Download DNA analysis in forensics, disease and animal/plant identification

DNA analysis in forensics, disease and animal/plant
identification
R Lynn Alford and C Thomas Caskey
Baylor College of Medicine, Houston, USA
During 1993, significant advances have been achieved in applications of
DNA analysis to forensic science, disease assessment, and animal/plant
identification. These advances include the development of simple and high
sample-throughput techniques for highly informative personal identification,
rapid screening for pathogens and the development of polymorphic genetic
markers in plants and animals.
Current Opinion in Biotechnology 1994, 5:29-33
Introduction
Table 1. DNA polymorph isms and techniques used for their detection.
Modem DNA analysis is the practical application of
two rapidly growing and progressing fields, genetic
research and biotechnology. As genetic research reveals new information, genes, markers and repetitive
sequence elements, the variabilities in DNA sequences
among individuals and populations are being revealed.
Meanwhile, new techniques for analysis of DNA are
being developed and the ability to study certain types
of DNA sequence variation is being gained.
DNA variation
Size of variation
Detection method
Base pair sequence
Single nucleotide
RFlp, ASO
Biallelic markers
PeR
Microsatellites
Minisatellites
DNA analysis is fast becoming a highly accurate and reliable tool for the physician, as well as for the criminal
justice and public health care systems. It is impossible
in the limited space of this review to provide an adequate historical and scientific account of the current
state of the art. Instead, the goal of this article is to provide a general review of the topic, and to point the
reader to some of the most interesting developments
from the past year.
DNA analysis in forensics
Recent years have seen the growing application of
DNA technologies to forensic science. The value of
DNA testing for forensic analysis depends on the utility
and informativeness of DNA markers and probe sequences. In recent years, we have seen the development of a variety of DNA variations useful for forensic analysis. Some of these variations are illustrated in
Table 1.
CA di-nucleotide
repeats
Tri-, tetra-, penta-,
nucleotide repeats
Ten(s) to a hundred
base pair repeats
PeR
Southern blot
The history of forensic DNA analysis has been most
noted for its application to criminology. Personal identification and forensic matching of DNA are useful in
cases of crimes of violence and kidnapping of children,
but are not limited to these applications. Additional
uses include the identification of crib switches in hospitals, laboratory quality control for sample switching,
paternity testing, immigration control, identification of
military personnel, and forensic matching of criminal
reoffenders. For those unfamiliar with such histories
of DNA forensic analysis, complete chronological and
scientific histories are provided by Jeffreys [Il, and by
Weedn and Roby [2]. The latter authors also provide an
in-depth look at the relevant issues and problems with
different foren'iic and forensic/genetic techniques.
The application of DNA analysis to criminal investigation is growing, but by far the most requested use of
this technology is in cases of disputed paternity. This
demand has required the development of methods that
are faster, more reliable, less expensive and automated.
Abbreviations
AIDS-acquired immune deficiency syndrome; ASO-allele-specific oligonucleotide; CMTlA-Charcot-Marie-Tooth disease type lA;
DMD/BMD-Duchenne/Becker muscular dystrophy; HIV-human immunodeficiency virus; HSV-herpes Simplex virus;
OTC-ornithine transcarbamylase; PeR-polymerase chain reaction; RFLP-restriction fragment length polymorphism;
SSCP-single strand conformation polymorphism; STR-short tandem repeat; VNTR-variable number tandem repeat.
Current Biology Ltd ISSN 0958-1669
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Analytical biotechnology
The older variable number tandem repeat (VN1R) and
restriction fragment length polymorphism (RFLP) systems are being challenged by newer technologies.
For example, short tandem repeat (STR) loci that are
amenable to polymerase chain reaction (PCR) amplification and automated allele size measurement are
being developed for personal identification and for
parentage testing ([3--]; HA Hammond, Llin, Y Zhong,
CT Caskey, R Chakraborty, unpublished data; RL Alford, HA Hammond, I Coto, CT Caskey, unpublished
data). The advantages of this system over older systems include minimal DNA requirements, simplified
rapid analysis, and the potential for non-isotopic detection and automation. This new system has withstood the legal requirements for discrimination among
individuals and several loci have already been widely
characterized.
The major drawback to the VNTR and STR systems
is the lengthy gel analysis that is required for allele
siZing. A novel approach to forensic DNA typing has
been presented by Syvanen et al. [4--], In their report,
DNA typing is accomplished with techniques based on
minisequencing protocols. This strategy uses multiple
biallelic DNA markers. PCR-amplified DNA is attached
to a solid support with a biotin tag, and, after one-step
sequencing, incorporation of isotope is measured for
marker typing [4--],
The increasing use of DNA fingerprinting and VNTR
analyses in the courtroom has resulted in an increased
demand on the mathematical parameters of the test systems. It is necessary to determine the likelihood of two
unrelated individuals exhibiting similar DNA profiles
or VNTR alleles by chance. Two recent analyses of
this probability are provided by Chakraborty et al.
