DNA Techniques for Forensic Entomology
From ForensicWiki
Edited by Jason H. Byrd James L. Castner
Introduction and Brief Review of Forensic DNA Typing
At a time when many aspects of forensic science are dominated by recent advances in the field of molecular biology, it is no surprise that DNA technology should also become a tool of the forensic entomologist. At present, efforts to develop these tools are still mostly at the research stage. However, they have the potential to move very quickly into widespread use by those who analyze insect evidence in forensic investigations.
Since 1985, DNA typing of biological material has become one of the most powerful tools for personal identification in forensic medicine and in criminal investigation (Benecke, 1997b). The advantages of using DNA are that it provides a huge amount of diagnostic information compared to some older techniques (such as blood-group typing), it is present in all biological tissues, and it is much more resistant to environmental degradation than most other biological molecules (e.g., proteins). The entire DNA content of an organism is termed its genome, and techniques that extract all regions of DNA from a biological sample are said to yield genomic DNA.
A region of DNA that is used for identification purposes is often referred to as a marker or a locus (pi. loci). It displays one of two general types of variation between individuals (or species). A locus may vary between individuals in its length or in its sequence (the arrangement of paired bases that make up the DNA molecule). These two types of variation
are measured using different techniques (see below). The variants at a particular locus are called alleles, and this variation is responsible for the differences observed between individuals. A general term for a genetic difference observed between individuals is a polymorphism. The allele, or combination of alleles at however many loci were examined, determined for a biological sample constitutes its DNA type.
In most cases the purpose of a forensic DNA test is to investigate the possibility that two or more biological samples originated from the same individual (or species). Identity between the DNA types of two samples, for example a hair found at a crime scene and a blood sample taken from a suspect, can be highly incriminating. Conversely, a mismatch between samples, such as a semen stain from a rape victim and a blood sample from a suspect, can be highly exculpatory. Interpretation of a match depends very much on how rare the DNA type is. If the type is common, then a match between evidence and suspect could easily occur even if the evidence came from someone other than the suspect (i.e., a random match). If the type is extremely rare, then it would be very unlikely for the evidence to match the suspect if it had come from someone else. It is often the job of a forensic scientist to estimate this random match probability of a DNA type in order to help determine the weight to be given to forensic genetic evidence during a trial. However, it is up to the judge or jury, and not the forensic scientist, to determine guilt or innocence.
A locus that is less than about 1000 base pairs (bp) in length can be easily amplified (many copies made) using a method known as the polymerase chain reaction (PCR) (for more details on PCR procedures see (Newton and Graham, 1997)). A key ingredient(s) in PCR is the primer(s). This is a short piece of DNA that anneals to the sample DNA, thereby creating a location where the "copying" can begin. Specific primers are selected by the investigator to anneal at known locations on the sample DNA. Thus, the resulting amplified PCR product will be from a known locus. In contrast, nonspecific primers anneal somewhere on the sample DNA, but the investigator has no idea where. Despite this fact, the resulting PCR product can still be used for some analyses even though one does not know what has been amplified.
The major advantages ofPCR-based techniques are that they are faster (requiring hours or days rather than weeks) and require much less sample DNA (see section on minisatel-lites) than methods without PCR (Hillis et al., 1996). Another benefit is that loci for PCR analysis have been developed that are less than about 350 bp in length, allowing the use of sample DNA that is very degraded and broken into short pieces.
Types of Loci
The science of forensic DNA typing is notable for its wealth of specialized jargon and acronyms, and this can be very confusing for the beginner. In order to clarify the manner in which forensic entomologists have adapted existing DNA methods for their own purposes, the following sections will describe the common genetic procedures and terms used in identity testing.
Human Leukocyte (or Lymphocyte) Antigen (HLA) DQa
This and some similar methods are PCR-based tests in which the loci show sequence variation (Helmuth et al., 1991). The PCR product is applied to a nylon strip with attached
probes designed to bind each of the possible alleles. A particular allele is detected by the location on the strip where binding takes place.
Satellite DNA
Satellite DNA is composed of a particular sequence repeated many times. The alleles differ from each other in the number of repeating units ("repeats"), and these alternate "head to tail" in what is called a tandem arrangement. Such loci also are called variable number tandem repeats (VNTRs). The large number of alleles shown by VNTR loci, and the fact that well established population genetics and statistical theory may be used to calculate the probability that two individuals selected at random will have the same VNTR type (Evett and Weir, 1998), has led to the great technical advances that have recently been achieved in identifying the individual source of a biological sample.
