A6 Murder DNA evidence

[Victor]

Just a minor observation here Victor. In post 12 you clearly state ....I'm a graduate chemist and capable of understanding the science.

regards,
James

Yes, I'm a graduate chemist - I graduated with a Chemistry degreee.

KR,
Vic BSc(Hons) Ebor.

[jimarilyn]

Oh look, some more massaging of the truth.* Let's have some FACTS shall we:-I have a degree in chemistry, but do not work as a chemist.

Just a minor observation here Victor. In post 12 you clearly state ....I'm a graduate chemist and capable of understanding the science.

regards,
James

[Victor]

So how could Whitaker in Hanratty(2002) suggest that the DNA was from sperm?

Simple - you need to read the link posted by DM a few pages back.

It's possible to separate sperm heads from every other source of DNA in a sample, and once isolated the sperm heads can then be profiled.

KR,
Vic.

Hi All
Another excellent scientific resource about the failings of LCN to elicit profiles correctly.

http://www.cstl.nist.gov/biotech/strbase/pub_pres/LCNintro_MAAFSworkshop_May2006.pdf

Check out slide 5 on page 9. I quote "LCN.....May not be able to associate DNA profile with bloodstain or other visual evidence".

And slide 3 on page 10 "Tissue source cannot be determined".

So how could Whitaker in Hanratty(2002) suggest that the DNA was from sperm?

If the less sensitive techniques used in 1995 proved inconclusive then considering the evidence was nigh on forty years old and with all the degredation that has taken place this makes the DNA evidence in Hanratty next to useless.

Regards
The abusive bully.

The truth about LCN

Hi All

An excellent scientific overview of the failings of LCN and it's unsuitability for use in the Criminal Justice System.

http://www.theforensicinstitute.com/PDF/Jamieson%20statement%202.pdf

Regards
Reg

[Victor]

Supra 150rfu's is the accepted, internationally validated threshold for SGM+. It is not at all arbitrary. It has been based on sound reasoned science that demands a sufficient amount of DNA to have been deposited that could quite possibly be detected within a reasonable rfu range. This is codified by the FBI's SWGDAM.

The above is bovine excrement.

If Reg had bothered to read the material in the links he posted, he would have understood that any threshold set is arbitary, as in, it is an informed guess assigned because it appears to give adequate results in the SGM+ process, but there is no explanation as to why 150rfu works and why the process shouldn't use 125 or 141.76983 or 175.

Of course he is now going to object to the above, because he doesn't know whether a threshold is used in the LCN process or not.

Do not be put off by Victor's dismissive attitude towards the findings of some of us here who have found very good reason for seriously doubting the DNA evidence put forward.

Erm... Reg has psuedo-scienfic-sounding reasons as to why the DNA evidence should be doubted because he needs it to be wrong for his faith in Hanratty's innocence to be justified. He has taken the conclusion he wants and manipulated the evidence to fit his pre-conceived ideas. This way of doing things is as logically-bankrupt as it is possible to get.

We have posted enough information here and on the other thread that if Victor and others had read would not just accept the court of appeal as being gospel and then do an ostrich impression when it is challenged.

Reg doesn't seem able to grasp the concept that argument involves the use of persuasion and that shouting and bullying are not good enough to convince reasonable people.

His ostrich analogy is particularly ridiculous because I have always challenged his warped logic and have not ignored him and left his lies to mascarade as the truth.

Victor says he is a chemist but I wouldn't expect my local chap at Boot's to explain the DNA evidence in Hanratty much farther than gattaca.

Oh look, some more massaging of the truth. Let's have some FACTS shall we:-
I have a degree in chemistry, but do not work as a chemist.
The person who works in Boots would be a pharmacist - a specific branch of chemistry.
Gattaca is a fictional film - probably where Reg gets his so-called facts from.
Hanratty is a rapist and murderer.

KR,
Vic.

Hi All
Ignore Victor when he posts stuff like

The Detection Threshold - tells us that Reg's holy grail the "supra-150rfu threshold" is just an arbitary value anyway.

in his post #131.

Supra 150rfu's is the accepted, internationally validated threshold for SGM+. It is not at all arbitrary. It has been based on sound reasoned science that demands a sufficient amount of DNA to have been deposited that could quite possibly be detected within a reasonable rfu range. This is codified by the FBI's SWGDAM.

Do not be put off by Victor's dismissive attitude towards the findings of some of us here who have found very good reason for seriously doubting the DNA evidence put forward. We have posted enough information here and on the other thread that if Victor and others had read would not just accept the court of appeal as being gospel and then do an ostrich impression when it is challenged.

Victor says he is a chemist but I wouldn't expect my local chap at Boot's to explain the DNA evidence in Hanratty much farther than gattaca.

