Nonhuman Dna Testing And Microbial Forensics

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We urge as rapid development of new systems as is consistent with their validation before they are put into general use.

One's ideas must be as broad as Nature if they are to interpret Nature.

(Sherlock Holmes, A Study in Scarlet)

While the vast majority of forensic DNA typing performed for criminal investigations involves human DNA, it is not the only source of DNA that may be useful in demonstrating the guilt or innocence of an individual suspected of a crime (Sensabaugh and Kaye 1998). Domestic animals such as cats and dogs live in human habitats and deposit hair that may be used to place a suspect at the crime scene. Demonstration that a botanical specimen came from a particular plant can aid the linkage of a crime to a suspect or help demonstrate that the body of a deceased victim may have been moved from the murder site. DNA testing can now be used to link sources of marijuana. A large area of future application for forensic DNA typing involves identification of bio-terrorism materials such as anthrax. This chapter will briefly discuss each of these topics and the value of non-human DNA testing in forensic casework.


The American Pet Products Manufacturers Association reported in April 2003 that over 64 million U.S. households own a pet (see Their survey found 77.7 million cats and 65 million dogs in these households, which make up at least one-third of all U.S. residences. Since many of these domestic animals shed hair, these hairs could be picked up or left behind at the scene of a crime by a perpetrator. An assailant may unknowingly carry clinging cat hairs from a victim's cat away from the scene of a crime, or hair from the perpetrator's cat may be left at the scene.

The Veterinary Genetics Laboratory at the University of California-Davis (see has been performing forensic animal

DNA analyses since 1996. They have found that there are three types of animal DNA evidence: (1) the animal as victim, (2) the animal as perpetrator, and (3) the animal as witness.

Animal abuse cases or the theft of an animal can sometimes be benefited by the power of DNA testing. The remains of a lost pet can be positively identified through genetic analysis. Typically genetic markers like short tandem repeats (STRs) and mitochondrial DNA (mtDNA) are examined in much the same way as with human DNA.

When animals are involved in an attack on a person, DNA typing may be used to identify the animal perpetrator (e.g., a Pit Bull). If the victim is deceased, then DNA evidence may be the only witness that an animal in custody committed the crime. Animal DNA testing can 'exonerate' innocent animals so that they are not needlessly destroyed.

Animal DNA has been used successfully to link suspects to crime scenes (see D.N.A. Box 11.1). A study on the transfer of animal hair during simulated criminal behavior found that hundreds of cat hairs or dog hairs could be transferred from the homes of victims to a burglar or an aggressor (D'Andrea et al. 1998). In fact, the number of hairs found was so high that the authors of this study felt that it is almost impossible to enter a house where a domestic animal lives without being 'contaminated' by cat and/or dog hairs even when the owner describes his or her animal as a poor source of hair (D'Andrea et al. 1998). Due to the fact that shed hairs often do not contain roots, nuclear DNA may not be present in sufficient quantities for STR typing. Mitochondrial DNA may be a more viable alternative for many of these types of shed hair transfers.

Snowball's DNA

The identity of white cat hairs found on a bloodstained leather jacket left at a murder scene became a turning point in the case of Douglas Leo Beamish versus Her Majesty The Queen in the Providence of Prince Edward Island, Canada. The victim, Shirley Duguay, was discovered in a shallow grave in a wooded area eight months after she disappeared. Beamish, her former common law husband was charged with the crime. At the time he lived with his parents and a white cat named Snowball. Laboratory analysis of the bloodstains on the recovered jacket contained the victim's DNA profile. The white cat hairs matched Snowball at 10 STR loci. The defendant was convicted of murder based in part on this evidence.


Menotti-Raymond, M., et al. (1997) Pet cat hair implicates murder suspect. Nature, 386, 774.


