Quantification of human mitochondrial DNA using synthesized DNA standards

摘要

A real-time quantitative PCR assay that utilizes a duplex, synthetic DNA standard to ensure optimal quality assurance and quality control. One embodiment of the invention facilitates amplification of mtDNA by focusing on a 105-base pair target sequence that is minimally homologous to non-human mtDNA. The present invention can also be used to identify the presence of PCR inhibitors and thus indicate the need for sample repurification.

主权项

1. A process for quantifying human mitochondrial DNA, said process comprising:
amplifying a region of human mitochondrial DNA by contacting a biological sample containing human mitochondrial DNA with an oligonucleotide primer pair having a nucleotide sequence complementary to a target sequence located within the NADH dehydrogenase subunit 5 gene of said mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of said primer pair to the mitochondrial DNA; detecting amplification following hybridization of the primer pair to the mitochondrial DNA; and quantifying the detected amplified product using a synthetic DNA standard comprising a signature nucleotide sequence, wherein the signature nucleotide sequence comprises nucleotide substitutions, which, if translated, would result in the introduction of a stop codon and an amino acid substitution in the target sequence.