Reg

[Victor]

And finally, you will be saying thank god for that, a last article by the aforementioned FI of Glasgow. This one is most interesting as it deals with the transfer and persistence of DNA between differring surfaces.

Which is all well and good, but it is completely irrelevant when you are talking about semen stains.

KR,
Vic.

[Victor]

Here is a paper by the Forensic Institute that I obtained from Professor Allan Jamieson.

Hi Reg,

Is there any point me replying? If you have me on ignore then I don't know whether there is.

For anyone else reading...From the first article...
Low Copy Number (LCN) DNA Profiling
Introduction - is some background info.
DNA and Distance - tells us Skin cells flake off your body all the time.
LCN Profiling and Control Samples - tells us sound science use blank controls or placebos
The Detection Threshold - tells us that Reg's holy grail the "supra-150rfu threshold" is just an arbitary value anyway.
Replicate Samples - tells us the more times you repeat a Statistical experiment, the better the combined result will be, but also that the individual experiments are likely to be different.

A nice example of this is - Toss a coin 10 times, note how many heads you got. Repeat it, did you get the same result? Try it again - did you get the same again? But you expect to get 5 heads each time don't you? You got different results from the same experiment!

KR,
Vic.

And finally, you will be saying thank god for that, a last article by the aforementioned FI of Glasgow. This one is most interesting as it deals with the transfer and persistence of DNA between differring surfaces.

Transfer and persistence of DNA
DNA is transferred from a person to other surfaces (including other people) inside skin cells and in other cells contained in body fluids such as blood, saliva, urine, and semen. Each of us has about 10^14 cells in our body (100,000,000,000,000), each with a full DNA profile packed inside them. We lose a number of these cells every minute of every day. Everywhere you go you probably leave your DNA. This is one of the main differences between DNA and fingerprints; DNA can be transferred from you to someone else and from that someone to somewhere else where you may have never been. Most contacts between people and objects are expected to result in the transfer of cells, and hence DNA, from one to the other and vice versa.

The amount of DNA transferred, and the ability for subsequent transfer to other people and objects, has been the subject of scientific research. The vast majority of this limited research has been conducted using only small numbers of individuals (about 4 – 10) and in a very limited range of scenarios. In summary, no clear picture has emerged that could enable a confident interpretation of the finding DNA that is not associated with any particular body fluid on an object. This discussion will simply highlight some of the comments and conclusions that feature in the published work. Operational scientists may attempt to make reference to their casework and experience as a source of informed data on these points, but it should be obvious that no matter how many profiles are observed or objects examined, without knowledge of what actually caused the DNA to be present it is impossible to use this as a substitute for controlled experiments or to draw any confident conclusion at all. Within this limited context, it can be reported simply how many objects submitted for such testing can yield profiles; even then, this is a very biased sample of objects.

The presence of DNA with a profile matching that found on an item does not necessarily show that the person ever had direct contact with the item. “It has also been shown that a full profile can be recovered from secondary transfer of epithelial cells (from one individual to another and subsequently to an object) at 28 cycles.”
“The full DNA profile of one individual was recovered from an item that they had not touched while the profile of the person having contact with that item was not observed. This profile was also detected using standard 28-cycle amplification.”

These statements were reporting experimental results performed in controlled conditions and so the extrapolation to casework is difficult other than to establish that such transfer is possible.

The authors state, “When 30 min and 1 h delays were incorporated between human contact and contact with the object, profiles from both individuals were detected. This suggests that for most casework situations where secondary transfer is concerned, mixed DNA profiles can be expected.”
This remains speculation.

They also state, “In the context of other cases or scenarios where the conditions are different to those tested within this work, specific experimentation would be required to generate the appropriate data on secondary transfer. It would be important
to hear what the ‘suspect’ says in the context of each case.”

One author concludes, “a detectable secondary transfer is very unlikely” although there is no quantitation of ‘unlikely’ nor supportive evidence from controlled trials. This paper has a number of logical and factual flaws. For example, “case experience has found that the handled object bears the profile of the most recent handler”; this is conclusively contradicted by other work. While the DNA of the last person to touch the item may, or may not, be on the item, the DNA of others who may or who may not have touched the item may also be present. There is no reliable way of ascribing a particular profile to a particular time.

The factors affecting transfer of DNA are not fully known, but there is work to suggest that, inter alia; the duration of contact (longer = more transfer), force (higher = more), friction (more = more), and movement (more = more), are important factors. None of these are unexpected.

The results obtained in some of the papers discussed so far did not replicate (agree) the work of another group who had published in 1999. The improving sensitivity of current techniques may explain this discrepancy. Another may be the variable influence of personal factors in the ability of the participants, of which there are usually few (less than 10), to shed DNA.

