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

Shanan S. Tobe, Adrian Linacre

Research output: Contribution to journalArticle

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.

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Mitochondrial DNA
Real-Time Polymerase Chain Reaction
Accidental Falls
Mammals
Cytochromes b
Mitochondrial Genome
Polymerase Chain Reaction
DNA
Genes

Keywords

  • quantification
  • human mtDNA
  • non-human mtDNA
  • human contamination

Cite this

@article{405e9342282142a6b607df9a7a855e9f,
title = "Quantification of trace amounts of human and non-human mitochondrial DNA (mtDNA) using SYBR Green and real time PCR",
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.",
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author = "Tobe, {Shanan S.} and Adrian Linacre",
year = "2008",
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doi = "10.1016/j.fsigss.2007.10.103",
language = "English",
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pages = "71--73",
journal = "Forensic Science International: Genetics Supplement Series",
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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.; Linacre, Adrian.

In: Forensic Science International: Genetics Supplement Series, Vol. 1, No. 1, 08.2008, p. 71-73.

Research output: Contribution to journalArticle

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AU - Tobe, Shanan S.

AU - Linacre, Adrian

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N2 - 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.

AB - 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.

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KW - non-human mtDNA

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