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Historical aspects of forensic DNA analysis
Forensic DNA analysis began with the work of Alec Jeffreys, a geneticist at the
University of Leicester, UK. In 1985 he described a technique which he called DNA
fingerprinting, the importance of which was immediately realised and within a very
short time was being applied forensically. Jeffreys was studying the human gene that
encodes the protein myoglobin, an oxygen binding protein in muscle. In common
with most human genes (see Figure 6.3 and Section 6.2.2) the protein information in
the DNA is broken up into exons by the presence of introns that carry no protein
information. In an intron of the myoglobin gene was a repeated sequence of DNA
that was 33 bp long – this is a minisatellite (Section 6.2.4). Using classic techniques
of molecular genetics (gel electrophoresis and Southern hybridisation) he was able to
show that amongst individuals the number of 33 bp repeats in the minisatellite varied
(i.e. it is a VNTR, Section 6.2.4). Further a related sequence to the repeat was present
elsewhere in the genome, not just in the myoglobin intron.
These discoveries led to the classic approach of forensic DNA analysis (originally
called DNA fingerprinting), employing Southern hybridisation, invented by Jeffreys.
This is outlined in Fig. 1.
DNA is purified from the sample, in the example a blood stain, the very long DNA
molecules are cut with a restriction enzyme (these are very important tools for
geneticists, they cut the DNA precisely, only at certain short DNA sequences
producing reproducible patterns of fragments). This step produces a huge number of
DNA fragments that are short enough to be separated by gel electrophoresis. After
running the gel the DNA fragments are transferred to a nylon or nitrocellulose
membrane to which the DNA sticks in the same pattern as it was on the gel. The
membrane is then placed in bag with buffer solutions and a radioactive probe. The
probe is a short section of single stranded DNA which is complementary to (its
sequence can base-pair to) the gene of interest. Jeffreys used a probe that would bind
to the 33 bp minisatellite. The probe binds in a process called hybridisation only to its
target sequence and when the hybridised membrane is placed on a photographic film,
the radioactivity fogs the film only at the positions where the probe has bound, that is
to the DNA fragments on which are the target sequences. In Fig. 1 if the probe binds
to a single locus and the number of repeats varies then up to two bands would be seen
for each sample as shown – the length of each band being related to the number of
repeats – each of the samples in Fig. 1 has two alleles and is therefore heterozygous.
If only one band was seen the person would be homozygous –both of the alleles they
carry are the same. This method where the probe recognises one locus is called
single locus probing.
However using a section of the 33 bp repeat as probe Jeffreys obtained a more
complex pattern because the repeat was found in a number of genes, not just the
myoglobin gene. The probe had hybridised to a number of loci and hence is referred
to as a multilocus probe. Many of these other loci also had a variable number of
repeats and the result was a complex pattern which became called a DNA fingerprint.
Further studies demonstrated that the pattern in terms of the position (length) and
number of bands was unique for any individual tested. This was the first method
clearly showing a distinct DNA pattern for an individual and its power for forensic
purposes was soon demonstrated (Box A).
Box A. Case studies. The first applications of DNA fingerprinting.
The first legal application in 1984 involved a Ghanaian boy returning, from Ghana, to
his family who were resident in Britain. He was refused entry because the
immigration authorities suspected he was not the son. Blood samples from the boy,
the mother and three of her other children were analysed by Dr. Alec Jeffreys using
his newly developed technique of DNA fingerprinting. The evidence argued very
strongly that the boy was the mother's son and the case against the boy was dropped.
The sexual assault, rape and murder of two schoolgirls, Lynda Mann in 1983 and
Dawn Ashworth in 1986 in the village of Narborough, near Leicester, UK became the
first cases where DNA evidence played a crucial role in a murder conviction. Semen
from both bodies showed two characteristics in common; it was from a Group A
secretor and carried the same enzymatic marker. This and the nature of the murders
suggested that the girls were murdered by the same man.
Circumstantial evidence led to a 17-year old local kitchen porter being arrested and
charged with the murder of Dawn Ashworth and he eventually confessed. However
he denied killing Lynda Mann. Application of DNA fingerprinting by Dr Alec
Jeffreys clearly demonstrated that the suspect was not involved with either murder
and the boy was cleared. This was the first time DNA evidence had absolved a
suspect.