'
Minisatellites
Minisatellites have a repetitive core in which the repeat is greater than -15 bp in length and can be repeated up to 10,000 times. Classical "DNA fingerprinting" or restriction fragment length polymorphism (RFLP) is performed by detecting very long minisatellite loci, up to 10 kilobase pairs (kb) in length (Jeffreys et al., 1985) (Figure 12.1). DNA from the sample is cut on either side of the locus with restriction enzymes: molecules that recognize a specific short DNA sequence and cleave the double strand. The resulting DNA fragments are separated according to length in a polyacrylamide gel by the forces of an electric field. This is commonly termed gel electrophoresis and it operates on the principle that during a given length of time a short molecule will migrate farther through the gel than will a long molecule. To detect the fragments that can vary in length between individuals, species, or
Figure 12.1'' Principle of RFLP typing. Genomic DNA is cut by use of restriction enzymes, separated by length in an electric field, blotted to a membrane, hybridized against fluorescent or radioactive oligonucleotide probes, and made visible on an x-ray film. The left column shows the use of a multilocus probe, which will produce multiple bands within a lane. The right columns show the use of single locus probes, which result in one or two bands per lane. This is because a locus is represented by two alleles, which may be the same or different lengths. Two biological samples that originated from the same individual will show the same alleles, as shown in the left column but not the center column. A child inherits one allele from each parent.
(Adapted from Benecke, M. 1997b. Naturwissenschaften, 84:181-188.) [High resolution pic in original article -- this is a web-based version.]
populations, the DNA is transferred from the gel to a nylon membrane (a Southern blot). Probes, molecules that bind specifically to the DNA of a particular locus (and which, unlike the sample DNA, can be detected because they contain fluorescent or radioactive molecules), are then used to detect the location and, therefore, the length of the allele.
The great range in lengths of minisatellites used for RFLP makes them the most variable of forensic loci (and, therefore, the most discriminating) because so many different alleles are possible. Unfortunately, their length also means that they are destroyed when DNA is broken up by ultraviolet (UV) light, bacteria, etc., as often happens to forensic specimens. Also, because it is only the original sample DNA that is detected, a relatively large amount (at least 5 to 10 u,g) must be obtained. This means that the equivalent of up to 400 ul of a 2-day-old dried blood stain on cotton, or 200 ul of dried blood on paper, glass, or wood are necessary for just one successful RFLP test. About 50 ul of fresh ejaculate contain enough genomic DNA for one RFLP test, but postcoital swabs, a single hair, or a small drop of dried saliva will usually not allow RFLP typing due to the low amounts of DNA found therein. One exception to this limitation is minisatellite locus D1S80 which has a repeat core only -220 to 650 bp in length and which can be amplified by PCR prior to electrophoresis. This variation on VNTR analysis is sometimes called amplified fragment length polymorphism (AMPFLP).
Microsatellites
Microsatellites, also called short tandem repeats (STRs), have repeating units < 7 bp and a total length of 400 bp or less. Consequently, they can be amplified and analyzed even from very small and degraded samples. The ability to conduct PCR on a single STR system locus requires only 50 pg of template DNA, the equivalent of around five cells. In rare cases, even a single cell can be DNA typed. Many forensic laboratories now simultaneously amplify many STR loci in the same reaction, a process called multiplexing. In practice, at least 100 diploid cells with nuclei must be recovered in order to succeed in obtaining a multiplex DNA type.
The use of STRs began to gain acceptance and widespread use in 1992 (Benecke, 1997b) and have since become the method of choice for identifying individual humans. The length of STR alleles is measured using electrophoresis in a polyacrylamide gel or capillary tube (Figure 12.2). The code names commonly used for STR loci reflect their location. For example, HUMTH01 is from an intron within the human tyrosine hydroxylase gene.
