The Use of PCR Technology for Routine Grouping in the Forensic Biology Laboratory.

Paul E. Roffey and Graham J. Harmon

John Tonge Centre
Forensic Biology Section
Queensland Health
Introduction
Forensic science is the application of scientific procedures to help solve criminal and legal
matters. At the scene of any crime a variety of physical evidence may be left behind that can
link a criminal to a crime, or help reconstruct the sequence of events which occurred during
that crime. As forensic biologists, a large part of our role is to examine articles and crime
scenes for evidence of biological material and attempt to determine the origin of that material.
We do this by using tests that allow us to biologically discriminate between individuals.
All individuals in a community have characteristics that allow them to be recognised and
distinguished from others, for example, visual characteristics such as height, facial
characteristics, skin, eye and hair colour and weight. Similarly individuals differ at the cellular
and subcellular level. Forensic biologists utilise these characteristics. The methods of
biological discrimination can basically be divided into four main categories:
Visual discrimination by microscopic and macroscopic comparisons. This mainly relates to
hair and fibre comparisons.
Immunological discrimination. A variety of molecules are present on the surface of cells or in
body fluids that label the cells and fluids within our body as "self". These are required to
allow the immune system to identify foreign molecules such as bacteria and viruses from "self"
cells and proteins. The best known of the antigenic markers is the ABO blood group system.
Other antigenic markers include the Rhesus, Kell, Duffy, Kidd, MN, Ss, Gm, Km, and Lewis
systems. These are all antigens found on the surface of red blood cells and/or in serum or other
body fluids. The major histocompatibility antigens which are largely responsible for organ
transplant rejection also fall within this category.
Discrimination based on protein variations. A variety of cellular and serum proteins have
polymorphic forms that can be electrophoretically separated and identified and hence used to
discriminate between individuals. For example group specific component (Gc), 2-human serum
glycoprotein (AHSG), phosphoglucomutase-1 (PGM-1), haptoglobin (Hp), adenylate kinase
(AK), erythrocyte acid phosphatase (EAP), glucose-6-phosphate dehydrogenase (GPD),
haemoglobin (Hb), 6-phosphogluconate dehydrogenase (PGD), carbonic anhydrase II (CA),
esterase D (EsD), peptidase A (PepA), glyoxalase I (GLO) and adenosine deaminase (ADA).
Discrimination based on genetic variations. Diversity at the genetic level is far greater than
visual, antigenic and protein variations. Indeed, all the visual, antigenic and protein variations
are a result of genetic variations. There are literally millions of genetic polymorphisms that
could potentially be used for the purpose of biological discrimination. In reality to date the
application of DNA technology to forensic science is only rudimentary. Only a very small
portion of the total genetic variations that occur are useful to forensic biologists using the
technology that is available today. However the systems that are available already provide far
greater discriminating power than any of the visual, antigenic and protein systems described
above.
The History of Application of DNA Technology to Forensic Science
In 1980 Wyman and White identified the first hypervariable locus in human DNA. The allelic
forms detected at this locus differed in size and were so-called restriction fragment length
polymorphisms (RFLP's). This procedure became the foundation for more exciting discoveries.
In 1985, Dr Alec Jeffereys, using this technology, found a DNA fragment that when used as a
DNA probe against human genomic DNA bound specifically to a number of different RFLP
sites on the genome producing a characteristic and reproducible banding pattern that was
specific for an individual (with the exception of identical twins). He found this banding pattern
was inherited, with half the bands coming from the mother and half from the father. This
procedure was aptly named "DNA fingerprinting". Initially, DNA fingerprinting was used to
determine family relationships in immigration applications. However, in September 1986 Dr
Jeffereys was requested by police to apply the genetic fingerprinting technology to aid a
rape/murder investigation. This highly publicised case, known as the Narborough Murder
Enquiry, was to be the first murder investigation to be resolved by DNA fingerprinting.