There is work that has discovered that people vary greatly in their ability to shed DNA;
“The observation made by other authors that individuals differ in their tendency to deposit DNA when in contact with an object has been confirmed through specific experimentation … The reason for this difference in shedding ability is as yet unknown.”
“It is well known that there are significant differences among individuals in terms of the amount of DNA that they deposit upon touching an object. The reasons for these differences remain unclear and need further investigation.”
“We have also determined that the quantity and quality of DNA profiles recovered is dependent upon the particular individuals involved in the transfer process. The findings reported here are preliminary and further investigations are underway in order to further add to understanding of the issues of DNA transfer and persistence.”

The literature contains preliminary studies or case histories on the possibility of recovering DNA from fingerprints left on the skin or in rope, cord, wire, etc., used for strangling, gloves, knives, solid parts of cars and other objects, and on the interference of substances used to highlight fingerprints during later genetic analysis.

These works report on isolated experiments, dictated by the need to resolve definite cases, but systematic studies on recovery techniques, interference by contaminants, the influence of individual and exogenous factors in the number of cells left …, and the percentage of success in PCR analysis of the genetic impression from nuclear DNA, have not been exhaustively carried out.” [my italics]

So, there is a lack of definitive work on the transfer of DNA, but there are nevertheless studies that show that DNA can persist for some time after contact.

The highly variable results obtained by experimenters in this area merely add to the uncertainty about any conclusion based on the finding of low amounts of DNA on an object.

All of the work cited refers to person to person, person to object, or person to person to object transfers. We can find no work referring to object to object transfer although this is clearly a possibility given the precautions taken by laboratories and other agencies in avoiding such.

Regards
Reg (ps.. I am available for childrens parties!)

Here is a very good article, again from the Forensic Institute in Glasgow on PCR.

DNA and the Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction
DNA is now profiled by using the feature that makes it the ideal genetic mechanism. That is, when you know one side of a DNA chain, you automatically know the other, because the bases which make up the ‘steps’ of the DNA double helix bind with each other in a very specific way (A binds only with T, and G binds only with C).

Using this information, the polymerase chain reaction (PCR) is a method of multiplying the number of DNA molecules to an amount that can be detected by the identification technique. This multiplication is called amplification.

Consider that you have one piece of double stranded DNA from a suspect (or indeed anyone), but you need to make more copies of it in order to use it for profiling. If you pull the strands apart like a zipper, you will have strings of bases (for example, one might be AAAGCCTT, and therefore the other will be its complement TTTCGGAA). If you then throw in a cupful of A’s, C’s, G’s and T’s, they will stick to their complement and you will now have TWO double stranded molecules exactly the same as you started with.

Now let’s unzip these two double strands to give us four single strands. We now throw in our nucleotides again and now we have FOUR double-stranded molecules. This is four times the original number, and identical in composition to the original double strand.

It does not take many repetitions to produce a very large number of copies of the original. If it doubles every time it will produce 2n copies, where n is the number of repetitions or ‘cycles’ (this just means multiply two by itself n times, so 23 is 2 x 2 x 2 = 8). Current techniques use 30 cycles. Assuming that the process is 100% efficient, how many copies will be produced from 1 original double strand? What if there were 4 original copies, how many after 30 cycles?


Applying PCR to Forensic Stains
DNA is usually extracted from a stain by washing with saline (0.8% salt solution), and the amount of DNA in this ‘extract’ is then assessed. A known, specific amount of DNA is then subjected to the process of PCR.

The real benefit of profiling is that it amplifies only very particular areas of DNA (the loci). This allows us to amplify and look at only those parts of the individual’s DNA that we are interested in. In forensic cases those parts are the short tandem repeats (STRs) at each locus.

Small sections of DNA that have been manufactured using a known sequence of DNA specific for each STR locus stick to the beginning of the loci that we wish to examine and initiate the sequence of events that duplicate the original DNA. Because these manufactured pieces get the whole thing going, they are called primers.

As we discovered, each cycle of amplification approximately doubles the amount of DNA at the loci. This goes on for about 30 cycles to produce enough DNA to put into the next phase.

Remember that what we have amplified are the STRs – short tandem repeats – and these differ only in their size. The amplification process will have produced millions of DNA fragments of different lengths depending on the alleles that the person had at each locus. So what we need now is a method to separate molecules of different sizes.

This is done using PCR, a method of multiplying (amplifying) the number of DNA molecules to an amount that can be detected by the identification technique.