Randomly Amplified Polymorphic DNA
Most DNA typing applications were developed for the specific detection of human DNA and, therefore, only a few VNTRs of invertebrate DNA are known. This limitation can be overcome with randomly amplified polymorphic DNA (RAPD), a technique that can be used on virtually any organism. RAPDs use nonspecific primers that can amplify many regions of a sample DNA at once. The resulting PCR products are separated by electrophoresis, and a "band" or "peak" of a particular length can be considered a locus even though the investigator doesn't know what portion of the sample DNA it represents (Figure 12.2). RAPDs allow the amplification of up to 100 loci in one PCR. The high number of amplified RAPD loci can make it difficult to sort out informative PCR polymorphisms from noninformative ones. Therefore, to detect and analyze the highest possible number of RAPD PCR products (and their length), a semiautomated electrophoresis unit that is directly coupled with a fragment size analysis software should be used (Benecke, 1998;
Figure 12.2 Principle of STR typing. Here, three STR loci were amplified and separated together in one run. DNA fragments are separated by size by electrophoresis as in Figure 12.1. In this case the analytical instrument has recorded the intensity of fluorescence of labeled DNA as a line. A peak corresponds to a band on the electrophoresis gel. An allele mixture (all possible alleles, "cocktail," top row) is compared to the alleles actually found in a specimen (bottom row). Once the alleles of an unknown sample are determined, they can be compared to other specimens and stored in databases. (Adapted from Benecke, M. 1997b. Naturwissenschaften, 84:181-188.) [High resolution pic in original article -- this is a web-based version.]
Moscetti et al., 1995). Although there are several reports that RAPDs are highly sensitive to small changes in experimental conditions (Albornoz and Dominguez, 1998), RAPD results can be exactly reproduced if stringent laboratory conditions are maintained (Benecke, 1998).
Mitochondrial DNA
All of the previously described techniques utilize nuclear DNA (nuDNA), which comes from the cell's nucleus. Another source of DNA in a cell is the mitochondrion (pi. mito-chondria). Mitochondria are the cellular organelles that are the site for the production of adenosine triphosphate (ATP), the molecule that provides the energy needed for many metabolic processes. Several lines of evidence support the theory that all mitochondria are descended from a free-living bacterium that formed a symbiotic relationship with a eukary-otic (nucleus-containing) cell in the distant past (Gray et al., 1999).
Although mitochondrial DNA (mtDNA) analysis is a relatively new aspect of forensic science (see reviews by Butler and Levin, 1998; Holland and Parsons, 1999), the techniques now used by forensic scientists are already a routine part of fields such as medicine (Wallace, 1999) and systematics (Hillis et al., 1996). Forensic scientists typically turn to mtDNA for:
(1) identification of an individual when the recovered specimen contains too little useful DNA for nuDNA analysis (e.g., a hair shaft or an old bone), (2) identification of remains using a maternal relative as a reference (see discussion of inheritance patterns below), and (3) identification of species.
Like a cell nucleus, a mitochondrion contains DNA that is copied and passed down through the generations. Mitochondrial DNA contains the genes for some of the protein and ribonucleic acid (RNA) molecules needed for mitochondrial function. Mitochondrial DNA differs from nuclear DNA (nuDNA) in several ways that determine the relative strengths and weaknesses of each as a forensic DNA marker. Although there are exceptions to almost all of the generalizations that follow, they are not likely to be encountered by a forensic scientist.
Nuclear DNA is diploid. There are two copies of each locus, one inherited from the mother and one from the father. Recombination, or the "shuffling" of nuclear loci between chromosomes during gamete formation, makes it very difficult to trace genealogical relationships across more than one generation. Mitochondrial DNA is haploid. There is usually only one version (sequence) in an organism that can be detected, and inheritance is strictly maternal. Because there is no genetic recombination, an organism has the same mtDNA haplotype as other members of its maternal line, e.g., its mother, its great grandmother, or its siblings.
The total amount of genetic information in mtDNA is much smaller than that of nuDNA. The typical animal (including human) mtDNA molecule is between 15 and 20 kb in length, while, for example, human nuDNA contains about 3 billion bp (Avise, 1994). Thus, a typing system based on nuDNA has the potential to be much more discriminating than one based on mtDNA. With the exception of identical twins, each human has unique nuDNA, while this may not be the case for mtDNA. On the other hand, while there are only two copies of each nuDNA molecule per cell, there are often hundreds or thousands of copies of each mtDNA molecule (Holland and Parsons, 1999). This is because each mitochondrion has several copies, and there are many mitochondria in a cell. This greater abundance of mtDNA in tissues means that mtDNA can often be extracted and analyzed from very small, degraded, or otherwise poor sources of DNA that are not suitable for nuDNA analysis (Holland and Parsons, 1999).
Animal mtDNA is a circular molecule and has very little DNA that does not contain the code for making a protein or RNA molecule (Avise, 1994). The one major "noncoding" region in vertebrates is called the control region (because it contains one site where DNA replication originates) or D-loop (because in electron micrographs the two DNA strands at this location are found to be "displaced" into a loop as part of the process of replication (Holland and Parsons, 1999). Invertebrates have a similar noncoding region usually called A+T-rich because of the high proportion of adenine and thymine bases (Zhang and Hewitt, 1997). The A+T-rich region is also thought to include the origin site for replication, although this function is less understood compared to the vertebrate D-loop.
