Methods and compositions for the quantitation of mitochondrial nucleic acid

A multiplex method for determining dosage of mitochondrial nucleic acid relative to genomic nucleic acid for a sample from a subject, comprising:

• a. amplifying sets of mitochondrial polynucleotides and genomic polynucleotides from nucleic acid of a sample under amplification conditions using amplification primers, wherein:
• (i) each set comprises a mitochondrial polynucleotide and a genomic polynucleotide;
• (ii) the mitochondrial polynucleotide and the genomic polynucleotide are native;
• (iii) the mitochondrial polynucleotide of a set differs from the mitochondrial polynucleotide of the other sets and the genomic polynucleotide of a set differs from the genomic polynucleotide of the other sets;
• (iv) the mitochondrial polynucleotide and the genomic polynucleotide of a set are defined by formula 5′X—V—Y3′;
• (v) 5′X—V—Y3′ is about 30 to about 300 base pairs in length and represents a contiguous sequence of nucleotides present in the mitochondrial polynucleotide and the genomic polynucleotide;
• (vi) X and Y of the mitochondrial polynucleotide are identical to X and Y, respectively, of the genomic polynucleotide in each set;
• (vii) V is one or more nucleotide positions at which a nucleotide of the mitochondrial polynucleotide differs from the corresponding nucleotide of the genomic polynucleotide in a set;
• and
• (viii) the amplification primers are selected such that:
• (1) the mitochondrial polynucleotide and the genomic polynucleotide of a set are reproducibly amplified relative to each other by a single pair of amplification primers that hybridize to a polynucleotide within X and Y;
• (2) the mitochondrial polynucleotide and the genomic polynucleotide of a set are amplified by different pairs of amplification primers that hybridize to flanking polynucleotides that are 5′ to X and 3′ to Y, wherein one pair of the amplification primers is specific for the mitochondrial polynucleotide and the other pair of the amplification primers is specific for the genomic polynucleotide;
• (3) the mitochondrial polynucleotide and the genomic polynucleotide of a set are amplified by an amplification primer that hybridizes to a polynucleotide within X for both the mitochondrial polynucleotide and the genomic polynucleotide and two amplification primers, wherein one of the amplification primers is specific for the mitochondrial polynucleotide and the other is specific for the genomic polynucleotide and each of the two amplification primers hybridizes 3′ to Y;
• or (4) the mitochondrial polynucleotide and the genomic polynucleotide of a set are amplified by an amplification primer that hybridizes to a polynucleotide within Y for both the mitochondrial polynucleotide and the genomic polynucleotide and two amplification primers, wherein one of the amplification primers is specific for the mitochondrial polynucleotide and the other is specific for the genomic polynucleotide and each of the two amplification primers hybridizes 5′ to X,
• thereby providing a plurality of amplified sets each comprising amplicons corresponding to all of the mitochondrial polynucleotide and genomic polynucleotide in the set, or amplicons comprising V and at least a portion of each of the X and Y regions of the mitochondrial polynucleotide and genomic polynucleotide in the set;
• b. comparing (i) the amplicons corresponding to the mitochondrial polynucleotide, to (ii) the amplicons corresponding to the genomic polynucleotide for each set, thereby generating a comparison;
• and
• c. determining the relative dosage of mitochondrial nucleic acid to genomic nucleic acid in the sample based on the comparison.