[S--] and Li et al. [6]. Chakraborty et al. [S--] describe
the mathematical derivation and statistical considerations of estimates of multilocus genotype probabilities
with reference to VN1R studies. Li et al. [6] study the
discriminatory powers of DNA fingerprint analysis and
a new method for deriving such estimates.
The utility of DNA fingerprinting techniques is emphasized by the numerous ways in which this technology is being applied. For example, Mehle et al. [7]
have found fingerprinting useful in describing intratumor heterogeneity found in renal cell carcinomas. This
usage has implications for the clinical management of
patients, and the mechanistic understanding of cancer
development and tumor progression, as well as for
gene mapping and gene discovery in cancer. Hersee
et al. [8] have found PCR fingerprinting to be useful in rapid, cost-effective crossmatching of unrelated
bone marrow donors. In their studies, PCR-amplification of RIA-DRB polymorphism typing was useful
in detecting polymorphisms in serologically matched
donor/recipient pairs. It has also provided the potential for multiplex amplification of RIA loci for rapid and
accurate crossmatching of donors [8].
Additional applications of polymorphic DNA characterization include linkage analysis in disease where polymorphic markers can be used to trace a known disease
gene through a family in the absence of a known gene
or mutation, and characterization of simple repeat diseases (see below).
DNA analysis in disease
In the race to develop viable methods for the detection and diagnosis of genetic disease of known etiology, several different techniques have been applied
to a variety of diseases, as a result, in part, of differences in mutational mechanisms. Diseases such as
cystic fibrosis, ornithine transcarbamylase (OTC) deficiency, and ~-thalassemia occur primarily as a result
of point mutations in genes. In diseases such as cystic
fibrosis and ~-thalassemia, where a few mutations account for the majority of disease, screening techniques
such as allele-specific oligonucleotide (ASO) hybridization and reverse dot blot typing have been successfully
employed [9,10]. The combination of ASO typing and
robotic methods, which allows screening for 22 mutations in cystic fibrosis, has recently been described
DeMarchi, CT Caskey, S Richards, abstract 1483,
American Society of Human Genetics Meeting, October, 1993).
a
In diseases such as OTC deficiency that arise from
new mutations, more generalized screening techniques
must be applied. In these cases, single strand conformation polymorphism (SSCP) has been a more effective method [1l]. PCR-SSCP has also been applied to
the detection of mutationl' in the cancer-causing pS3
gene [12] and the adenomatous polyposis coli gene
[13]. (PCR-RFU> analysis has also been useful in detecting mutations in the latter condition [14].) In addition,
PCR using primers specific for expected mutations has
been used to preferentially amplify mutant products for
detection of K-ras mutations in patients with pancreatic
adenocarcinoma [IS], Another method, denaturing gradient gel electrophoresis, is often the method of choice
when screening genes for new point mutations. This
technique and some of its applications are described
in detail in Cariello and Skopek [16-]. It has been applied to mutation detection in pS3 [17], K-ras2 [18], and
human immunodeficiency virus (HIV) [19].
In contrast to the diseases mentioned above, some disorders are caused primarily by deletion mutations. In
Duchenne/Becker muscular dystrophy (DMD/BMD),
where a large proportion of mutations are deletions, multiplex PCR/quantitative PCR deletion scanning techniques are being developed and are proving
effective [20], The current procedure involves the amplification of 22 exons in the DMD/BMD gene in two
multiplex PCR reactions, and is capable of identifying
more than 99% of deletions (S Richards, M Morsy, P
Ward, P Watson, S Smith, CT Caskey, abstract 1221,
American Society of Human Genetics Meeting, October, 1993).
Despite the progress that has been made in genetic research, the etiology of hundreds of genetic conditions
DNA analysis in forensics, disease and animal/plant indentification Alford and Caskey
remains a mystery. One of the most exciting developments of recent genetic research has been the discovery of new mechanisms of mutation. A new class of
mutation that has been described is the germ-line expansion of simple repeat sequences within genes. This
mechanism of mutation has been associated with myotonic dystrophy, fragile X syndrome, and Kennedy disease [21-23]. In the past year, two new fragile sites in
Xq28, near the original fragile X site, have been described [24,25]. In addition, two other diseases, Huntingtons disease [26--] and spinocerebellar ataxia type I
[27--], have been determined to be caused by expansion of trinucleotide repeats.
Another novel mutation mechanism has been described by Lupski et al. [28--]. Charcot-Marie-Tooth
disease type 1A (CMTlA) is a common inherited neuropathy that is caused, in some cases, by a gene dosage
effect in which patients carry a duplication of a region
on chromosome 17. A more recent report describes
mutations within the PMP22 gene locus, which maps
to the duplicated region of chromosome 17 [29]. These
mutations segregate with the disease and affirm PMP22
as the genetic location of the disease in which mutation or duplication is capable of causing the CMTlA
phenotype.
