Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications.
Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.
Short
A D
AD
Centre for Integrated Genomic Medical Research, Stopford Building, University of Manchester, Manchester, UK. andrea.short@manchester.ac.uk
Kennedy
L J
LJ
Forman
O
O
Barnes
A
A
Fretwell
N
N
Wiggall
R
R
Thomson
W
W
Ollier
W E R
WE
D000818:Animals; D002610:Cheek; D002851:Chromatography, High Pressure Liquid / Q000662:veterinary; D004247:DNA / Q000097:blood / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004285:Dogs / Q000235:genetics*; D016678:Genome / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000662:veterinary
D017931:DNA Primers; D004247:DNA