Validation of a short tandem repeat multiplex typing system for genetic individualization of domestic cat samples

Aim

To conduct developmental validation studies on a polymerase chain reaction (PCR) based short tandem repeat (STR) multiplex typing system, developed for the purpose of genetic individualization and parentage testing in domestic cat samples.

Methods

To evaluate reproducibility of the typing system, the multiplex was amplified using DNA extracted from hair, blood, and buccal samples obtained from the same individual (n = 13). Additional studies were performed to evaluate the system’s species’ specificity, using 26 North American mammalian species and two prokaryotes Sacchromyces and Escherichia coli, sensitivity, and ability to identify DNA mixtures. Patterns of Mendelian inheritance and mutation rates for the 11 loci were directly examined in a large multi-generation domestic cat pedigree (n = 263).

Results

Our studies confirm that the multiplex system was species-specific for feline DNA and amplified robustly with as little as 125 picograms of genomic template DNA, demonstrating good product balance. The multiplex generated all components of a two DNA mixture when the minor component was one tenth of the major component at a threshold of 50 relative fluorescence units. The multiplex was reproducible in multiple tissue types of the same individual. Mutation rates for the 11 STR were within the range of sex averaged rates observed for Combined DNA Index System (CODIS) loci.

Conclusion

The cat STR multiplex typing system is a robust and reliable tool for the use of forensic DNA analysis of domestic cat samples.In the field of forensic DNA analysis, new methods and technology have revolutionized the analysis and detection of genetic variation for human identification (1-3). These advances have been extended to the analysis of DNA extracted from non-human specimens such as plants, bacteria, viruses, and domesticated animals (4). Genetic individualization of animal specimens has increasingly been included as key evidence in criminal investigations (5-11). It has been reported that pet hairs are invariably transferred to the clothing of those visiting the home of a pet owner (12). With approximately 73 million cats residing in one third of households in the United States (13), it is not surprising that cat hairs are often part of the physical evidence associated with crime scenes.Recently, a polymerase chain reaction (PCR) based short tandem repeat (STR) multiplex typing system has been developed for the use in genetic individualization and parentage testing of domestic cat specimens (14). The system simultaneously amplifies 11 polymorphic tetranucleotide STR loci and one gender identifying sequence tagged site on the Y chromosome Sex-Determining Region Y gene (SRY gene). The STR markers have been mapped in radiation hybrid and/or genetic linkage maps of the domestic cat (15,16) and were selected for forensic analysis as they are unlinked, amplify robustly, exhibit Mendelian inheritance, and exhibit a high degree of heterozygosity in cat breeds (14).Following the standard 8.1.2.2 of the quality assurance standards for DNA analysis recommended by the DNA Advisory Board (DAB) (17) and recommendations made for animal DNA forensic and identity testing (18), developmental validation studies were conducted on the cat STR multiplex typing system. These validation studies are required prior to the application of a new genetic forensic typing system to analysis of evidentiary samples, to ensure the accuracy, precision, and reproducibility of the system.