KING SOLOMON S STORY is well known for his famous decision

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1 DNA AND CELL BIOLOGY Volume 25, Number 3, 2006 Mary Ann Liebert, Inc. Pp DNA Fingerprinting in the Criminal Justice System: An Overview ABSTRACT DNA fingerprinting is a powerful technology that has revolutionized forensic science. No two individuals can have an identical DNA pattern except identical twins. Such DNA-based technologies have enormous social implications and can help in the fight against crime. This technology has experienced many changes over time with many advancements occurring. DNA testing is a matter of serious concern as it involves ethical issues. This article describes various trends in DNA fingerprinting and the current technology used in DNA profiling, possible uses and misuses of DNA databanks and ethical issues involved in DNA testing. Limitations and problems prevailing in this field are highlighted. INTRODUCTION KING SOLOMON S STORY is well known for his famous decision in a maternity dispute, but today, parentage decisions are relatively easy with the assistance of DNA technology. DNA technology was first developed in England in 1985 by Sir Alec Jeffreys. This technology, so named because DNA is used for identification rather than latent physical fingerprints, gives a unique and specific profile similar to a thumb impression (Jeffreys et al., 1985). DNA fingerprinting technology today has made it possible to identify the source of biological samples found at a crime scene and also to resolve disputes of paternity and other criminal cases. Ninety-five percent of the human genome is noncoding DNA, with only 5% coding for protein region. These noncoding regions are also called junk DNA, and are abundant where the number of repeats show variations among individuals. These DNA polymorphisms change the length of the DNA fragments produced by the digestion of restriction enzymes. A DNA fingerprint is constructed by first extracting a DNA sample from biological sample. The sample is then cut at specific sites with restriction enzymes, and the segments are arranged by their molecular size using electrophoresis. The segments are labeled with probes and exposed to X-ray film, where they form a characteristic pattern, that is, the DNA fingerprint. If the DNA fingerprints produced from two different samples match, the two samples probably came from the same person. The exact number and size of the fragments produced by a specific restriction enzyme digestion varies from individual to individual. In 1985, police in the UK for the first time used forensic DNA profiling ( and in the United States, the first criminal conviction based on DNA evidence occurred in In 1992, the National Research Council of the United States found that DNA testing was a reliable method for the identification of criminal suspects, and the technology rapidly entered the mainstream court system. The DNA evidence obtained is powerful, since it can implicate or exonerate a suspect. Currently, DNA testing is routinely used in civil and criminal cases. DNA ANALYSIS Early markers Karl Landsteiner, in 1901, discovered ABO blood grouping in humans (Landsteiner, 1901). A set of blood group markers (red cell antigens and serum protein) were also used to identify victims and suspects in crime cases. As the polymorphism was very limited in ABO blood grouping, it was neither informative nor very suitable for excluding a person as a suspect in a criminal case. The advent of DNA-based markers has now revolutionized the field of forensic science, as it can measure an unlimited extent of polymorphisms sufficient to differentiate one individual from another. Laboratory of DNA Fingerprinting Services, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India. 181

2 182 Hybridization based multilocus and single locus DNA profiling The use of DNA fingerprinting in forensic science had been suggested in the early 1980s by Prof. Alec Jeffreys using restriction fragment-length polymorphism (RFLP). There was great enthusiasm among the forensic science community for the discovery of the minisatellite approach for specific identification (Wong et al., 1987). Eukaryotic genomes contain a large number of repeated DNA sequences. These regions are often referred to as satellite DNA. The core repeat unit for a medium-length repeat, sometimes referred to as a minisatellite or variable number of tandem repeats (VNTR) is in the range of approximately bases in length. VNTR patterns are inherited and are unique (Weising, et al., 1995). The more VNTR probes used to analyze a person s VNTR pattern, the more distinctive and individualized that pattern, or DNA fingerprint, will be. The children will inherit the different combinations of paternal and maternal VNTRs, and therefore their profile will be different from each other except identical twins as shown in Figure 1. DNA regions with repeat units that are 2 6 bp in length are called mcrosatellites, simple sequence repeats (SSRs) or short tandem repeats (STRs). STRs consisted of microsatellites of small repeating units, two to five bases in length, with a maximum length of around 300 