In most cases mtDNA analysis involves the comparison of the mtDNA sequences of two or more specimens. The procedure for determining the sequence usually involves some version of the Sanger reaction (Hillis et al., 1996). Investigators do not typically sequence the entire mtDNA molecule, but instead they examine a region that shows variation that is appropriate for their purposes. D-loop sequences are used to distinguish individual humans (Holland et al., 1995) and also may be used to separate very closely related species, while protein-coding genes are most often used to recognize species. Although there is little evidence that any of the protein-coding genes are (for a given number of base pairs) a more effective marker than any of the others (Caterino et al., 2000), the need to compare new results with previous work has led to a concentration of studies on just a few regions. For vertebrate species, this is the cytochrome b gene (Cyt b) (Johns and Avise, 1998), whose forensic utility has been clearly recognized (Barallon, 1998; Zehner et al., 1998). For insects it is most common to sequence some or all of cytochrome oxidase subunits one and two (COI+II), although this is not the overwhelming choice that Cyt b is for those who study vertebrates (Caterino et al., 2000; Simon et al., 1994). Once sequence data are obtained from an unknown specimen, they can be compared to a huge number of identified sequences by performing a BLAST search of the GenBank database.
Figure 12.3 RAPD pattern (peak view) from a case in which the question was asked if a pupa found outside of a body bag could be connected to maggots found inside the body bag. (A-D) four individual Lucilia sericata (Meigen) maggots from the inside of the body bag, (E) pupa found near the corpse, (F) Calliphora erythrocephala (Meigen) (= C. vicina Robineau-Desvoidy) from another case, and (G) Carrion beetle (Oiceoptoma thoracicium} from another case. Above a certain threshold, the peaks of (A-D) show a high similarity. Due to its different DNA profile, the pupa is unlikely to belong to the same species as (A-D). DNA profiles of (F) + (G) differ from all others. (Modified from Benecke, M., Foren. Sci. Int., 98:157-168.) [High resolution pic in original article -- this is a web-based version.]
'''Forensic Entomology Applications'''
There are few published reports on the use of DNA techniques by forensic entomologists. However, there are several ongoing research programs that will soon expand the available applications. Most efforts have been directed at improving our ability to identify the insect specimens. It is also possible to identify the gut contents of blood or carrion feeding arthropods and, thereby, associate an insect with a living or dead human even when contact between the two is not observed.
Nuclear DNA
As previously described, nuDNA was used earlier than mtDNA for forensic genetic testing, and this also has been the situation for forensic entomological applications. Apart from numerous applications for species identification (see overview in Benecke, 1998), Replogle, et al. (1994) demonstrated that human DNA from dried crab louse, Pthirus pubis (L.), feces could be successfully typed using AMPFLP. The excreta were obtained from adult crab lice that fed on human volunteers, and the alleles of the human specific loci D1S80 and HUMTH01 in the insect feces matched those of the volunteers. Victims of sexual assault may acquire lice that have fed on their attacker, and it is now possible to link louse and human host in such situations. Benecke (1998) used RAPD profiles to identify a puparium collected during a death investigation (Figure 12.3). In this case investigators needed a PMI estimate right away, and the puparium could not be identified by conventional means.
Figure 12.5 UPGMA phylogram (Hillis et al., 1996) of Chiysomya blow fly specimens described in Wells and Sperling (1999). Numbers on each branch indicate genetic distance, i.e., there is 1.1 % sequence divergence within this part of the mtDNA COII gene between C. albiceps(Wiedemann) and C. rufifacies (Macquart) from a variety of geographic locations. These two species are difficult and sometimes impossible to distinguish for all life stages using morphological criteria. [High resolution pic in original article -- this is a web-based version.]
Mitochondrial DNA
One of the potentially greatest benefits mtDNA offers the forensic entomologist is in species determination. The eggs or larvae of many forensically important dipteran species are particularly difficult to distinguish morphologically (Benecke and Seifert, 1999; Greenberg and Singh, 1995; Liu and Greenberg, 1989), and an incorrect or uncertain identification can seriously harm or impede an investigation. This is because adult arrival times, egg duration, and larval growth rates can vary dramatically between species. Proper species identification is usually an essential first step in the use of entomological evidence in a legal investigation.