Cats have 18 pairs of autosomes and the sex chromosomes X and Y and genetic markers have been developed on each of the Felis catus chromosomes (Menotti-Raymond et al. 1999). A panel of STR markers dubbed the 'MeowPlex' has been developed that contains 11 STRs on nine different autosomes (Butler et al. 2002). A gender identification marker was also included in this assay through the addition of PCR primers that are specific for the SRY gene on the cat Y chromosome. The PCR products for this 12plex amplification fall in the size range of 100 bp to 400 bp and use three dye colors (Figure 11.1).

Feline STR allele frequencies from domestic cats have been published (Menotti-Raymond et al. 1997) for the purpose of demonstrating uniqueness of DNA profiles in forensic investigations, such as used in the Beamish case (D.N.A. Box 11.1). Population studies on over 1200 cats from 37 different breeds have been conducted by the Laboratory of Genomic Diversity at the National Cancer Institute-Frederick Cancer Research and Development Center in Frederick, Maryland. In an initial study of 223 cats from 28 different breeds, the MeowPlex exhibited an average composite locus heterozygosity of 0.73 across the breeds (Menotti-Raymond et al. 2003). The power of discrimination with this 11plex feline STR multiplex ranged from 5.5 X 10-7 to 3.3 X 10-13 across the various breeds.

A real-time quantitative polymerase chain reaction (PCR) assay (see Chapter 4) for estimating the DNA yield extracted from domestic cat specimens has been developed (Menotti-Raymond et al. 2003). This assay is capable of detecting down to 10 femtograms of feline genomic DNA and uses high-copy number short interspersed nuclear elements (SINEs) similar to the Alu repeats described in Chapter 8. Feline STRs and mtDNA testing is performed by Joy Halverson of QuestGen Forensics (, which also does canine STR and mtDNA testing to aid forensic investigations.

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Figure 11.1 DNA profiles produced from male (top panel) and female (bottom panel) cat DNA using a multiplex STR typing assay dubbed the 'MeowPlex' (Butler et al. 2002). This test examines 11 autosomal STRs and a region of the SRYgene contained on the Y chromosome that can be used for sex determination.


While cat DNA testing may be involved in situations where the animal hair acts as a silent witness to connecting a perpetrator to a crime scene, evidence from dogs is more frequently linked to situations where the animal is the perpetrator. Rottweilers, German Shepherds, Doberman Pinchers, and Pit Bulls can be trained as security animals and may attack, injure, or even kill people.

Canine mitochondrial DNA possesses two hypervariable regions (HV1 and HV2) similar to the human mtDNA described in Chapter 10. Savolainen et al. (1997) found 19 sequence variants across a 257 bp segment of the hypervariable region 1 of mtDNA control region in 102 domestic dogs of 52 different breeds. They concluded that on average 88 out of 100 tested animals could be excluded with this mtDNA sequence analysis. Another study that used a larger portion of the dog mtDNA control region found the overall exclusion capacity to be 0.93 in 105 dogs tested. By way of comparison in 100 British white Caucasians an exclusion capacity of 0.97 was observed (Piercy et al. 1993). Therefore, domesticated dog mtDNA is not as variable as human mtDNA yet it can still provide helpful clues in forensic cases (Savolainen and Lundeberg 1999, Schneider et al. 1999).

A number of STR markers have been mapped and characterized on the 38 pairs of autosomes and the X chromosome of Canis familiaris, the domestic dog (Neff et al. 1999). Recently, 15 canine STR loci have been characterized with sequenced alleles to define nomenclature for future work (Eichmann et al. 2004). A set of 10 dinucleotide repeat STRs has been used to aid investigations in illegal animal deaths (Padar et al. 2001) and a dog attack that resulted in the death of a seven-year-old boy (Padar et al. 2002). In addition, DNA profiling of human blood recovered from a dog's fur can associate or exonerate the animal from connection to an attack (Brauner et al. 2001).