bases, which show a high degree of polymorphism. Specific microsatellites can be isolated using hybridized probes followed by their sequencing. SSRs can be detected by specific dyes or by radiolabeling using gel electrophoresis. The advantage of using SSRs as molecular markers is the extent of polymorphism shown, which enables the detection of differences at multiple loci between strains (Butler, 2001). Hybridization-based DNA fingerprinting involves the use of two types of probes called multilocus probes (MLP), which identify repeating sequences in genomic DNA digested by restriction enzymes followed by Southern blot analysis at low stringency conditions. The radio labeled probes hybridize with a series of tandemly repeated sequences with a size range greater than 20 kb. The resulting autoradiographs consist of a ladderlike series of bands that will be identical only for identical twins as shown in Figure 2. The fact that DNA is a very stable molecule makes this technology more effective, and even old cases also can be reviewed. This is equally good for archived specimens. The MLP system, however, was not very successful. The method was demanding and the results were not suited for inclusion in computer databases. After the advent of the single locus probe (SLP) the technology substantially progressed. In the SLP system probes were isolated, which identified repeat sequences in individual minisatellites using high stringency conditions to produce a maximum of two bands per probe. Although the targeted minisatellites showed considerable variation in their repeat numbers, a single locus was not sufficient for individualization. A number of such loci were analyzed (Lalji, 1991). PCR-based DNA profiling FIG. 2. Multilocus DNA profiling of (a) Identical and (b) nonidentical twins. In the early 1990s, the DNA fingerprinting technology took a dramatic change with the advent of the polymerase chain reaction (PCR), a technique that enabled amplification from tiny amounts of template DNA. The method was fast, reliable, and was efficient to work even with degraded samples. In the PCRbased technique the time required to analyze a sample was significantly reduced (Weber and May, 1989). It was found to be more suitable than the previously used MLP and SLP systems. There was a period when both SLP and PCR-based methods were used to identify a person in parallel. PCR technology was used to type many VNTRs region for forensic purposes. Many polymorphic loci like D1S80 and HLADQ were used for analysis. There were many reports claiming that it was not much useful for forensic purposes (Varsha et al., 1999). In 1992, the use of a new compatible form of genetic profiling called STRs was proposed. Due to their small size they were extremely stable, and could be detected even with old and degraded samples. In 1998, STR analysis became an established feature of forensic sciences, which basically involved study of a panel of polymorphic loci comprising THO1, VWA, FGA, D21S11, D8S1179, and D18S51, as well as D7S820, D13S317, D5S818, CSF1P0, D16S539, D2S1338, D19S433, TPOX, with recently introduced Penta E and Penta D (Edwards et al., 1991). A flow diagram (Fig. 3) and Table 1 show the progressive advancements in forensic genetics. FIG. 1. VNTR distribution in a family. HOW IS DNA TYPING PERFORMED? In criminal cases, samples obtained from crime scene evidence and a suspect are used for DNA extraction and analysis for the presence of a set of specific DNA markers. DNA markers are small DNA probes that bind to a complementary sequence in the DNA sample. A distinctive pattern for an individual is created by a series of probes bound to a DNA

3 DNA FINGERPRINTING 183 FIG. 3. The progress in Forensic genetics. (Diagram reprinted by permission of Mark A. Jobling and Tanita Casci from paper Encoded evidence: DNA in Forensic analysis, Mark A. Jobling, Nature reviews/genetics, Vol 5, Oct 2004.) sample. Comparing such DNA profiles determines whether the suspect s sample matches the evidence sample. If the patterns match, the suspect may have contributed the evidence sample and if profiles do not match, the person is assumed innocent. Scientists believe that DNA evidence is far superior to eyewitness accounts, where the chance for correct identification is about 50:50. A combination of probes used in DNA analysis, gives greater reliability for profiling. Four to six probes are recommended for a good DNA profile. The recent new innovation in DNA chip technology (in which thousands of short DNA sequences are embedded onto a tiny silicon chip) enables a more rapid, and less expensive analysis using many more probes (Linacre and Graham, 2002). A flowchart is presented in Figure 4, which shows the steps involved in DNA profiling. DNA TECHNOLOGIES CURRENTLY USED IN FORENSIC INVESTIGATION RFLP is a technique for analyzing the variable lengths of DNA fragments that result from digesting a DNA sample with a special kind of restriction endonucleases. Amplification fragment Length polymorphism (AFLP) is a PCR-based derivative method of RFLP in which sequences are selectively amplified using primers. It is a reliable and efficient method of detecting molecular