So now we know how to produce enough copies of DNA so that we can actually ‘see’ it. What are we looking for? Short Tandem Repeats, called STR’s.
During evolution it appears that little pieces of DNA joined themselves together like the beads on a necklace. This ‘tandem replication’ produces necklaces with different numbers of beads. It is the different numbers of beads that we are trying to establish – the different number of repeats in the STR.

We will consider just one, but the current system looks at ten areas of the genome.
We then use the molecular equivalent of a sieve. A gel is prepared. It has the consistency of table jelly but is between two vertical glass plates about a foot square and is only about a millimetre thick. The plate sits in a machine called a sequencer.
Little troughs are cast at the top of the gel and the DNA mixture is placed in these.
To pull the molecules through the sieve a high voltage is applied. The smaller molecules (the fragments with fewer repeats) move faster than the larger ones causing a ladder effect, with the rungs farthest down the gel being the smallest molecules.

Nowadays, this aspect is computer controlled. We do not actually look at gels any more. The molecules are allowed to run out of the bottom of the gel and are detected by a laser that sends a signal to the computer. The computer then creates a picture that looks just like a gel. A little chemical trick applied during the amplification process causes the alleles to glow different colours in the laser light and these are reproduced on the gel picture and the densitometric scan (how dense the bands or rungs of the ladder are). This makes it easy to see the different loci.
Each allele has a specific identifying number. It is this ability to convert the data to numbers that allow the DNA database to be created and to be quickly checked against any crimestain. A computer can easily compare two numbers, but it is harder to compare two photos.

regards
Reg

Here is some really good basic information about DNA analysis, its inherent properties and its place in the CJS (criminal justice system) which I am sure that you will all be reading this bedtime!

Again from the Forensic Institute.

DNA

Introduction
It would not be an understatement to argue that the advent of forensic DNA analysis has revolutionised the place of scientific evidence in court. The public perception, judged by a wholesale acceptance of the National DNA Database (NDNAB) as ‘A Good Thing’, underlines the need to remind people, and especially those involved in the investigation and prosecution of crime, of the potential dangers that accompany the undoubted benefits of this technology.

DNA is arguably one of the most scientifically robust techniques to be placed before a court. A series of legal and scientific challenges has honed the collection, processing, analysis and evaluation of DNA evidence to reduce the possibilities for erroneous results. Already some other evidence types have benefited from the advances in evidence evaluation that followed the challenges to DNA. On the other hand, some identification disciplines with a long pedigree in court, but less so in science, have signally failed to catch the wind that is now blowing through forensic science. DNA has reminded us that no matter how small or large the numbers, scientific evidence is probabilistic, and only an exclusion is practically certain.

DNA, or deoxy ribonucleic acid, is the molecule that nature uses to transfer the genetic information from one generation to the next. It has been likened to the blueprint for the body, inasmuch as it is not of the same form or material as the body, but is primarily an information medium.

To understand DNA it is almost inevitable that you will have to at least have a nodding acquaintance with the terms used.

Chromosomes
The human being begins with a fertilised egg. This egg has had a contribution of 50% of its genetic information from the mother, and 50% from the father. DNA is packaged within the cells of the body within complex structures that are normally encapsulated in the nucleus of the cell. This is why DNA is a ‘nucleic’ acid. These structures are called chromosomes. The chromosomes are designed to contain 23 chains of DNA in each sperm or egg.

These 23 chromosomes are numbered (in approximately decreasing order of size) 1 to 22 and collectively termed the autosomes. Number 24 is the so-called sex chromosome, and this can be of two types, either X or Y. The complete genetic complement of the individual is therefore composed of two of each of the autosomes plus two sex chromosomes. The genetic notation for the normal human female is 23XX, the male 23XY. Remember that, for the autosomal chromosomes there are still two of each; one contributed by mother and one by father.

The entire genetic content of your chromosomes is termed your genome.

Replication
What makes DNA different from most other molecules is that the term covers an infinite number of molecular types which simply have the same basic constituents and, usually, the same shape (a double helix).

This would represent a handicap if it were not for the fact that the main building blocks of DNA number only four. These four nucleotides (deoxyadenosine-5’ phosphate (A), deoxyguanosine-5’ phosphate (G), deoxythymidine-5’ phosphate (T), and deoxycytosine-5’ phosphate (C)) are linked linearly via their phosphate and ribose (sugar) parts to create a DNA molecule of almost any length. Length is therefore one way of creating different types of DNA. The other is to vary the sequence of each of the four subunits (A, C, G, and T).