Shortly after other forms of DNA typing were being reported. Unlike DNA fingerprinting these
procedures detected polymorphisms at single loci on the genome; these allelic variations were
due to variations in the number of tandemly repeated core units of DNA present. Nakamura, et
al (1987) coined the term variable number tandem repeats (VNTR's) to describe these
polymorphic loci. VNTR's were quickly recognised to be extremely useful in forensic
investigations, largely because the methods were more sensitive and the patterns less confusing
to interpret than the multilocus multiallele system described by Jeffereys. For some loci more
than 70 different allelic forms have been identified (Wong, et al, 1987). The probability of
different individuals having the same alleles is low. Additionally, three or four probes can be
used consecutively, each detecting hypervariable VNTR loci, yielding an extremely powerful
method of discrimination.
PCR technology (polymerase chain reaction) was conceived in 1987 and developed by Mullis
and co-workers at Cetus Corporation. Though this is relatively new technology it has rapidly
gained acceptance and been adapted to a variety of applications in research, medicine,
industry, agriculture and justice. It has revolutionised the approach to the recovery of DNA
from a variety of sources through the specific amplification of informative gene sequences and
has been rapidly adapted for forensic purposes.
Polymerase Chain Reaction
PCR is a procedure used to enzymatically amplify a specific DNA locus in vitro. Any locus of
DNA can be amplified so long as the DNA sequences flanking the region are known. The
concept is remarkably simple and is analogous to the replicative processes that are used by all
living cells to replicate their own DNA.
Deoxyribonucleic acid (DNA) is a double stranded molecule with a basic structure similar to a
ladder. If a ladder were to be cut longitudinally down the middle each half would represent a
strand. Each strand is made up of a sugar backbone with the informational molecules called
bases protruding inwards like the rungs in a ladder. The bases on one strand electrically bind
to the bases on the opposite strand to create the basic ladder like structure. It is the sequence of
bases along the length of the DNA strand that defines the genetic code. During DNA synthesis
the strands are peeled apart or so-called denatured. Each strand is used as a template for the
synthesis of the opposite strand such that upon completion of synthesis two identical double
stranded DNA molecules result.
DNA synthesis in vitro by PCR is kick-started by short segments of DNA called primers.
These primers are designed to bind to the template DNA strands so they flank each end of the
target sequence and delimit the region that will be amplified, hence the need to know the
sequences flanking the region of interest. The synthesis is performed by a heat resistant enzyme
called Taq polymerase that has been cloned and purified from the thermophilic bacterium
Thermus aquaticus. The synthesis reaction must be performed in a defined chemical
environment (the reaction buffer) for maximum polymerase activity.
The PCR amplification process consists of a three step cycle:
1. The double-stranded DNA molecule is denatured into single strands by incubating at
high temperature (94
0
C).
2. The temperature is lowered to allow the primers to specifically bind to their
complementary sequences flanking the target site (typically 55
0
C-65
0
C).
3. The temperature is raised to 72
0
C, the optimum temperature for the activity of Taq
polymerase, to allow the synthesis of the DNA region delimited by the primers.
After completion of synthesis the temperature is again raised to denature the DNA strands so
the next cycle can begin. The process is repeated for about 30-35 cycles. In theory the
completion of each cycle doubles the number of target DNA molecules, causing the target to
amplify in a logarithmic fashion, however in practice this does not occur as the efficiency of
amplification is not 100%. A 30 cycle amplification process generally amplifies the target
between 100,000-10,000,000 fold. In theory, it should be possible to amplify DNA sequences
of any length, however in practice the amplification of sequences greater than 2000 base pairs
(bp) is inefficient. Subsequently target sequences from 100-2000 bp in length are most
commonly used for PCR amplification.
The application of PCR technology to forensic science
For PCR to have useful application in forensic science the selected target sequences should
have several allelic variations, preferably all less than 1000 bp in size. A variety of
polymorphic loci that are useful for biological discrimination purposes fall within these
parameters. The best known of the PCR systems used for forensic science are the HLA DQA1
and the D1S80 systems. Both these systems are sold as kits by Perkin Elmer Cetus or may be
typed independently by similar or alternative methods.
The HLA DQA1 kit uses a series of allele specific probes to detect the various allelic forms.
An alternative kit, marketed by Merck, uses a variety of allele specific primers to identify the
allelic forms. This procedure is not as sensitive and is far more time consuming. Operators not
wishing to use the HLA DQA1 kit technology can identify the HLA DQA1 alleles by
performing RFLP analysis of the PCR products or by DNA sequencing of the PCR products,
however both are technically difficult and time consuming.