markers. Random amplified polymorphic DNA (RAPD) is one of the most commonly used primary assays for screening the differences in DNA sequences of two species of plants. It involves random amplification (Cooper, 2000). HLA- DQ analysis was also used for identification for quite long time (Saiki et al., 1986). With the advancement, these technologies became obsolete and were replaced by various techniques like microsatellite analysis, single nucleotide polymorphisms, etc. Capillary electrophoresis Capillary electrophoresis, or CE, is a group of techniques used to separate a variety of samples. These analyses are performed in narrow tubes, and can result in the rapid separation of different compounds. DNA typing with STR markers is now widely used for a variety of applications including human identification. CE instruments, such as genetic analyzers, are the method of choice for many laboratories which perform STR analysis. The Genetic Analyzer (Applied Biosystems, Foster City, CA) is a fluorescence based DNA analysis system utilizing capillary electrophoresis. It has multiple color fluorescence detection capability. This capillary machine is capable of running sequence and fragment analysis (Butler et al., 2004) and is a very popular method for STR typing. ABI 310 is a single-capillary Year TABLE 1. YEAR-WISE ADVANCEMENT IN FORENSIC DNA TYPING Developments 1985 RFLP approach was developed by Alec Jeffreys 1986 Automated DNA sequencing was first described 1988 FBI started using RFLP probes for case studies 1989 TWGDAM (Technical Working Group on DNA Analysis Methods) was established 1995 O.J. Simpson case, which spread the awareness of DNA; UK DNA database established 1997 Y chromosome STR marker was first described Source:

4 184 A Genescan program was used to compare the individual s profile (Fig. 5). In genotyping the alleles are denoted by peaks rather than bands unlike Genescan and peak height is also taken into consideration while analyzing the results (Fig. 6, Probability of paternity is %). The match probabilities obtained with STR multiplexes are very low, and is more than the world population. The preferred system for STR genetic typing was via fluorescent dye-labeled primers and automated DNA sequencers. Multiplexes are analyzed and typed using automated sequencing equipment. First multiplex used was quadruplex, which had four STR regions. The recent multiplex amplifies for 16 loci in a single reaction one out of that is Amelogenin, a gender marker. STR base provides complete information about different STR markers available (Butler, 2001). Simultaneous amplification and separation of a number of STR loci in a single step is possible here. Other methods include the use of single nucleotide polymorphs (SNPs), DNA amplification fingerprinting (DAF) and their offshoots. DNA amplification fingerprinting (DAF) involves amplification of genomic DNA, which is directed by only one oligonucleotide primer of arbitrary sequence (Caetano- Anolles et al., 1991). While the major efforts were made for detecting markers in genomic DNA, there has also been a considerable effort expanded on methods for typing sequences within the mitochondrial DNA (mtdna). Although these results are not as discriminating as those of the STRs, they can be very useful in cases where skeletal remains, hairs, etc., are the only samples available. There have also been advances in the use of markers which are made specific (Y chromosome), and these have been used successfully particularly in cases of sexual assault. Mitochondrial DNA analysis Degraded samples such as hair, bones, and teeth quite often lack nucleated cellular material and cannot be analyzed with STR and RFLP. Mitochondrial DNA analysis (mtdna) can be used to examine such DNA samples. mtdna is extremely valu- FIG. 4. Flowchart of DNA profiling. instrument. Similarly ABI 3100 is a 16-capillary instrument and is used in large-scale applications. Short tandem repeat analysis STRs have discrete alleles. Variability in STR regions can be used to distinguish one DNA profile from another (Edwards et al., 1991). The chance that two individuals will have the same 13-loci DNA profile is about one in one billion. Microsatellite typing can be multiplexed and semiautomated. Sophisticated software has been developed for STR analysis to take sample electrophoretic data through the genotyping process (Ziegle et al., 1992). This is done in two steps by two different software programs. Genescan software (Applied Biosystems) is used to spectrally resolve the dye colors for each peak and to size the DNA fragments. The resulting pherograms are then imported into the second software program, Genotyper. This program determines each samples genotype by comparing the sizes of alleles observed in a standard allelic ladder sample to those obtained at each locus in the DNA sample. Genotyping can be performed on the Genetic Analyzer with different available models (Ziegle et al., 1992). These are capillary electrophoresis systems. FIG. 5. Genescan analysis of STR markers in a paternity case. Lane 1. DNA profile of the mother; Lane 2. DNA profile of the child; Lane 3. DNA profile of the father.