DNA analysis in animal/plant identification
DNA analysis has shown increasing utility in infectious
disease epidemiology for acquired immune deficien<.:y
syndrome (AIDS), in control of infected hospital areas
and supplies, and in identification of contaminated
foods for control of epidemic outbreaks. (An excellent review has recently been provided by Lupski
[30].) DNA fingerprinting techniques have also been
used for the determination of relatedness of animals
both in breeding programs and in the wild.
Techniques for the detection of HIV in environmental
samples such as hospital wastes, raw wastewater, soil
and pond water will both impact epidemiological studies on the transmission mechanisms of HIV and facilitate the tracing of relevant sources of environmental
contamination [31]. In addition, methods for the analysis and control of antibiotic-resistant Staphylococcus
aureus infections in hospitals have recently been reported [32]. Techniques have also been described for
the rapid, non-isotopic, and direct detection of hepatitis B virus in human blood products [33,34]. PCRbased detection of human papillomavirus in urine as
a potentially useful clinical screening technique for
women at risk for developing cervical cancer has also
been reported [35], Finally, PCR-based techniques have
been successfully applied to the diagnosis of a number of conditions: herpes simplex virus (HSV)-induced
encephalitis by PCR amplification of HSV from cerebrospinal fluid [36]; meningococcal meningitis by amplification of Neisseria meningitidis from cerebrospinal
fluid [37]; and HSV or varicella-zoster infection by amplification of viral sequences [38].
Forensic DNA techniques are impacting on patient
management in cancer treatments to identify the source
of malignant relapse after autologous bone marrow
transplant [39]. Marker systems such as these are important for therapy regimens by identifying sources of
therapy failure and sites for future modification [39].
The application of forensic DNA technology to the fingerprinting of animals is becoming more prominent as
new markers are defined and characterized. This technology has the potential to affect breeding programs
by measuring the relatedness of individuals and by
mapping genetic traits through linkage analysis. These
techniques have relevance for both organized breeding programs and sociobiology. Lang et al. [40] have
described the successful use of DNA fingerprinting to
study the relatedness of crocodile populations in the
wild and in structured breeding programs.
These data have impact not only on the purposeful
breeding of endangered species, but also on the sociobiological examination of animal social structures in
the wild (40). In addition, DNA technology is playing a
role in the breeding of livestock. For instance, Trommelen et al. [41] describe the identity and paternity testing
of cattle in an organized breeding program.
In an interesting twist to the application of DNA forensic technology, Guglich et al. [42] have reported the use
of DNA fingerprinting in deer and moose specimens in
Ontario for the prosecution of suspected illegal hunting activities. This was achieved by matching samples
found at illegal kill sites to samples found in the possession of suspected poachers.
Picard et al. [43] report techniques for extracting DNA
from soil samples for the specific detection of different types of bacteria. In addition, Muyzer et al. [44]
have described denaturing gradient gel techniques
for profiling the genetic diversity of microbial populations. Techniques have also been developed for
the detection of Vibrio cholerae in foods [45-] and
Aeromonas salmonicida in large fish cultures [46-].
Both V cholerae and A. salmonicida are of interest
to public health and agriculture, and the techniques
described in these reports represent a rapid, cost-effective way of identifying these pathogens.
The application of DNA technology to plants also has
relevance for breeding programs and genetic engineering by linkage analysis of traits. protocols have been
developed for genotyping plants [47] and for identification and discrimination of plant DNA profiles [48].
The latter precedent-setting article is of particular interest as it details the use of DNA profiles from seed
pods to link a murder suspect to the scene of a crime
[48].
Conclusions and future directions
Genetic research and technology development have resulted in a variety of DNA methods that allow the iden-
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tification of individual animals and plants for forensic,
paternity, sociobiological and breeding purposes. DNA
analysis is also of potential interest in the diagnosis of
human disease and the detection of pathogens in our
environment.
Widespread application of these techniques awaits several advances. The techniques must be made simple,
reliable, and inexpensive. They must also be made
amenable to automation for high sample-throughput
and laboratories must be organized for the implementation of DNA analytical protocols. Once these
objectives are achieved, DNA analysis for a variety of
concerns will become widespread.
Acknowledgements
The authors thank Dr Belinda Rossiter for critical review of this
manuscript. The authors' work noted in this article was supported
by a gr,mt from the National Institute of Justice. C Thomas Caskey
is an Investigator with the Howard Hughes Medical Institute.
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RL Alford, Institute for Molecular Genetics, T813, Baylor College of
Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
CT Caskey, Howard Hughes Medical Institute and Institute for Molecular Genetics, T809, Baylor College of Medicine, One Baylor Plaza,
Houston, Texas 77030, USA.
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