D1S80 is a VNTR locus. The allelic forms are simply separated and identified by size. The
term amplified fragment length polymorphisms (AMP-FLP's) has been used to describe D1S80
and other VNTR loci that can be amplified by PCR. Other AMP-FLP's that have also proved
useful PCR systems are D17S30, the 3' HVR region of the apolipoprotein B gene and Col1A2.
A third PCR system, known as the mitochondrial D loop region, has more recently attracted a
deal of interest by forensic scientists. This is a highly polymorphic DNA locus on the
mitochondrial genome (mitochondria are commensal living units inside the cells of plants and
animals, and other higher organisms, that provide the energy requirements for cellular activity).
The D loop locus once amplified is best analysed by direct sequencing of the PCR products.
With the aid of modern automated DNA sequencers this system is likely to receive a great deal
more attention.
Advantages of PCR technology
PCR has a number of advantages over RFLP technology.
1. As it involves an amplification process it is more sensitive than the single-locus and
multi-locus probe technologies and the protein and antigenic systems.
2. The technology is potentially very cheap, unless kit technology is used.
3. The PCR procedure is much faster than RFLP technology.
4. The technology is simple to understand and much easier to perform than RFLP
technology.
5. The amplification process is largely automated, and therefore should not be subject to
human error.
6. Radioactive isotopes are not required.
7. The PCR products do not appear to be altered by degradation of the template DNA
caused by decomposition of the sample.
8. The PCR process can be tailored for a particular locus.
9. Results can often be obtained from crude DNA preparations.
Disadvantages of PCR technology
PCR has a number of disadvantages over RFLP technologies that must be considered before
adopting the PCR system.
1. PCR has relatively low discriminating power in compared to RFLP technology.
2. PCR cannot be used for the efficient amplification of DNA segments in excess of 2000
bp. The vast majority of the extremely powerful VNTR's have allelic forms in excess of
2000 bp.
3. The PCR process may be inhibited by various chemicals present in the DNA extract.
For example various organic acids in soils, metabolites released by vaginal flora and
haemoglobin are powerful inhibitors of the PCR process.
4. False negatives may rarely occur due to genetic variations within individuals that
prevent the binding of one or both of the primers.
5. False positives can occur if a great deal of care is not taken to prevent the
contamination of samples, DNA extracts or reagents from amplified DNA or other
sources of foreign human DNA.
Cost considerations of PCR technology
A well-equipped basic PCR laboratory can be established for approximately A$100,000
provided suitable accommodation is available. Equipment is necessary for the preparation of
samples, reagents and reaction mixes; the amplification and analysis of PCR products; and the
storage of samples, DNA extracts, PCR products and reagent preparations.
The reagents for the preparation of samples, the PCR reactions and the analysis of the PCR
products are also relatively inexpensive. The most expensive components are the Taq
polymerase and the primers. Most PCR systems require reagents which cost about A$5 per
test. Labour costs are largely dependent on the PCR systems used, how often the tests are
performed and how many tests are performed concurrently. Samples can generally be
extracted, amplified and analysed in batches. When performed in batches tests generally take
between 3 and 5 days to complete, from sample to result.
Kit technology versus do-it-yourself
Most of the forensic laboratories performing PCR techniques in Australia have adopted the use
of kits for at least some of their PCR systems. At the moment Perkin Elmer Cetus has two kits
available in Australia that are designed for forensic purposes, the HLA DQA1 and the D1S80
kits, with a third kit to be released in the near future. Kits have several advantages that make
them seem at least initially very attractive. Most of the reagents are prepared. The procedures
have already been optimised, meaning developmental work required to introduce the systems is
reduced. The procedures are generally very simple; take extract, add reagents A and B, mix
and amplify according to procedure C, analyse products according to procedure D and read.
Known positive controls are included to monitor test batches. The tests and reagents are quality
controlled by the manufacturer and are guaranteed to work. It also reduces the possibility of
human error in reagent preparations.
