The early success of Operation Advance shows that techniques for characterizing
human DNA have improved significantly during the past several decades. Forensic
DNA analysis has evolved from an examination of cleaved DNA fragments to the
characterization of target nucleotide sequences amplified from an infinitesimal
quantity of DNA. As the Forensic Science Serviceís Alan Matthews told
the BBC, ìInstead of looking previously for amounts of blood, perhaps
the size of a coin, to even begin to start looking for DNA, weíre now
looking at microscopic amounts which amount to a few cells.î Further
advances in DNA forensics may shift analysis from the lab to the crime scene.
The RFLP Riff
The story of DNA profiling begins in the early 1980s with Dr. (now Sir) Alec
Jeffreysí investigation of genetic stutters. The Leicester University
geneticist had been interested in regions of human DNA - known as minisatellites
- that contain core nucleotide sequences repeated end-to-end, often up to hundreds
of times. As indicated in Figure 1, the number of reiterations contained within
any particular minisatellite varies among individuals. And this means that
distances between the nucleotide sequences at either end of one of these genetic
stutters also vary, a phenomenon called length polymorphism.
Hereís an analogy. If a core nucleotide sequence is a boxcar, then
a segment of tandem repeats would be a line of identical boxcars. And the number
of boxcars - residing between the engine and the caboose - determines the length
of the train. The trick is to visualize this length. (See Figure 1)
Jeffreys visualized length polymorphisms by treating DNA samples with restriction
enzymes, which cleave DNA to produce fragments that contain a repeating segment.
He then employed the molecular sieving technique of gel electrophoresis to
separate DNA fragments by size. After transferring the separated DNA fragments
from the gel to a nylon membrane, Jeffreys treated the membranes with a radioactive
probe that binds with a selected repetitive nucleotide sequence. When the nylon
sheets were placed against X-ray sensitive film, the positions of DNA fragments
carrying the radioactive markers appeared as a series of lines that resemble
bar codes. These bar codes have two important qualities: the pattern varies
from person to person, enabling the characterization of an individualís
DNA; and the patterns are inherited, providing information about family relationships.
In the spring of 1985, Jeffreys published the first report on his DNA fingerprinting
method. The study came to the attention of Sheona York, a lawyer who represented
a Ghanaian family of UK citizens entangled in an immigration dispute. The familyís
youngest son had recently returned from a trip to Ghana, but the Home Office
detained him, alleging that he held a forged passport. York asked Jeffreys
for his help. Jeffreys compared DNA from the boy, the woman who claimed to
be his mother, and her other children. In this first application of DNA fingerprinting,
the technique revealed that the boy was the womanís son and that all
of her children shared the same father.
A year later, the Leicestershire Constabulary asked Jeffreys to analyze DNA
evidence from two rape-homicides. Jeffreys knew, however, that he would need
to find a different strategy to scrutinize DNA.
Why not apply DNA fingerprinting to the murder investigation? Among other
things, the technique required large amounts of DNA, a significant constraint
when dealing with crime scene samples. Also, DNA fingerprinting relies on multi-locus
probes that detect multiple nucleotide sequences throughout the genome and
on numerous chromosomes. While the complex pattern produced by multi-locus
probes is ideal for characterizing a pure DNA sample, DNA fingerprint results
are difficult to interpret if an evidence sample contains DNA from two or more
individuals.
To overcome the limitations of the multi-locus probe approach, Jeffries devised
a panel of single locus probes. When a single locus probe attaches to a target
minisatellite region, it produces a pattern of one or two bands, representing
nucleotide sequences inherited from the mother and father. The simple pattern
created by a single locus probe is no longer specific to an individual. However,
the probability that DNA from two people would produce the same DNA profile
drastically shrinks if an investigator combines results from numerous single
locus probes. For a criminal investigation, the single locus probe tactic also
offered the advantage of requiring about 10 to 100 nanograms of DNA, or one-tenth
the amount needed for a multi-locus probe examination.
During the course of the Leicester murder investigation, 5,000 men provided
blood or saliva samples. The Forensic Science Service analyzed DNA from the
ten percent who had the same blood type as the killer. Meanwhile, two men confessed
to the crimes; DNA profiling exonerated one and confirmed the guilt of the
other.
A Rising STR
The RFLP technique became the first scientifically accepted forensic DNA analysis
method in the United States. But it had a few drawbacks: the single locus probe
strategy still required relatively large amounts of DNA, and that DNA had to
be recovered from a crime scene in a high quality form. DNA degrades when exposed
to the environment, breaking into fragments that may be too small for RFLP
analysis.
Researchers overcame these limitations in two stages. In the first, scientists
applied the new polymerase chain reaction (PCR) technique to forensic analysis.
The PCR method can duplicate, or amplify, a billion copies of a target nucleotide
sequence, even a target sequence buried in a mixture of millions of other sequences.
And PCR duplication can be performed with a nanogram of DNA.
But there was a hitch. PCR could duplicate DNA segments of about 1000 to 2000
nucleotides, a length much shorter than numerous minisatellite targets. Incorporation
of PCR amplification into forensic DNA analysis required a switch to small
targets that offered the same type of tandem repeat variation. And hereís
where stage two comes in: the use of microsatellites. While minisatellites
have sequences of six to 100 nucleotides that repeat two to several hundred
times, microsatellites have repeat unit lengths that typically peak at seven
nucleotides, repeated five to 100 times.