The structures of the nucleotides also facilitates the main feature of any genetic mechanism; that is, reproduction or copying. The ability to form weak bonds (called hydrogen bonds), between the bases A and T, and between the bases C and G, causes single-stranded DNA to be sticky for other pieces of DNA. Because of the A-to-T and C-to-G binding, this stickiness is fairly specific. DNA that was all A’s would bind only to a string of T’s, and would not bind to DNA that was all G’s, or C’s, or even A’s. Each nucleotide (A, T, G, or C) binds only to it’s ‘other half’ or complement. A chain of nucleotides will easily and fairly rapidly form pairs (dimers) with its so-called complementary strand.

This complementarity leads to the ability to efficiently create copies of the molecule. In essence, a linear sequence of, for example, AAAGCCTT will create hydrogen bonds to the complementary sequence TTTCGGAA. (The ‘original’ strand can of course be reproduced by repeating the pairing exercise with the complementary strand.) It is this feature that causes the DNA molecule to exist as a two-chain molecule resembling a twisted ladder with the hydrogen bonds forming the steps and the phosphate-ribose backbone the sides.

Even if the AAAGCCTT strand is in a solution with other individual molecules of A,T, C, and G, the same pairings will form. In the DNA molecule the ladder twists itself to create a helix; the well-known ‘double helix’.

These features of replication are the basis of practical DNA profiling.

Each chromosome has one long double-stranded molecule of DNA. The genetic information of the individual is arrayed along these strands. There are, however, regions of the strands that have no known function (other than perhaps structural). This ‘junk’ DNA features prominently in the forensic application of DNA.
Genes
Through the processes of transcription and translation, which are not essential for our purposes to know, the genetic information within the chromosomes is converted to all of the inherited characteristics of the individual. These characteristics are called genes. Each gene is located on a specific chromosome and in a particular place on that chromosome called the locus. The task of identifying where the genes reside is called gene mapping. This task is complicated by the fact that many, if not most, genes may have several types. These types are termed alleles.
Alleles
Alleles are the existing variations of each gene. For example, the inherited disease Sickle Cell Anaemia is the consequence of a change in just one base of the long string of bases that comprise the gene for the protein haemoglobin (Hb). One allele of the Hb gene produces normal adult Hb, (HbA), the other allele produces the aberrant sickle-cell Hb, (HbS).

There are several variants of the Hb gene. However, as there is only one Hb gene locus within an individual’s genome (the entire repertoire of genes in an individual) and only two contributors of the alleles of the individual (mother and father), each individual can possess a maximum of two Hb alleles (e.g. HbS/HbS, HbA/HbS, HbA/HbA).

Phenotype and Genotype
When an individual possesses two identical alleles (e.g. HbS/HbS) they are described as being homozygous at that locus – they are homozygotes. When they have different alleles (e.g. HbA/HbS), they are heterozygous at the locus – they are heterozygotes. There are only a few genes where the type of alleles that the individual possesses are actually known (especially without testing). The allelic composition of the locus is known as the genotype.

For some combinations of alleles it is not always possible to distinguish the genotype between the combinations. For example, in the inherited condition Cystic Fibrosis (CF), individuals may have two CF alleles (i.e. are homozygous for CF), in which case they will have the disease. In comparison, a person who has one CF allele and one normal allele (i.e. heterozygous) can not be distinguished from those who have two normal alleles (i.e. homozygous). The observed feature or state is called the phenotype. In this case the hetero¬zygous and homozygous-normal individuals have the same phenotype (i.e. they have no disease), despite their different genotypes. Their phenotype is different from the person with the homozygous-CF alleles. Depending on the gene, you may be able to tell the genotype (their actual alleles) from the phenotype (what you see), but for most genes you cannot.

Again Kindest Regards
Reg

Here is more from the Forensic Institute

DNA – Basic Probability

Differentiating Between Groups
DNA profiling separates the population into groups. In criminal work the aim is to end up with a group in which there is only one person – the suspect. It’s easy to get confused with all of the science involved in DNA profiling, so a model may help.

Consider how a general description differs from a description that is more specific. The more detailed a description of an offender is, the more likely it is that we will identify him. For example, if witnesses identify a black-haired man, with a hooked nose, two missing teeth, three fingers on his right hand, one eye, and a bite out of one ear, then it is likely that there will be very few people who fit that description. You may have thought as you read the description, “Black hair, hmmm a lot of people have black hair. OK, a man, so that cuts that lot about in half. Only three fingers on his right hand… not many people with that. Two missing teeth? Not many of those…” and so on. By adding more and more requirements to the description, we reduce the number of people left in our ‘suspect’ group. That is the objective in DNA profiling.

Using this example, we can also illustrate how statistics are used in DNA profiling and how they are the key to understanding the significance of DNA evidence.