However, there are a number of disadvantages to kit technology that may be initially
overlooked. These kits are expensive. The HLA DQA1 and D1S80 kits cost approximately
A$30 and A$15 per test respectively for the components of the kit only. Neither kit includes the
reagents required to extract the sample. The D1S80 kit does not include the reagents required
to analyse the PCR products, whereas most are supplied in the HLA DQA1 kit. The protocols
are inflexible, as the companies will not guarantee the results that are obtained from
procedures other than those quoted by the company. The validation of the kits prior to their
implementation as a routine test is an extremely expensive process. Additionally once the kits
are implemented then laboratories are bound to continue the use of that technology simply
because of the expense involved in its implementation. As a result the laboratories are bound to
the company and any problems that may be associated with that company; for example any
price increases or supply/demand problems.
Laboratory design and other precautions
The PCR process is an amplification process. Literally millions of copies are synthesised for
each reaction. Logically the possibility exists for carry-over contamination and it must be
addressed. It is extremely important to ensure the samples destined for PCR analysis and the
reagents used in the PCR reactions are not contaminated by PCR products from previous
reactions, so-called carry-over contamination. The most effective ways of preventing carry-
over contamination are all logical.
The PCR process should be physically separated from the rest of the laboratory by dedicating a
separate and isolated room for the PCR process and the analysis of the PCR products. Air
pressure in the PCR room should be neutral or preferably negatively pressurised to prevent the
movement of PCR products out of the room as aerosols. A second separate area should be
provided for sample and reagent preparations. This area is best positively pressurised to
reduce possible inflow of contaminating DNA molecules. The article examination and sample
collection area should also be separated from the DNA preparation and the PCR rooms.
Equipment within the PCR room must be dedicated to that room. Access to the PCR room
should be restricted as much as possible to the staff directly involved in its function. PCR room
staff must wear dedicated laboratory coats and disposable gloves, both of which must be
removed before leaving the room. Amplification products should be stored within this room
until they are no longer required. Waste should be transferred directly out of the building.
Cleaners and security workers should not have access to the PCR room except in emergencies
or by request. Dedicated cleaning equipment should be provided in the room.
The reactions should be prepared in a sterile environment such as a biohazard hood. Reagents
should be aliquoted into use-once volumes, so if contamination does occur it will not affect a
number of batches. And finally, positive and negative controls should always be included in
every batch to monitor for possible contamination and to monitor the success of the PCR
process.
Conclusions
It is unfortunate but true that most forensic laboratories in Australia have been forced to decide
whether to implement RFLP technology or PCR technology. It would be better to be able to
have both however the public purse simply will not allow it. In Queensland we have chosen to
implement PCR technology largely because of the expected monetary savings and the increased
sensitivity of this technology as compared to RFLP technology. We are satisfied with the
technology although the power of discrimination provided by PCR technology is less than
RFLP technology. The introduction of PCR technology has substantially increased our powers
of biological discrimination particularly in rape cases. However the savings have not been as
substantial as expected. This is largely because we have chosen to utilise kit technology. This
is an expensive practice.
It is nearly impossible to compare PCR technology with RFLP technology and conclude which
is the best technology. PCR technology is relatively new whereas the RFLP technology has
been used in forensic examinations for a longer period of time and has been exposed to a great
deal of legal argument and statistical examination and is still considered a popular, reliable
and valid technique. PCR has not yet been exposed to these elements. It will be interesting to
see how it fares.
References.
Jeffereys, A.J. 1987. Hypervariable "minisatellite" regions in human DNA. Nature vol.
314, pp. 67-73.
Mullis, K.B. and F.A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase
catalysed chain reaction. Methods in Enzymology vol. 155, pp. 335-350.
Nakamura, Y., M. Leppert, P. O'Connell, R. Wolff, T. Holm, M. Culver, C. Martin, E.
Fujimoto, M. Hoff, E. Kumlin and R. White. 1987. Variable number tandem repeat (VNTR)
markers for human gene mapping. Science vol. 237, pp. 1616-1622.
Wong, Z., V. Wilson, A.J. Jeffereys and S.L. Thein. 1986. Cloning a selected fragment from a
human DNA "fingerprint": isolation of an extremely polymorphic minisatellite. Nucleic Acids
Research vol. 14, pp. 4605-4616.
Wyman, A.R. and R. White. 1980. A highly polymorphic locus in human DNA. Proceedings of
the National Academy of Science USA vol. 77, pp. 6754-6758.