Fortunately, the chance of an improper identification on morphologically similar species is likely to be diminished in the very near future. The protocols for analyzing calliphorid mtDNA have been extensively demonstrated from experiments done on species of veterinary importance (Azeredo-Espin and Madeira, 1996; Gleeson and Sarre, 1997;Narang and Degrugillier, 1995; Roehrdanz and Johnson, 1996; Stevens and Wall, 1997; Taylor et al., 1996; Valle and Azeredo-Espin, 1995). In terms of both size and structure, the mtDNA of a calliphorid fly closely resembles that of Drosophila yakuba (Buria) (Diptera: Drosophilidae), the first fly species for which the entire mtDNA sequence was described (Clary and Wolstenholrne, 1985). Therefore, there is a great deal of basic biological information available concerning fly mtDNA, which makes it easier to design PCR primers and to interpret the results of any study on a new fly species.
Sperling et al. (1994) were the first to demonstrate how mtDNA sequence data from (easy to identify) adult specimens of forensically important flies could be used to identify immature forms of the same species (Figure 12.4^). Using the same techniques, Wells and Sperling (1999) found that calliphorid species that can be difficult to separate taxonomi-cally even in the adult stage have distinctly different mtDNA (Figure 12.5). (See color section insert following page 78.)
This author (J. D. Wells), in collaboration with Felix Sperling, has now obtained COI+II sequence data for more than 20 species of carrion flies found in North America, and these data will soon be publicly available. In almost all cases, closely related species can be separated using a short (-300 bp) region such as can be obtained from even very degraded DNA (see Figure 12.4 in color insert). Because there is often some intraspecific variation in mtDNA haplotypes, two samples from the same species may not match exactly. Only experience with the taxonomic group in question will allow an investigator to know if the differences observed between two samples fall within the range of normal variation for that species. Even if identification is uncertain, phylogenetic analysis can be used to reveal the specimen's closest relative(s) and narrow the choice of species to which it belongs.
Sequence data also can be used to design a restriction fragment length polymorphism test for PCR product (PCR-RFLP) (Sperling et al., 1994) (Figure 12.6). This is a faster and less expensive method than DNA sequencing. However, we believe that large samples will be needed to validate PCR-RFLP for this purpose. A single point mutation can eliminate a restriction site, so a reliable PCR-RFLP test must utilize restriction sites that are fixed or nearly fixed for a given species.
Figure 12.6 Restriction site maps for the 2.3 kb COI+II region of mtDNA for three closely related blow flies. Sequence data were searched using the computer program GeneJockeyII (Taylor, 1993) for sites that will be cleaved by a particular restriction enzyme. [High resolution pic in original article -- this is a web-based version.]
As described in the previous section, insect specimens can be a source of noninsect DNA, that of the organism upon which they have fed. Lord et al. (1998) typed human mtDNA from the gut contents of a crab louse. Similar results using the gut contents of blow fly maggots have been produced (Introna et al., 1999). Such analyses may prove to be crucial evidence in the creation of victim/suspect associations. Investigators that find maggots but no body, now have the potential to identify the insect's last meal. There also are occasions, particularly if the scene has been disturbed, where both maggots and a corpse are present but not in physical contact. DNA analysis of maggot gut contents provides an independent means for associating larva and victim. This ability also may prove invaluable in cases of multiple homicides or mass burials.
Comments
Just as with other aspects of a forensic entomological investigation, it is difficult to predict in advance which DNA methods will be most useful for investigators. Instead we need to develop and evaluate as many techniques as possible, and to make such information widely available. In addition to the methods described, new techniques are constantly appearing in one professional field and quickly spreading to others.
It is essential that DNA samples be obtained from insects likely to be encountered during forensic investigations all over the world. We strongly encourage all practicing forensic entomologists to expand their normal specimen collection techniques (Benecke, 1997a) to include freezing or preserving in 95% ethanol, and to either undertake molecular genetic studies themselves or to make this material available to others willing to characterize the DNA for identification purposes.
Acknowledgments
We thank Felix Sperling (University of California at Berkeley) for providing a manuscript in press. The work of J. D. Wells was supported in part by a grant from the Pathology/Biology Section of the American Academy of Forensic Sciences, and by grants from the National Institute of Justice to J. D. Wells (1999-1 J-CX-0034) and to Dr. Sperling (97-IJ-CX-0035). The views expressed by the authors are not necessarily those of the U.S. Department of Justice.
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