Let’s say that 20% of the population has black hair, 1% has a hooked nose, 5% has two missing teeth, 0.1% has three fingers, 0.5% has one eye, and 0.01% has a bite out of one ear. What is the chance that if you pick a random person from the population, they will have black hair? 20%, or 1/5. What about the chance of picking someone who has a bite out of 1 ear? 0.01%, or 1/10,000.

If you were in two separate court cases, one where the perpetrator had been identified as having black hair, and the other where the perpetrator had been identified as having a bite out of one ear, in which case you would be more confident that the accused was the perpetrator (assuming that the accused had black hair in the first case, and a bite out of one ear in the second)? The one with the lowest probability, of course (the ear, for clarity!).

The Product Rule
Now what would be the chance of finding someone with both characteristics?

Let’s start with an easy example. Consider the odds of throwing a tail on a fair coin, getting a five on a dice, and choosing the king of diamonds from a pack of cards. The odds of each event occurring separately are ½, 1/6, and 1/52. To calculate the odds of them occurring together we use what is called the product rule – simply multiply them all together;
½ x 1/6 x 1/52 = 1/624 = 0.0016

So, to go back to our identification example, we simply apply the product rule using the frequency data for each of the characteristics (black hair and a bite out of one ear);
1/5 x 1/10,000 = 1/50,000
This result means that if we were to pick a random person from the population, the chance that they would have black hair and a bite out of one ear is 1 in 50,000.

To calculate the probability of randomly finding someone with all of the features in the original example would similarly give;
1/5 x 1/100 x 1/20 x 1/1,000 x 1/5,000 x 1/10,000 = 1/50,000,000,000 or 1 in 50 billion

These examples demonstrate how combining a number of characteristics which, on their own, do not have very good discriminating power, can provide very discriminating evidence. This is why you will frequently see DNA statistics of the order of one in a billion.

Notice, because it will be important in understanding the correct interpretation of the number, that we have not calculated the probability that this person is the suspect, merely the probability that this set of characters would match anyone else in the population. This is called the match probability.

Characteristics of DNA
The earliest form of DNA profiling was called Restriction Fragment Length Polymorphism (RFLP). A restriction enzyme is one that cuts only a specific sequence of DNA. This cut produces at least two fragments of different lengths. If there is a mutation at the site, then the enzyme won’t cut the DNA there and therefore this shows up as a genetic polymorphism (meaning ‘many shapes’). Different polymorphisms for different individuals allows their DNA to be differentiated by this process of RFLP. And that’s where the name comes from! This was the technique used the first time that DNA was used forensically in the UK; the case against Colin Pitchfork (e.g. http://members.iinet.net.au/~sphinx/html/dnacase.html ).

The current technology used for DNA profiling in the UK is more specific and sensitive. It is based on Short Tandem Repeats (STRs) of DNA. The most widely used method is a trade name called SGMPlus (SGM+), which replaced an earlier version called SGM (Second Generation Multiplex). During evolution, pieces of ‘junk’ DNA duplicated themselves like a row of beads on a necklace. These short pieces are then said to be duplicated in tandem and repeat alongside each other. Scientists believe there is only a small range of numbers of repeats amongst the population, so again this provides the opportunity to differentiate DNA from different individuals.

When analysing DNA forensically, scientists used RFLP (and now use SGM+) techniques to look at several areas (loci) of the DNA and see what version of DNA (allele) is there. In a similar fashion to the identifying characteristics discussed in section one, we use the frequency of the particular alleles in the population to calculate the odds of finding that particular combination of alleles by chance.

Originally, the SGM technique examined 6 loci. The current technique used in the UK (SGM+), examines the short tandem repeats at 10 different loci and establishes which alleles are present at each loci. At each of the 10 loci there are two alleles (one from each parent), and in a given population there may be several possible alleles to choose from for that loci.

For a simple illustration, let’s assume that there are only two possible alleles at each of the ten loci. This means that everyone in the population has some combination of only those two alleles at each locus (this is a very simplified example!). For the sake of simplicity, let’s also say that the alleles are called a and b. So at each locus, a person will have either have two a’s, two b’s, or one a and one b. Even with only these two choices at each locus, the number of possible combinations for the 10 loci is 3^10, or 59,049 (3 possibilities, 10 times). In reality there are several alleles to choose from at each locus and that is part of the reason why the odds of any particular combination being found are frequently in the billions.

Kindest Regards
Reg

LCN DNA was used to provide the DNA evidence in the appeal court hearing R vs Hanratty (2002). I spoke to Professor Allan Jamieson of the Forensic Institute in Glasgow on the telephone and he confirmed that this was correct. The BBC Horizon programme on the case in 2002 also recorded that the narration, following a clip of Dr Johnathan Whitaker, said that the DNA samples were exposed to 34 cycles of PCR amplification, thus also confirming the use of LCN DNA.