5 185 DNA FINGERPRINTING FIG. 6. Electropherogram of genotype of STR markers in samples of a paternity case. 1. Genotype of the mother; 2. Genotype of the child; 3. Genotype of the father. able in such cases, as it has high copy number and has no recombinational events. Mitochondrial DNA is inherited maternally and does segregate independently. Therefore, it gives an excellent record of matrilineage. The father s sperm contributes only nuclear DNA. Occurrence of heteroplasmy is the special feature of mtdna. Comparing the mtdna profile of unidentified remains with the profile of a potential maternal relative can be an important technique in investigations on missing persons. Simultaneously, it can also be used as an important tool for matrilineage and evolutionary studies. Mitochondrial DNA can also help in studying the variety of human diseases, associated with mitochondrial disorders (Wallace, 1999). SNP outside the hypervariable segments along with mtdna can provide us a better tool for typing. Y-chromosome analysis Analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors as in rape cases. There are a total of 219 useful STR markers available on Y chromosome (Kayser et al., 2004). Single nucleotide polymorphism typing SNP are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is changed. SNPs create mutations in human and can be used as markers. SNPs have lower heterozygosity compared to STR. It will be not of much use when the samples are mixed, as SNP is a binary marker. Forensic SNP typing provides considerable assistance to forensic scientists in major disasters (Budowle et al., 2005). Many polymorphic sites need to be analyzed for the identification of SNPs. Mutation analysis is one of the major thrusts of functional genomics. Validation of a 21-locus autosomal SNP multiplex has been performed for forensic identification purposes (Dixon et al., 2005). Microarrays are used very effi- ciently for mutation analysis, which helps in monitoring the whole genome on a single chip and for studying the interactions of thousands of genes simultaneously. In the scientific literature various terminologies have been used to describe this technology as such as DNA chip, DNA microarray, etc. (Linacre and Graham, 2002). Forensic studies are mainly done by commercially developed autosomal STR multiplexes, autosomal SNPs, and markers on the Y chromosome and mitochondrial DNA (Jobling and Gill, 2004). ESTABLISHMENT OF DATABANKS DNA intelligence databases are modern tools for the executive force investigating crime (Werret, 1997; Hoyle, 1998; Parsoh et al., 1998; Peeren boom, 1998; Schneider, 1998). DNA forensics databases The first of the national DNA databases involving STR data was formed in the UK in The usefulness of such types of databases was established during the first 5 years of use of this technology (Werrett and Sparkes, 1998). By 1998, three other European countries, Austria, Germany, and The Netherlands had introduced national DNA databases (Martin et al., 2001). The STR loci used to form these databases were THOI, VWA, FGA, D8S1179, D18S51, and D21S11. Finland and Norway have introduced a national database in Sweden has already a database of unknown crime samples. Belgium, Denmark, Switzerland, France, Italy, Spain, and some east European countries also took steps in this direction. In 1989, the FBI proposed a national DNA database for North America. The Austrian DNA intelligence database was inaugurated in In the United States, the Federal Bureau of Investigation (FBI) has created a national database of genetic information called the National DNA Index System. The database contains

6 186 DNA obtained from convicted criminals and from evidence found at crime scenes. National DNA databank: CODIS The COmbined DNA Index System, CODIS, utilizes computer and DNA technologies together. All DNA profiles stored in CODIS are generated using STR analysis. The current version of CODIS uses two indexes where biological evidence is recovered from the crime scene. The Convicted Offender index contains DNA profiles of individuals convicted of sex offenses and other violent crimes. The Forensic index contains DNA profiles developed from crime scene evidence. A computer software will search these two indexes for matching DNA profiles. Law enforcement agencies compare DNA from biological evidence collected from crime scenes that have no suspect with the DNA profiles stored in the CODIS systems. If a match is made between a sample and a stored profile, CODIS can identify the culprit. This technology is authorized by the DNA Identification Act of As of January 2003, the database contained more than a million DNA profiles in its Convicted Offender Index and about 48,000 DNA profiles collected from crime scenes but which have not been connected to a particular offender. This database can also be used to identify criminals who are responsible for crime in other countries. The FBI uses a standard set of 13 specific STR regions for CODIS (Budowle et al., 1998). CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted criminals and missing persons. Advantages of DNA databanking One advantage of a comprehensive and