The remains of stolen animals or illegally procured meat (e.g., endangered species or poaching) can be identified through DNA testing (Giovambattista et al. 2001, Poetsch et al. 2001). The U.S. Fish and Wildlife Service have a forensic laboratory in Ashland, Oregon that does some species identification using DNA (see Sequence analysis of the mtDNA cytochrome b gene is effective at identifying the species of origin for a biological sample (Bartlett and Davidson 1992, Parson et al. 2000, Hsieh et al. 2001, Branicki et al. 2003). Bataille et al. (1999) developed a multiplex amplification of a portion of the human mtDNA control and the cytochrome b gene to enable simultaneous human and species identification.


In the area of plant DNA testing, there are primarily two areas being investigated currently. The first is the linking of plant material to suspects or victims in order to make an association with a particular area where a crime was committed. The second is in linking marijuana to aid in forensic drug investigations. A review of some of the applications of forensic botany was published recently (Miller-Coyle et al. 2001).


Crimes often occur in localized areas containing a unique combination of botanical growth. If these plants, algae, or grass are sufficiently rare, then recovery of trace evidence from the clothing of a victim or the personal property of a suspect may be helpful in making an association that can link them to a crime scene (Szibor et al. 1998, Norris and Bock 2000, Horrocks and Walsh 2001).

Although it is not yet used routinely (Bock and Norris 1997), non-human DNA has helped link suspects to crime scenes and aided important investigations. In the first use of forensic botanical evidence, two small seedpods from an Arizona Palo Verde tree found in the back of pick-up truck were used to place an accused murderer at the crime scene (Yoon 1993). Genetic testing on the seeds showed that in a 'lineup' of 12 Palo Verde trees near the crime scene, DNA from the seeds matched only the tree under which the victim's body had been found. In State versus Bogan, the jury found the accused guilty based in large measure on the plant DNA evidence.


The Connecticut State Forensic Science Laboratory has developed a sensitive DNA test for Cannabis sativa (marijuana) because it is an illegal substance associated with many crime scenes. In fact, marijuana is the most commonly identified drug tested by U.S. forensic laboratories in criminal investigations (see Marijuana DNA testing can link an individual to a sample, link growers, and help track distribution networks (Miller-Coyle et al. 2001). However, it is important to keep in mind that if the marijuana plants were propagated clonally rather than by seed, then they will have identical DNA profiles. Clonal propagation in marijuana is performed by taking cuttings from a 'mother' plant and rooting them directly in the soil to create large numbers of plants having identical DNA (Miller-Coyle et al. 2001).

Efficient extraction protocols have been developed that yield 125-500 ng of DNA per 100 mg of fresh plant tissue (Miller-Coyle et al. 2003a). DNA testing of marijuana as with other plants has traditionally been performed with one of three methodologies: randomly amplified polymorphic DNA markers (RAPDs), amplified fragment length polymorphisms (AFLPs) or short tandem repeats (STRs). These techniques and their specific application to marijuana DNA typing have been reviewed (Miller-Coyle et al. 2003b).

Randomly amplified polymorphic DNA marker analysis utilizes short PCR primers consisting of random sequences usually in the size range of 8-15 nucleotides in length. Complex patterns of PCR products are generated as these random sequence primers anneal to various regions in an organism's genome. RAPD suffers from poor reproducibility between laboratories largely because of the requirement of consistent PCR amplification conditions including thermal cycler ramp speeds. The complex patterns of RAPD also prevent mixture interpretation and provide challenges in consistent scoring of elec-trophoretic images even in single source samples.

Patterns from amplified fragment length polymorphism markers can be generated with greater reproducibility compared to RAPDs. AFLPs are generated by first cutting a double-stranded DNA sample with one or more restriction enzymes (Vos et al. 1995, Ranamukhaarachchi et al. 2000). Specific 'adaptor' sequences are then ligated to the restriction cut sites. PCR primers that recognize these ligated adaptor sequences are used to amplify different sized DNA fragments that can then be separated using electrophoresis. The final result is a complex series of peaks usually in the 50-400 bp size range that can be scored with computer software and compared with other AFLP patterns from different marijuana plants. Even highly inbred individual plants can be distinguished by their AFLP patterns (Miller-Coyle et al. 2003b).

