Less is More--Optimization of DNA Extraction from Canine Feces.

Although most DNA crime laboratories may not encounter fecal samples often, they are a familiar sample type in non-human forensic laboratories due to their prevalence in the environment. Fecal matter can be challenging due to low numbers of nucleated cells and the presence of inhibitors that impede amplification success. Sampling location (internal vs. external), sampling quantity (10-200 mg), and various extraction protocols (silica matrix, bead beating, and clean-up column) were evaluated to maximize DNA yield. The greatest yield of intact DNA was obtained using a modified silica matrix extraction protocol (VGL-Fecal) on 30-50 mg of fecal matter collected from the external surface of a stool that had been dried for 24 h. This optimized sampling and extraction protocol was applied to a pilot study where DNA yield and genotyping success were evaluated. By optimizing our collection, sampling, and extraction procedures, a reliable method for maximizing the yield of canine fecal DNA was developed.

Mitochondrial DNA sequence heteroplasmy levels in domestic dog hair.

To assess the level of mtDNA sequence heteroplasmy in dog hair, we sequenced a 612 base pair fragment of the hypervariable region 1 (HVI) in 576 hairs from six dogs representing a range of age, sex, breed, and hair color. Blood and buccal samples were collected from each dog for reference. Three instances of sequence heteroplasmy were observed at nucleotide positions 15627 (G/A), 15628 (T/C) and 15639 (G/A) in two hairs from different dogs. An HVI sequence heteroplasmy frequency of 0.0034 was obtained. The Probability of Identity (PI) value, or probability that two random, unrelated dog hairs share an HVI sequence, and the Power of Discrimination (PD), or probability that two random unrelated dog hairs have different HVI sequences, were determined from the 88 HVI haplotypes represented in the Veterinary Genetics Laboratory database (n=1006) and found to be 0.086 and 0.914, respectively.

Developmental validation of Mini-DogFiler for degraded canine DNA.

Dogs (Canis lupus familiaris) are kept as pets in 39% of American households and are, therefore, a significant source of potentially probative biological evidence. As with any biological evidence, degradation can occur as a consequence of environmental exposure causing fracturing of the DNA and a resulting loss of intact template. Degraded human DNA analysis has benefited from the application of primer sets that amplify shorter nuclear sequences for core STR loci (miniSTRs), resulting in improved DNA profiles. This same approach was applied to our core canine STR loci. The 16-locus "DogFiler" panel was redesigned into three panels of miniSTRs for analysis of degraded canine DNA, with all primer pairs producing amplicons below 205 base pairs in length. These new miniSTR marker panels - known as Mini-DogFiler - were validated according to SWGDAM guidelines, and concordance with the original 16-locus multiplex was demonstrated through genotyping 1244 samples. The combination of these miniSTRs and a half-volume reaction increased the amplification success of degraded and low copy number canine biological samples resulting in a near three-fold increase in reportable alleles. This assemblage of miniSTRs along with the DogFiler panel and associated allelic ladder are the first non-human DNA profiling system to parallel the human forensic paradigm.

Identification of single nucleotide polymorphisms within the mtDNA genome of the domestic dog to discriminate individuals with common HVI haplotypes.

We sequenced the entire ∼16 kb canine mitochondrial genome (mtGenome) of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. We then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. Haplotype frequencies in our data set were highly correlated with previous ones (e.g., F(ST)=0.02, r=0.90), suggesting the total data set reasonably reflected the broader dog population. A total of 128 HVI haplotypes was represented. The 10 most common HVI haplotypes (n=184 dogs) represented 53.3% of the sample. We identified a total 71 SNPs in the mtGenomes (external to the control region) that resolved the 10 most common HVI haplotypes into 63 mtGenome subhaplotypes. The random match probability of the dataset based solely on the HVI sequence was 4%, whereas the random match probability of the mtGenome subhaplotypes was <1%. Thus, the panel of 71 SNPs identified in this study represents a useful forensic tool to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).

Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays.

Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA that acts as an internal PCR control for detecting inhibition. Developmental validations were carried out following the revised guidelines of the Scientific Working Group on DNA Analysis Methods with modifications necessary for validation of nonhuman qPCR assays. All assays demonstrated the specificity, sensitivity, stability, reproducibility, accuracy, and precision required for forensic casework. The application of these assays to animal forensic DNA analysis has both conserved laboratory resources and improved genotyping results.

Real-time polymerase chain reaction quantification of canine DNA.

The accurate quantification of target DNA is an important step in the short tandem repeat analysis of forensic biological samples. By utilizing quantification data to control the amount of template DNA in the polymerase chain reaction (PCR), forensic scientists can optimize testing and minimize the consumption of limited samples. The ability to identify and quantify target DNA in mixed-species samples is crucial when it may be overwhelmed by nontarget DNA, as in cases of dog attack. We evaluated two quantitative real-time PCR assays for dynamic range, species specificity, and inhibition by humic acid. While both assays proved to be highly sensitive and discriminating, the Melanocortin-1 Receptor (MC1R) gene Taqman assay had the advantages of a shorter run time, greater efficiency, and safer reagents. In its application to forensic casework, the MC1R assay has been advantageous for quantifying dog DNA in a variety of mixed-species samples and facilitating the successful profiling of individual dogs.