modern national DNA databank is the capability to perform DNA identification tests of victims and missing persons in the case of disaster. It is just a matter of comparison of the profile of the sample with the databank, as the data is already available and the results are more reliable. Time of investigation as well as cost is reduced. It is also beneficial to society as it helps the investigator to catch the criminal and exonerate the innocent person. APPLICATIONS OF DNA FINGERPRINTING TO PLANTS AND ANIMALS Plant DNA fingerprinting is defined here as the application of molecular marker techniques to identify cultivars, but this also can be helpful in providing evidence in crime cases. RAPD can be a better choice for identification of plant strains. DNA profiling of plants can be used in solving civil disputes over the identity of commercially important cultivars (Kumar et al., 2001). The genotypic information is used for identification of animals (Barallon, 1998), forensic analysis (Zehner et al., 1998) and for tracing specific animals to finished meat products (Varsha et al., 2003), pedigree analysis, selection, and breeding programs. Forensic analysis can be of immense help for poaching cases and to check the illegal trade of endangered species. The cytochrome b region had been used for identification of the endangered animal species (Branicki et al., 2003). DNA typing in plants and animals is again a vast topic, and needs a separate TABLE 2. discussion. DNA fingerprinting has tremendous application especially in forensic sciences, which is mentioned in Table 2. ETHICAL, LEGAL, AND SOCIAL CONCERNS INVOLVING DNA FINGERPRINTING The primary ethical, legal, and social issues concerning DNA fingerprinting is privacy. DNA profiles are different from latent fingerprints, which are useful only for identification. DNA can provide insights into many aspects of a person and their families including susceptibility to particular genetic disorders, legitimacy of birth, and fertility. This increases the potential for genetic discrimination by government, society and others. Therefore, DNA typing should be carried out in a very sophisticated way, and should meet all the international standards and follow and abide all the ethical, legal, and social concerns involved with DNA typing. Databanking also involves some ethical issues and requires a balance between human rights and justice. Retention of samples for long periods of time is also risky, since this provides considerable accessibility to private genetic information. RELIABILITY OF DNA TECHNOLOGY SOME EXAMPLES OF DNA USE FOR FORENSIC IDENTIFICATION Criminal identification. Exonerate persons wrongly accused of crimes. Identify crime and catastrophe victims in mass disasters. Establish paternity and other family relationships. Identify endangered species and can help in wild-life poaching cases. Match organ donors with recipients in organ transplantation as kidney transplantation. Determine pedigree for seed or livestock breeds. To identify different strains and cultivars and can help in protecting the rights of plant breeders. Personal Identification like identification cards, etc. Generally, the legal system and courts have accepted the reliability of DNA testing everywhere, and admitted DNA test results as evidence. However, DNA fingerprinting is still controversial because of some important aspects. One is the accuracy of the results, as result will always be in the form of some probability value. Another aspect is the high cost associated with analysis. Some of the other important aspects such as the possibility of misuse of the technique, chances of manual error, and contamination cannot be ignored. The accuracy of DNA fingerprinting has been challenged because DNA segments rather than complete DNA strands are analyzed, which may not be necessarily unique. More research work is needed to confirm the uniqueness of DNA fingerprinting has not been conducted. In addition, DNA fingerprinting is often performed in private laboratories that may not follow uniform testing standards and quality controls. Therefore, human error could lead to false results. Awareness is still lacking about this technology among law enforcing agencies and judiciaries, requiring a lot of interaction between these and forensic scientists. DNA identification can be quite effective if used intelli-

7 DNA FINGERPRINTING 187 gently. Portions of the DNA sequence that vary the most among humans must be used; also, the number of loci studied should be sufficient enough to make the results more reliable. PROBLEMS WITH DNA FINGERPRINTING DNA fingerprinting is not fully assured. The term DNA fingerprint is, in a true sense, a misnomer. We cannot compare it with latent fingerprinting, as it is not as specific as fingerprints. Actually, a VNTR pattern gives a probability value that the person in question is indeed the person to whom the VNTR pattern (of the child, the criminal evidence, or whatever else) belongs. The probability decides whether the person can be reasonably matched with the DNA fingerprint. 