Here is a paper by the Forensic Institute that I obtained from Professor Allan Jamieson.

Low Copy Number (LCN) DNA Profiling

Introduction
Low Copy Number (LCN) DNA is NOT a different technique from SGM+. It may increase the number of amplifications of the sample, or lower the threshold normally required to show an allele (the method in this case), and is therefore claimed to detect lesser amounts of DNA than the conventional process.

Sensitivity is a measure of how little DNA we need to perform analyses and produce a profile. Until recently, DNA was recovered from visible stains like blood splashes or semen stains. Then we started to collect samples, usually by swabbing, from areas where we may expect to find DNA from body fluids e.g. cigarette ends, cutlery, spectacle frames. The introduction of Low Copy Number DNA (LCN) has seen us now enter an era where single pieces of DNA may produce profiles; that is, less than 100 picograms (0.0000000001g) of DNA.

DNA and Distance
To understand why that may be a problem it is useful to consider that each of us has about 10^14 cells in our body, each with a full DNA profile packed inside them. We lose a number of these cells every minute of every day. Everywhere you go you leave your DNA, and your DNA goes places you’ve never been. This is probably one of the main differences between DNA and fingerprints. A correct fingerprint identification, on a fixed object, may establish that you were at a particular point, but DNA can be transferred from you to someone else, and from that someone to somewhere else where you may have never been.

When forensic scientists used only blood or semen stains that could be seen, we were perhaps a little more confident that this established a link between the source of the stain and the location of the donor. But because you can walk through a supermarket and so easily have one of your cells blows into a vehicle or onto a surface a distance away, your DNA has literally distanced itself from you.

LCN Profiling and Control Samples
As much as the DNA technologies created controversy and challenges when they were introduced, LCN DNA has produced its very own set of problems.

A key scientific practice is the control sample. This is an experimental technique used to establish that the effect that you observe is caused by the material under test and not some other component of the system. The purpose is illustrated well in clinical trials where some people are given a tablet that looks like the drug under test but does not contain the drug. This is because some people get better simply because they think that they have received the drug (the placebo effect). The scientist looks for a difference between the control group and the test group to establish whether it is the drug that is causing the improvement.

In forensic science the fact to be established is that the DNA profile originated from the material recovered from a crime scene or a suspect; not from the investigator, the laboratory, the packaging, or the analytical instruments. A ‘negative control’ is set up by simply processing a ‘blank’ sample that has had no DNA added to it. All being well, this control will not show any DNA at the end of the process. If, however, there is DNA in the negative control, this illustrates that there has been a source of contamination in the analytical method. It does not, of itself, show where that contamination occurred, merely that it has occurred. The tradition over many years has been, for very sound reasons, that anything found in the ‘negative control’ invalidates the analysis.

There are now some who argue that this principle cannot be applied to LCN DNA analysis because even in a tightly controlled analytical procedure a significant number of supposedly negative controls (usually unknown, but has been reported in at least one paper as up to 70%) give a positive result i.e. they indicate the presence of DNA.

The issue for scientists and lawyers, of course, is that if it can not be established where or when the DNA was introduced during the analysis, we can not be confident that any of the DNA found in the crime stains did not come from the procedure rather than the scene.

Proponents of LCN argue that the appearance of such spurious alleles in negative control samples is not significant because it only occurs only occasionally. It may be that tiny fragments of DNA from any material or piece of apparatus used in the analysis could have had the DNA on it, but it would be easily identifiable in a series of runs. This undesirable appearance of spurious alleles has become known as allelic drop-in.

Drop-ins which produce spurious alleles in crimestain samples may be difficult or impossible to identify. One method of ‘identifying’ them is to simply subtract the profile of a suspect from the stain profile, and if there are only one or two alleles left, to claim that they must be due to drop-in, thus leaving the suspect still a suspect!

The Detection Threshold
These effects can be seen as a consequence of pushing the detection threshold downward. This is illustrated in the following sequence.

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Figure 1: Threshold at 60 rfu

Figure 1 shows a sample from a profile. Traditionally, 150 rfu’s (the left hand scale) have been regarded as the cut-off – anything above 150 is considered a peak, but anything else is not. In figure 1 the threshold of 60 is shown by the horizontal line. Four peaks are clearly above 60, (which remember is still an arbitrary threshold). As an analyst, there may be a belief that there is at least a good chance that there is at least one other, and possibly two, other peaks. But if we reduce the threshold to include those, others will seem very close too.

As the threshold is adjusted upward, the confidence that everything above the threshold is a ‘real’ peak (and hence an allele) improves.

LCN DNA moves such decisions from the practically certain area in the supra-150 rfu regions to areas where not only is there no good scientific data supporting an actual numerical probability, but where one analyst may identify a peak and another may not.

