The Science Behind PooPrintsPowered by BioPet Laboratories
Electronic pipettes, analytical balances, reagent cold storage, etc. must pass maintenance, yearly calibration, and certification from an ISO/IEC 17025:2005 accredited outside provider. Considered the industry gold standard in medium to high throughput in genetic analysis, our Applied Biosystems/ThermoFisher 3730 DNA Analyzer is also certified yearly by an AB service engineer.
Our laboratory analyst must complete training, evaluation, and on-going proficiency testing in each category before moving on to analyze our customer samples.
These standards allow BioPet Laboratories to offer precise and reproducible DNA fragment analysis.
To begin PooPrints analysis, we extract genomic DNA and use microsatellite (STR) fragment analysis to establish genotypes of 15 loci (each with 2 possible alleles) plus a gender marker. In reference, the FBI core panel for its CODIS database consist of 13 loci plus a gender marker (1). Our 15 loci, each with hundreds of allele combinations, give us the high probability of producing a unique genetic ‘fingerprint’ from any sample type – buccal swab, feces, tissue, semen, etc.
A genotype yielded from a waste sample is simply compared to all buccal swab genotypes from that property. For a Match to be resulted, the genotypes MUST completely match on every available allele with no discrepancy. For this reason, a false positive match is extremely unlikely.
BioPet’s statistics are calculated from a population of over 15,000 individual dogs with unique genetic profiles. This allows us to accurately predict the allele frequency, genotype frequency, and thus profile frequency. For every Match, there is a probability based on the profile frequency within the population. This will determine the likelihood that another matching genotype exists in addition to the reported match. This should not be confused with the idea that another dog matches instead of the reported match.
In all of our calculations, these probabilities are upwards of 1 in 850,000,000,000 chance that another dog matches in addition to the reported match.
How does the lab differentiate samples that are contaminated?
Cells in the body contain the same nuclear DNA. The exceptions to this being the male and female gametes as well as mature red blood cells. When we extract DNA from feces, we are extracting DNA from cells. These cells are readily sloughed off during excretion as the feces moves through the intestines. Therefore, in the most basic of comparisons, DNA from cells from the intestines should be identical to DNA extracted from cells in the urine, buccal cells found in saliva, and tissue and hair cells found in fur.
Cross contamination of a fecal sample is possible in highly used green spaces. In these rare cases, we will detect at least 2 genotypes within one sample. Alleles amplify in different ways; some have lower intensity than others. Therefore we make no attempt to decipher what DNA profile is at a higher concentration. We will not discern what allele belongs to what dog, and will therefore fail the sample as contaminated. We receive thousands of waste samples each year and this occurrence is extremely rare.
For our purpose, urine is not a reliable source for nuclear DNA. Urine from healthy animals should not contain enough cells to produce a strong genetic profile using our current extraction protocols. Certain sample sources, like urine and saliva, require more concentration of the eluate to produce quality template DNA for amplification. Products are available that work to duplicate and increase the starting template DNA. We do not use such kits because they may add a nucleotide randomly in the process. Because we work with STRs, which are repeats of nucleotides, an insertion at an amplification site may alter a genotype and we are not willing to compromise our data.
I hope these explanations have addressed any questions you have about PooPrints DNA Matching Accuracy. If not, we would be more than happy to answer any more concerns and questions you may have.
Animal Waste has been used as a means of DNA collections for many years. It is one of the best methods to study animal populations in field studies as it allows for a low stress, non-invasive means of DNA collections. We have listed a few references below that use DNA retrieved from waste as key components for wildlife studies.
- Wasser S., Houston C., Koehler G., Cadd G., Fain S. (1997) Techniques for application of fecal DNA methods to field studies of ursids. Molecular Ecology 6: 1091-1097.
- Kohn M.H. and Wayne R.K. (1997) Facts from feces revisited. Tree 12: 223-227.
- Taberlet P., Waits L.P. and Lutkart G. (1999) Noninvasive genetic sampling: look before you leap. Tree 14: 323-327.
- Frantzen M.A., Silk J.B., Ferguson J.W., Wayne R.K., Kohn M.H. (1998) Empirical evaluation of preservation methods for fecal DNA. Molecular Ecology 7 (10): 1423-8. Abstract.
- Zhang, X. et al. Extraction of DNA and PCR analysis of DNA from free-ranging howling monkey (Alouatta palliata) feces. University of Pittsburgh. Abstract.
- Mowat, G. et al. (1999) Using genetic tagging to estimate animal population parameters.