1. Generation and determination of high probability is a requisite for forensic cases. The probability of a DNA fingerprint belonging to a specific person needs to be reasonably high, particularly in criminal cases. High probability can be achieved by increasing the number of polymorphic loci analyzed. Population structure can cause variation in allele frequencies between subpopulations (Lander and Budowle, 1994). VNTRs are not distributed evenly across all of human population. A given VNTR does not have a stable probability of occurrence, and not enough is known about the VNTR frequency distributions among ethnic groups to determine accurate probabilities for individuals within those groups. Some technical difficulties, like errors in the hybridization and probing process, will also affect the probability. Population studies should be carried out and reproducibility of the results should be confirmed. 2. DNA testing faces important problem of impurities of the sample. The sample collected from a crime scene most of the time is mixed with soil and other potential contaminants and considerable time and effort in purification processes purification on samples not directly amenable to analysis. Another important challenge to amplifying DNA samples from crime scenes is the fact that the PCR amplification process can be affected by inhibitors present in the samples themselves. The sensitivity of PCR with its ability to amplify low quantities of DNA can be a problem if proper care is not taken. Mixed samples can be challenging to detect and interpret. If mutation occurs at the primer binding site it can result in failure to amplify, which is known as null allele or allele dropout. Sometimes, stutter bands are noted in the electropherogram of STR analysis, which is nothing but small peaks several bases smaller than STR allele peaks, which occurs because of strand slippage. These bands effect the interpretation of DNA profiles specially in mixed samples, which requires good understanding of the behavior of stutter products (John M. Butler, forensic DNA typing). Proper training, better understanding, extensive experience, and careful analysis are required. 3. Very often investigators are not properly trained for sample collection and encounter problems in identification and collection of proper DNA evidence from the scene of crime. Contamination of the samples and using the correct sample, proper labeling, etc., are a few important aspects, which should be taken into consideration. The DNA laboratory should follow the international standard and should be accredited. 4. Proper awareness about the subject is required among the investigators, but unfortunately, interaction and proper communication is lacking between law enforcement agencies and DNA laboratories. 5. Limited resources and poor infrastructure facility of different laboratories effects the efficiency of the DNA typing laboratories. 6. Risk of false or misleading results from DNA testing and chances of tampering with the evidence cannot be ignored. Strict confidentiality is required as far as identity of the sample is concerned, and each sample should be tested by more than one examiner to confirm the results. Irrespective of having all these limitations, DNA fingerprinting is a very reliable technique for identification if used efficiently and intelligently. There are many international organizations, which are involved in quality control and uniformity of DNA fingerprinting such as the Technical Working Group on DNA Analysis Methods, The DNA Advisory Board, National Institute of Standards and Technology in United States, and International Society for Forensic Genetics in Europe. SOME HIGH-PROFILE CASES SOLVED BY DNA FINGERPRINTING 1. Identification of the remains of the last Russian Czar. 2. O.J. Simpson case in Los Angeles. 3. Clinton-Lewinsky affair. 4. Thomas Jefferson-Sally Hemings affair. 5. Identification of the bodies in World Trade Center attack: A mass disaster. 6. Assassination case of Sri Rajeev Gandhi, Prime Minister of India. CONCLUSION AND FUTURE DIRECTIONS The power of DNA technology will be of immense help in the future. The limitations of the technique have to be given serious thought. Forensic DNA testing will improve day by day, and will be benefited with the outcome of the Human Genome Project. Microarray-based analysis will be in much use for its more reliability and fast analysis. This technology has the potential to review the cases, which were previously reported with older methods. There is a real need for public awareness of the new DNA technologies and their implications. For instance, the collection of the correct sample and its proper preservation is very crucial. Productive interactions are required between the forensic scientists and the law enforcement and other investigators. It is necessary to make this technology more popular and cost effective so as to enable even developing and undeveloped third world countries to take advantage of the technology as well. ACKNOWLEDGMENTS My special thanks to all those authors whose names are not quoted here because of space constraints, and those who have made the information freely available on the internet. I am

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