Several STR markers have been recently reported for Cannabis sativa (Hsieh et al. 2003, Gilmore et al. 2003, Alghanim and Almirall 2003). As with human STRs, marijuana STR markers are highly polymorphic, specific to unique sites in the genome, and capable of deciphering mixtures. A hexanucleotide repeat marker showed repeat units ranging from 3-40 in 108 tested marijuana samples, and primers amplifying this locus produced no cross-reactive amplicons from 20 other species of plants tested (Hsieh et al. 2003).

All of these molecular techniques for identifying marijuana plants need comparative databases to be effective tools for law enforcement purposes. In order to determine the possibility of a random match with marijuana seizure samples, it is important to have a database of seizure samples so their DNA profiles can be used for comparison (Miller-Coyle et al. 2003). More information regarding on-going research in the field of forensic botany and its application may be found at and


Unfortunately microbial forensics will likely become a larger part of DNA testing in the future with the threat of terrorism and the use of biological warfare agents. Microbial evidence can be from either real terrorist events or hoaxes. The efforts in this area will likely require forensic laboratories to build strong collaborations with academia, private sector and national laboratories. Important requirements of bio-threat detection assays are high sensitivity, high specificity in complex samples, fast measurement, compact design for portability and field use, and internal calibration and reference to ensure reliable results (Ivnitski et al. 2003).

In October 2001 a bio-terrorism attack impacted the United States as government offices and media outlets received anthrax-laden letters sent anonymously through the postal service. This attack resulted in 22 anthrax cases and five deaths. In addition, many people were afraid to open their mail for months afterwards. More than 125 000 samples were processed as part of this case in the two years following this attack and yet no one has been charged with the crime to date (Popovic and Glass 2003).

Several challenges arise when trying to gather evidence, identify the biocrime organism(s), and trace the source of the organism(s). First responders to crime scenes where biological weapons have been dispersed have to be concerned about their own safety and the safety of others while maintaining chain of custody of any evidence collected from the crime scene, all the while trying to prevent contamination of the evidence and the environment. Databases need to be established for intrinsic background species and bio-threat strains. Reliable reference material is needed for comparison purposes. Proficiency and validation testing are necessary to estimate false-positive and false-negative rates (Kiem 2003).

The U.S. efforts in building a response to bio-terrorism have been announced in a policy paper (Budowle et al. 2003). The FBI has initiated a Scientific Working Group on Microbial Genetics and Forensics (SWGMGF) that will help develop guidelines related to the operation of microbial forensics (SWGMGF 2003). Currently there are an insufficient number of validated analytical tools to characterize and identify biological agents that might be used in a terrorist attack (Budowle 2003). Research efforts will continue to be made in this area.

Comparative genome sequencing promises to be a powerful tool for investigating infectious disease outbreaks as was performed with the whole-genome sequencing of Bacillus anthracis (anthrax) (Read et al. 2002a, 2002b). Phylogenetic analyses of viral strains of HIV have been admitted and used as evidence in court (Metzker et al. 2002). However, since bacteria and viruses reproduce asexually, clones are prevalent. A perfect match between evidence collected and a reference sample is much less definitive than with human identity testing where sexual reproduction shuffles genetic material each generation.


Sensabaugh and Kaye (1998) consider several issues regarding whether a given application with non-human DNA is ready for court use. These issues include the novelty of the application, the validity of the underlying scientific theory, the validity of any statistical interpretations, and the relevant scientific community to consult in assessing the application. Many times new methods are applied for the first time in microbial forensics or animal or plant DNA testing that have not yet undergone the scrutiny of regular forensic DNA testing techniques. Reference DNA databases for comparison purposes and use in calculating the probability of a chance match take time to develop and may not be in place prior to an investigation. Finding appropriate experts to review the scientific soundness of a novel application can also be challenging. Nevertheless, the power and influence of forensic DNA testing will continue to grow as it is used in more and more diverse applications to solve crimes that were previously inaccessible.


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  • azelio
    What primers do fbi use for dna testing?
    6 months ago

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