In early 1987 a massive screen of about 5000 local men was undertaken. This
enormous effort involved taking the blood or saliva samples, blood typing them and
then determining the DNA fingerprint of the samples with the same blood type as the
semen evidence. No match was found.
In a conversation in a pub, later in the year, a bakery worker Ian Kelly mentioned to
female colleague that he had donated a blood and saliva sample in the screen using
the name of a fellow worker Colin Pitchfork who had persuaded him to do so. The
woman contacted the police and Colin Pitchfork was arrested. DNA tests from him
matched the scene of crime evidence. He pleaded guilty to both murders in January
1988.
Details of the case are described at the Forensic Science Service Casefiles at the
website http://www.forensic.gov.uk as the Narborough murders.
This pioneering work cannot have its importance over-stated; it showed that it was
possible to use molecular genetics to uncover individual variation in DNA sequences
in a robust way applicable to forensic work. Early work also demonstrated limitations
of the technique but prompted rapid developments.
Multilocus probing required a large amount of DNA (about 200 ng) not always found
at a crime scene and the patterns produced were complex to interpret in detail; the
large number of bands were not easily attributed to individual alleles of particular
genes making analysis in terms of population genetics difficult.
Fairly soon after the description of DNA fingerprinting the use of single locus probes
was developed these detected minisatellites (VNTRs) as before but only at specific
well characterised loci – the pattern for an individual thus consists of a single band if
an homozygote for the number of repeats or two bands if heterozygous – this is shown
in the figure. The use of single locus probes increased the sensitivity of the technique
requiring about 10 ng of DNA. Another advantage is that if it is known how common
certain alleles are in the population, careful analysis of probabilities that two people
could share a given genotype can be made. Population genetics and the interpretation
of DNA information is discussed in Section 6.4. Further use of single locus probes
allows the ease of comparisons between different laboratories and the conversion of
the data to an electronic form allowing data bases to be assembled in and searched by
computers.
Another major development from the original methods has been the incorporation of
the technique called the polymerase chain reaction (Section 6.3.4) which makes the
blotting and hybridisation stages redundant and further increases the sensitivity of
analysis, allowing profiles to be produced from incredibly small amounts of material.
DNA evidence presented in the O.J Simpson trial of 1995 (Block B) was from early
PCR based systems. The importance of this case in the history of forensic DNA
analysis was the attention it drew from the public.
Box B. Case Study. The O.J Simpson trial
The trial of American football star, sports commentator and celebrity O.J Simpson in
1995 for the murders of his ex-wife Nicole Brown Simpson and her acquaintance
Ronald Goldman in June 1994 in Los Angeles became one of the most high profile
cases in recent years. It attracted huge media interest throughout the U.S as well as in
the rest of the world and became one of the longest trials in California State history. A
wide variety of evidence was used in the case, that of witnesses, blood, hair, fibre, and
shoe-prints. DNA evidence from blood spots at the scene of crime indicated with a
very high probability that O.J Simpson was the source. DNA spots found on O.J.
Simpson's socks, not at the crime scene, matched with a high likelihood that of the
murdered woman. There was further bloodstain evidence. In the history of forensic
DNA analysis the importance of this case was bringing the technique to the attention
of a very wide public. The basis of DNA typing, its precision and reliability were
discussed in the media and in many learned journals. These issues had been discussed
before; the difference was the level of attention. DNA evidence was partly
undermined by the defence using arguments related to contamination and the
suggestion of fraud. The trial became very complex and the outcome was O.J
Simpson being found not guilty. A civil trial brought by the relatives of the victims
followed shortly afterwards. The result of this, in early 1997, being that O.J. Simpson
had been liable for the deaths.
Classical DNA profiling using based on Southern hybridisation of probes is not used
routinely anymore; it has been supplanted by the multiplex PCR amplification of
STRs as described in Chapter 6, section 6.3.5. However it is occasionally used when
there is old DNA evidence produced by these methods which needs to be compared
with new evidence - a proper comparison can only be made if the DNA is analysed in
the same way as the original material
Web sites – Alec Jeffreys describes his work and the moment of the key discoveries
at http://abc.net.au/science/sweek/ausprize/alecj.htm.
The Animation Library at the Dolan Learning Centre at the Cold Spring Harbour
Laboratory at http://www.dnalc.org/resources/BiologyAnimationLibrary.htm contains
an animation called DNA Detective – this is an excellent animation showing “DNA
fingerprinting” by the classic approach using Southern Hybridisation.