN analysis is rather recent, the earliest case is from 1999, but the concept of DNA transfer is older, but has not been studied in great detail. There are plenty of articles out there though.

One of the issues brought up in the Ramsey discussion was that the DNA could have been transferred by JonBenét herself. Theoretically this is possible, but there is limited evidence that it did occur.

It depends on how it transferred, and also how much DNA was found. If we were to see transference we would see much DNA under her fingernails, and less on the clothing in the order that she put them on.

Now DNA transference can be ordered. Firstly we have primary transfer. This is DNA transferred when when you touch a doorknob, or a mouse, or shake hands with someone. The DNA is being transferred directly from you to the person or object. This is essentially a given.

Next is secondary transfer. This is when DNA is transferred to a third party. So let's say Person A shakes hands with Person B. Some of Person A's DNA will be transferred to Person B. This is primary transfer. Then Person B uses the same hand to open a door. Some of his DNA will be transferred (again primary), and some of Person A's DNA will be transferred. The latter is secondary transfer. Person A didn't touch the doorknob but some of his DNA is on it.

There have been studies that examine this. Lowe et al. examined this in the 2002 paper The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces (abstract here). They started by determining how good a DNA shedder the subjects were. Once they determined who was a good shedder and who was a poor shedder they washed the hands of the poor shedder and got them to shake hands with the good shedder. The poor shedder then touched an object and they used LCN analysis to see which profiles they found.

Turns out the poor shedder is a poor shedder for 6 hours after they wash their hands, and they managed to find the good shedder on the object.

However, Ladd et al. found in A systematic analysis of secondary DNA transfer (1999) (here) found no transfer, they also found that primary transfer was:
[H]ighly dependent upon the individual handling the objects or performing the handshaking.
Next is tertiary transfer. In the example with Person A and Person B tertiary transfer would be if Person C touched the same doorknob that Person B did. Some of Person B's DNA would be transferred to Person C's hand, and so would some of Person A's. The former is secondary transfer, while the latter is tertiary transfer.

I have not found any papers that deal with tertiary transfer. The closest that I could find was from G. N. Rutty in An investigation into the transference and survivability of human DNA following simulated manual strangulation with consideration of the problem of third party contamination (abstract) . The article doesn't examine tertiary transfer but uses it as a theoretical pathway as to how the unrelated DNA got on the "victim's" throat. Beyond that I've found nothing.

Parameters and Logic.

There are quite a few parameters that need to be examined. So beyond the level of DNA transfer, we would need to look at shedder type, so the odds of being a good/poor shedder, washing in general, how the people touch the object, and I think it would be fair to say the kind of object that they touch.

We are looking at a vast number of possibilities, and the reason for that is just to determine whether a sample is coincidental or not.

But we also have to look at the evidence. There is very little evidence for transfer beyond secondary. Anything above that appears to be theoretical.

But we must remember that logic would need to be applied based on the evidence.

So one possibility is that JonBenét came in contact with some guy's DNA. That somehow got under her fingernails. One suggestion for that is "dirty hands". She then touched her clothes with these fingers and the DNA transferred. So clothes wise we are looking at DNA that is at least secondary transfer DNA, but could actually be in levels that haven't been examined yet. The other thing is that when stuff gets under fingernails it usually stays there until removed.

The stranger possibility was that the DNA was on the panties when some guy at the factory touched them. I don't see that as logical at all, especially considering that the DNA under the fingernails was found and analysed far earlier then the leggings.

My own opinion is that it was a bunch of primary transfers. The DNA got under her fingernails by scratching, which is primary transfer. The DNA got on the leggings and the panties when the person touched them, which is more primary transfer.

But now I think it's time to answer the question.

Is a baseline even possible?

In my opinion it's simply no. LCN DNA already relies on standards set by the relevant government. It still needs to match 13 loci in the US. In the UK they would still need to match the 10 loci, and according to the University of Leicester the last 6 cycles are amplified in duplicate and alleles that appear in both can be used in a profile.

The legal standards will not change because the analysis is different.

One thing that I have noticed is that there are no baselines for other pieces of trace evidence. This is probably the most damning factor in the argument for a baseline. Why should one type of trace evidence have a baseline while all other types of trace evidence be exempt from such a baseline.

It's simply because there is no way to statistically discriminate between relevant and coincidental evidence. In a crime everything collected is potential evidence. It doesn't matter if it's touch DNA, a fibre, or blood spatter on a carpet, it's all evidence until it can be determined that it isn't. In a way it's backwards to what we would think. It's evidence until it isn't, not it's not evidence until it is.

Once we know that, say the vomit in the sink, actually belongs to a paramedic who didn't tell anyone that he threw up, it's potential evidence and should be processed accordingly. That's the reason why it's called an investigation. Because you investigate leads. There are no statistics that will allow you to discriminate evidence.

Lastly there is one other reason why a baseline is stupid. It's just one big red herring.

1 comment:

halides1 said...

I just have time for a quick comment tonight. But I wanted to point out one or two things about LCN analysis. First, it is more prone to alleles dropping in or dropping out. Therefore, most labs run the analysis twice, and score the alleles that show up both times. Second, due to stochastic effects, the peaks within a locus, which should be equal in height, are often quite different. This is also the situation when one has a mixture arising from two people. My bottom line is that LCN is qualitatively different from standard testing.