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Quantification of trace amounts of human and non-human mitochondrial DNA (mtDNA) using SYBR Green and real time PCR

Tobe, Shanan S. and Linacre, Adrian (2008) Quantification of trace amounts of human and non-human mitochondrial DNA (mtDNA) using SYBR Green and real time PCR. Forensic Science International: Genetics Supplement Series, 1 (1). pp. 71-73. ISSN 1875-1768

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Abstract

There are currently no tests available to quantify total non-human mammalian mtDNA. Standard universal DNA quantification tests are unsuitable due to the large size difference between nuclear and mitochondrial genomes and the ubiquity of human mtDNA. A method has therefore been developed to quantify total mammalian mtDNA and total human mtDNA present in a sample using SYBR Green. Mammalian primers designed to react with all mammals were designed on the 12S and human specific primers were designed on the cytochrome b gene. Each primer set was reacted separately with sample and SYBR Green and detected using RT-PCR. A standard curve was developed using dilutions ranging from 1 billion copies to 100 copies of mtDNA. Twenty-four human samples were analysed and an average log (copy number) human/universal ratio of 1.00 was obtained. Samples falling below this ratio will contain some non-human mtDNA while samples falling above this ratio contain human mtDNA only. Twenty-nine mammal samples were also tested. 96.6% of these showed human contamination to some extent. This test is able to quantify mtDNA down to the femtogramme (10E−15g) level.