Genotyping horse epithelial cells from fecal matter by isolation of polymerase chain reaction products.
Abstract: To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands were captured on streptavidin-coated plates, where everything but the dye-labeled target sequence was washed, eliminating all the background noise, released, and run on a genotyping instrument in a single-strand configuration. Results: The IPCRp-based multiplex kit (6 loci) revealed equine DNA full genotype profiles, ie, appearance of all six loci, when sampled from fresh feces in 87% of the samples and partial genotype profile (appearance of one to five loci) in 13% of the samples, for a total of 100% genotyping success rate. Conclusions: These results indicate that the IPCRp amplification method, coupled with the Qiagen DNA Stool Mini Kit extraction can maximize the likelihood of obtaining horse DNA genotypes from fecal samples.
Publication Date: 2017-06-15 PubMed ID: 28613041PubMed Central: PMC5470126DOI: 10.3325/cmj.2017.58.239Google Scholar: Lookup
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- Journal Article
Summary
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The research article focuses on a modified version of the Polymerase Chain Reaction (PCR) method known as Isolation of Polymerase Chain Reaction Products (IPCRp) that effectively improves the accuracy and reliability of obtaining genotypes from horse fecal DNA in comparison to earlier methodologies.
Methodology
- The researchers extracted DNA from horse fecal matter using a modified protocol of the Qiagen DNA Stool Mini Kit.
- After the DNA extraction, genotypes were obtained from the fecal samples using IPCRp, an advanced version of the PCR amplification method.
- The biotin-labelled strands amplified by the IPCRp-based multiplex kit were captured on streptavidin-coated plates.
- The coating process allowed for the elimination of everything but the dye-labeled target sequence by effectively washing away all the background noise, once all the non-target elements were removed, the target sequence was then released.
- Finally, the clean, isolated target sequence was then run on a genotyping instrument in a single-strand configuration.
Results
- The IPCRp-based multiplex kit (consisting of 6 loci) was able to extract full genotype profiles of equine DNA when sampled from fresh feces in 87% of the cases – meaning that all six loci appeared as expected.
- In the remaining 13% of samples, partial genotype profiles were achieved, with the appearance of one to five loci, but not all six loci.
- The overall result was a 100% success rate in genotyping based on the appearance of at least some loci.
Conclusion
- The research concludes that the IPCRp amplification method, combined with the Qiagen DNA Stool Mini Kit extraction technique, optimizes the chances of successfully obtaining accurate horse DNA genotypes from fecal samples.
- The success of this research lays the groundwork for further investigation into genetic analysis from fecal samples, not only in equine species but potentially across a wider range of animals.
Cite This Article
APA
Dimsoski P.
(2017).
Genotyping horse epithelial cells from fecal matter by isolation of polymerase chain reaction products.
Croat Med J, 58(3), 239-249.
https://doi.org/10.3325/cmj.2017.58.239 Publication
Researcher Affiliations
- Pero Dimsoski, Department of Chemistry and Biochemistry, Florida International University, Miami, USA, dimsoski@yahoo.com.
MeSH Terms
- Animals
- DNA / genetics
- DNA Fingerprinting / methods
- Epithelial Cells / chemistry
- Feces / cytology
- Genotype
- Horses / genetics
- Humans
- Polymerase Chain Reaction
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