Quantification of cattle DNA using quantitative competitive PCR with sheep DNA as competitor.
A novel method was developed to enable accurate and high-throughput measurement of cattle DNA concentration using quantitative competitive PCR, with sheep DNA as competitor. While quantitative competitive PCR has been used extensively for the quantification of specific RNA or DNA molecules, they have required development of internal standards with matching primer binding sites and similar amplification efficiencies to the target molecule. To develop such as assay can constitute a significant work-up. Instead, by utilizing the tendency of microsatellites developed in one species to amplify homologous loci across closely related species removes the need for internal standard development. Two cattle microsatellite markers were identified that produced distinct sheep specific peaks in an electropherogram. A standard graph was plotted for various dilutions of a cattle standard and a constant amount of sheep competitor. The sheep DNA, which is co-amplified with the cattle template in the PCR reaction served as the internal standard. The cattle DNA concentration of an unknown sample was determined by relating the ratio of sheep to cattle PCR product peaks to the standard curve. The standard deviation between replicate measurements of cattle DNA was 0.52 ng/microl using this method.
Mariasegaram
Maxy
M
Institute of Land and Food Resources, University of Melbourne, Victoria 3010, Australia.
Robinson
Nicholas Andrew
NA
Goddard
Michael Edward
ME
D000818:Animals; D002417:Cattle / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep / Q000235:genetics*
D004247:DNA