In 1991, Baylor College of Medicineís Thomas Caskey suggested using
a type of microsatellite called short tandem repeats (STRs). Peter Gill, then
a researcher at Britainís Forensic Science Service, devised the PCR-STR
method, which enabled the simultaneous analysis of multiple STRs, a tactic
called multiplexing. With this technique at hand, investigators could analyze
DNA recovered from minute and even partially degraded samples. Forensic DNA
analysis no longer depended upon the availability of tissues and fluids, such
as blood and semen. Now, DNA analysis could be performed with PCR-amplified
biological traces acquired from dental molds, cigarette butts, eating utensils,
chewing gum, postage stamps, ski masks, licked envelopes, toothbrushes, razor
shavings, band aids, and fingerprints.
The FBI favored the STR approach, and in 1997 the agency devised a standard
series for DNA typing that consisted of 13 STRs, each with a sequence of four
nucleotides. For example, the STR called D16S539 has the sequence ìGATA.î In
2000 the FBIís Bruce Budowle and his colleagues published their conclusion
that the probability that DNA from unrelated individuals would generate the
same 13 STR profile would be less than one in a trillion. That same year, the
FBI discontinued RFLP analysis in favor of STR multiplexing. Figure 2 depicts
a representation of a 13 STR-based analysis. (See Figure 2)
The U.S. forensic science community has adopted the FBIís 13 STR series.
According to Dr. Richard Saferstein, a forensic science consultant and former
chief forensic scientist of the New Jersey State Police, the 13 STR array will
remain the standard. One reason for this is that the FBI selected the 13 STRs
to serve as the core genetic markers for its national database structure, the
Combined DNA Index System Program. Dr. Saferstein also says that ìDNA
STR technology has become a mature science that will yield little in the nature
of significant scientific breakthroughs in the next 5-10 years.î
For now, forensic DNA analyses will remain focused on particular STR targets.
But the strategy for duplicating those targets may soon change.
Taking the Heat Out of DNA Amplification
In 2000, the U.S. Justice Department predicted that, within ten years, investigators
will be using portable, miniaturized instrumentation that will enable DNA characterization
at a crime scene. This prediction may soon be realized thanks to a new strategy
for amplifying DNA.
As shown in Figure 3, the polymerase chain reaction is initiated by heating
DNA to separate the two strands of double-stranded target DNA molecules (3A).
The sample is then cooled in the presence of DNA primers that bind with selected
nucleotide sequences (3B). After reheating the solution to activate a polymerase,
the enzyme extends the primers, synthesizing complementary copies of each DNA
strand (3C). Copies of target nucleotide sequences are generated exponentially
by repeating these cyclic heating steps, a process that also binds DNA amplification
to a lab setting. (See Figure 3)
Huimin Kong and colleagues at New England Biolabs (Beverly, MA) published
a report in July 2004 on a technique that may liberate DNA amplification from
the laboratory. In their helicase-dependent amplification method, helicase,
rather than high temperature, uncoils DNAís double helix, enabling investigators
to duplicate a DNA target at a uniform 37ƒC. Dr. Mark Griep, a helicase
expert at the University of Nebraska-Lincoln, says that the New England Biolabs
researchers ìhave eliminated the need for the energy-hungry heating-plate-type
PCR thermocyclersî and have taken ìa major step in the development
of hand-held PCR devices for use in the field.î
Helicase-dependent amplification requires some fine-tuning before miniature
DNA analyzers enter the marketplace. Dr. Griep notes that the helicase technique
duplicated a 123 nucleotide sequence fragment 10 billion times in three hours. ìA
useful hand-held device,î he says, ìwould be able to amplify such
a fragment in minutes, not hours.î
Getting SNPy: A Different Tactic for Identifying Variation
RFLP and STR techniques focus on length polymorphisms. That is, only size
matters. Yet about 90 percent of human genetic variation arises from the substitution
of one nucleotide for another. Single nucleotide polymorphism (SNP) analysis
focuses on these differences in nucleotide sequence. While STR analysis requires
duplication of DNA fragments containing about 200 to 400 nucleotides, SNP analysis
requires DNA fragments in the 50 to 90 nucleotide range. Consequently, the
SNP technique can be used to analyze DNA that may be too degraded for STR analysis.
Despite this advantage, the Forensic Science Serviceís Dr. Peter Gill
says that North American and European forensic science groups have taken the
position that SNPs are unlikely to replace STRs in the near to medium future.
This reluctance, he says, stems partly from the fact that SNPs ìindividually
have limited discriminating power - this means large multiplexes (about 50
or so) need to be constructed.î Since typical assays are limited to analyzing
about 12 SNPs per reaction, ìup to 5 different reactions would be needed,
which is not feasible when there is very limited sample available.î
Another reason why SNP technology wonít dominate forensic DNA analysis
in the near future is that national DNA databases store STR data. Since STR
and SNP analyses examine different regions of the human genome, a SNP profile
cannot be searched against an STR profile. Conversion to a SNP-based system
would require re-analysis of all DNA samples. Not only would such an effort
be cost-prohibitive, but DNA samples are not routinely retained after analysis.
Nevertheless, SNP technology has a place as an adjunct to STR analysis.
SNP analysis is not new. Alec Jeffreys examined SNPs in the late 1970s as
a means to study inherited variation. His search for DNA regions more variable
than SNPs led him to tandem repeats and, eventually, DNA profiling. Since then,
DNA analysis has become an indispensable tool for criminal investigations.
As David Coleman, chief constable of the Derbyshire Constabulary, declared
in the Operation Advance press release, ìthe clear message to criminals
is that there is no hiding place from the power of forensic science.î
