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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 29 30 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- 31 32 Analytical biotechnology 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. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest • •• of outstanding interest 1. ]EFFREYS A: 1992 William Allan Award Address. Am] Hum Gen 1993, 53:1-5. 2. WEEDN V, ROBY R: Forensic DNA Testing. Arcb Patbol Lab Med 1993, 117:486-491. KIMPTON C, GILL P, WALTON A, URQUHART A, MILLICAN E, ADAMS M: Automated DNA Profiling Employing Multiplex Amplification of Short Tandem Repeat Loci. PeR Metbods Appltc 1993, 3:13-22. These authors describe the protocols and many of the loci available for STR analysis using fluorescent detection systems. Multiplex PCR reactions simplify the procedure, and a chardcterization of 14 loci is completed. 7. MEHLE C, LjUNGBERG 13, STENUNG R, Roos G: DNA Fingerprillting of Renal Cell Carcinoma with Special Reference to Thmor Heterogeneity. Genes Cbromosomes Cancer 1993, 6:86-91. 8. 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Cancer Res 1993, 53:2472-2474. 3. •• SYVANEN A-C, SAjAN11LA A, LUKKA M: Identification of IndividuaIs by Analysis of Biallelic DNA Markers, using PeR and Solid-Phase Minisequencing. Am] Hum Gen 1993,52:46--59. This forensic DNA typing method immobilizes PCR-amplified DNA surrounding biallelic markers on a solid support with biotin. The incorpordtion of labeled nucleotides provides ~ker typing. Despite the necessity to type multiple loci as a result of the relatively low informativeness of each independent locus, the approach is much simpler and faster than gel-based systems and is amenable to automation and non-isotopic detection systems. 16. • CARlEU.O N, SKOPEK, T: Mutational Analysis using Denaturing Gradient Gel Electrophoresis and PeR. Mutation Res 1993, 288:103-112. An excellent summary of denaturing grddient gel electrophoresis and some of its applications. 17. 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