There are no set rules for such interpretations, and those that exist have most often been developed within the laboratory doing the testing. This differs from ‘normal’ SGM+ where there are well-established guidelines for allelic attributions. These guidelines would not be applicable to DNA amounts used in LCN analyses, because given the low input amount of DNA that is used for LCN profiling, the results will not usually produce a signal sufficient to meet the guideline criteria.

Replicate Samples
Lastly, the very small amounts of DNA deemed acceptable for use in LCN profiling and the vagaries of the method mean that it is frequently the case that replicate samples, (that is, samples which scientifically should produce the same results), don’t.

The current theory is that these so-called ‘stochastic’ effects are a consequence of the very small numbers of molecules in the stain.

As an example, consider a 1 litre pot of pea soup. Any normal sampling of this, by spoon, cup, or ladle, will contain peas. Now consider reducing the number of peas in the pot. If the pot contains 1000 peas then (assuming proper mixing) we have about 1 pea per millilitre. If we reduce this to just 10 peas then there is only 1 pea in every 100ml of soup, or 50% chance of getting a pea in 50ml, 5% in 5ml. So, only 1 in 20 spoonfuls of soup would be expected to be ‘positive’ for a pea. Even though the peas were there, some ‘tests’ (spoonfuls) would not find any.

In the LCN process this is claimed to be the cause of the phenomenon of allelic drop-out. This is the apparent lack of an allele in some replicates of stains that, ideally, should produce identical results.

The process purports to get around this difficulty by simply taking a vote of two test replicates. DNA types found in both runs are regarded as real and counted in the ‘consensus’ profile. So, for example, if a stain is extracted and divided into three parts (we call them aliquots), and subject to the same analytical procedure the results could be;
1. AB
2. C
3. A
The consensus result would be A. B and C would be regarded as “inconsequential”.
From these results, how confident can we be that A is the accurate or true result?

Given this data, we must at least concede the possibility that after 5 replicates we may find more B’s or fewer A’s, so we are not 100% confident that A is the true result. We use the consensus result as the basis of the statistical calculation of how rare this combination is in the population at large; in effect the probative value of the DNA evidence.

Now imagine that we take ten of these consensus results for different areas of DNA to calculate the match probability. This process will yield a statement of the form, “the probability of this profile coming from X rather than some unknown, unrelated person is …” and then a number that is frequently of the order of billions, but with no statement of the confidence that we can place in that result despite the clear, and probably measurable, uncertainty that must exist.

If even one of these is wrong then the opinion, as well as eliminating the real perpetrator, will be wrong by a factor determined by how rare the wrongly ascribed DNA type is. So if the profile includes a type that is present in 1/10th of the population the match probability will be wrong by a factor of 10. So a match probability of a million to one will become 100,000 to 1; a significant difference.

The scientific issue is the degree of confidence that can be placed in the results and the consequent opinion. The legal issue is whether the destructive techniques meet the requirements of physical evidence acceptable in court.

I hope that this pamphlet will enlighten anyone who's curiousity has not atrophied to that of an ostrich.

Regards
Reg

Didn't happen, another week living out of boxes and suitcases - hopefully moving next weekend now!



Contamination is not definite. If it was definite then you could predict it and account for it and therefore it wouldn't be an issue.

It is an issue because it is unpredictable. There is a probability associated with contamination and the control is there because the probability of the control being contaminated at the same time as the sample is very large.

In other words the existance of the blank control is further evidence that contamination is probability based.


OK, so 70% of the time the blank control is contaminated. One could infer that the probability of contamination is therefore 70%


94% of DNA analyses are not used in evidence, but that says nothing about whether those analyses were useful or not. Whether they were mixed profiles and therefore inconclusive. Whether they only gave results of the victim's DNA. Whether they gave negative results.

Where's the comparable data for another technique? For example:-
What proportion of fingerprint analyses are used in evidence?
How many fingerprint analyses show no fingerprints?


That tells me that statistics can be massaged to suit any situation, and that the same statistics can be used to prove opposite sides of the same argument...for example at University we were asked to look at the case of Gregor Mendel and his work on genetic inheritance in pea plants - some have argued that his results are too accurate and are therefore fraudulent.



It's a bit difficult to get a confession from a dead man! But that's just argumentative, what I think you are saying is that there needs to be corroborative evidence, like VS's identification, and that all the evidence should be considered as a whole, whereas the Hanratty appeal only considered NEW evidence like the DNA and non-disclosure of materials to the original defence team.

KR,
Vic.

Hi Vic
Take it to the main thread.
This thread is over. It was good while it lasted.

Reg