Methods and compositions for the quantitation of mitochondrial nucleic acid
Provided herein are products and processes for the quantitation of mitochondrial nucleic acid in a sample from a subject. In certain aspects are multiplex methods for determining dosage of mitochondrial nucleic acid relative to genomic nucleic acid for a sample from a subject including amplifying sets of mitochondrial polynucleotides and genomic polynucleotides from nucleic acid for a sample under amplification conditions. In certain aspects are multiplex methods for determining dosage of mitochondrial nucleic acid relative to genomic nucleic acid for a sample from a subject including amplifying sets of mitochondrial polynucleotides and amplifying sets of nuclear polynucleotides from nucleic acid for a sample under amplification conditions.
1 . 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—Y 3′;
(v) 5′ X—V—Y 3′ 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.
2 . The method of claim 1 , wherein the comparison in (b) is a ratio of (i) the amount of the amplicons corresponding to the mitochondrial polynucleotide, to (ii) the amount of the amplicons corresponding to the genomic polynucleotide, in each set and determining the relative dosage of mitochondrial nucleic acid to genomic nucleic acid in the sample in (c) is based on the ratio.
3 . The method of claim 1 , wherein V is a single nucleotide position.
4 . The method of claim 1 , wherein the lengths of the amplicons are about 30 base pairs to about 300 base pairs.
5 . The method of claim 1 , wherein the plurality of amplified sets is about 2 sets to about 20 sets, or about 2 sets to about 10 sets, or at least 5 sets.
6 . The method of claim 1 , wherein the mitochondrial polynucleotide and/or the genomic polynucleotide of a set comprise polynucleotides or portions thereof chosen from SEQ ID NOS: 1-691.
7 . The method of claim 1 , wherein the amplification primer or primers that are specific for the mitochondrial polynucleotide hybridize less efficiently than the amplification primer or primers that are specific for the genomic polynucleotide in a set, whereby the amplification of the mitochondrial polynucleotide is reduced relative to the amplification of the corresponding genomic polynucleotide in each set.
8 . The method of claim 1 , wherein the amplification primer or primers that are specific for the mitochondrial polynucleotide are provided at a lower concentration than the concentration of the amplification primer or primers that are specific for the genomic polynucleotide, whereby the amplification of the mitochondrial polynucleotide is reduced relative to the amplification of the corresponding genomic polynucleotide in each set.
9 . The method of claim 1 , wherein (b) comprises:
(1) determining the amount of a nucleotide at V in the amplicons corresponding to the mitochondrial polynucleotide of a set and the amount of a nucleotide at V in the amplicons corresponding to the genomic polynucleotide of a set; or
(2) determining the amount of amplicons corresponding to the mitochondrial polynucleotide of a set and the amount of amplicons corresponding to the genomic polynucleotide of a set by a qPCR process comprising two fluorescent probes each specific for either the mitochondrial or genomic nucleotide at V or a digital PCR process.
10 . The method of claim 9 , wherein determining the amount of a nucleotide at V in the amplicons corresponding to the mitochondrial polynucleotide of a set and the amount of a nucleotide at V in the amplicons corresponding to the genomic polynucleotide of a set is by a massive parallel sequencing process or by a nanopore process.
11 . The method of claim 9 , wherein the comparison in (b) is a ratio of (i) the amount of the amplicons corresponding to the mitochondrial polynucleotide based on the amount of a nucleotide at V in the amplicons corresponding to the mitochondrial polynucleotide of a set, to (ii) the amount of the amplicons corresponding to the genomic polynucleotide based on the amount of a nucleotide at V in the amplicons corresponding to the genomic polynucleotide of a set, and determining the relative dosage of mitochondrial nucleic acid to genomic nucleic acid in the sample in (c) is based on the ratio.
12 . The method of claim 1 , wherein (b) comprises contacting the amplicons with extension primers under extension conditions comprising a chain terminating reagent that is specific for the amplicons corresponding to the mitochondrial polynucleotide and a chain terminating reagent that is specific for the amplicons corresponding to the genomic polynucleotide, wherein:
(1) the chain terminating reagent that is specific for the amplicons corresponding to the mitochondrial polynucleotide is not specific for the amplicons corresponding to the genomic polynucleotide;
(2) the chain terminating reagent that is specific for the amplicons corresponding to the genomic polynucleotide is not specific for the amplicons corresponding to the mitochondrial polynucleotide; and
(3) the concentration of each of the chain terminating reagents is known and the concentration of the chain terminating reagent specific for the mitochondrial polynucleotide is less than the concentration of the chain terminating reagent specific for the genomic polynucleotide,
whereby the primers are extended up to V, thereby generating chain terminated extension products corresponding to the mitochondrial polynucleotide and the genomic polynucleotide, respectively.
13 . The method of claim 12 , wherein (b) comprises determining a ratio of the amount of extension product corresponding to the mitochondrial polynucleotide to the amount of extension product corresponding to the genomic polynucleotide; and (c) comprises determining the amount of mitochondrial nucleic acid relative to the amount of genomic nucleic acid in the sample based on the ratio of (b).
14 . The method of claim 12 , wherein V is a single nucleotide position at which a nucleotide of the mitochondrial polynucleotide differs from the corresponding nucleotide of the genomic polynucleotide and the primers are extended up to the single nucleotide.
15 . The method of claim 12 , wherein the concentration of the chain terminating reagent specific for a mitochondrial polynucleotide is between about 1% to about 20% of the concentration of the chain terminating reagent specific for a genomic polynucleotide.
16 . The method of claim 2 , wherein the ratios for a plurality of sets are combined and the relative dosage of mitochondrial nucleic acid to genomic nucleic acid for the sample is determined based on the combined ratio.
17 . The method of claim 16 , wherein the combined ratio is an average ratio or a median ratio.
18 . The method of claim 2 , wherein:
(1) the ratio of each set is compared to an average or median ratio based on the plurality of sets and an outlier or cluster that deviates from the average or median ratio is an indication of a mitochondrial deletion; or
(2) the ratio of a set representing one region of the mitochondrial genome is compared to the ratio of each of the other sets representing different regions of the mitochondrial genome and the presence of one or more deletions in the mitochondrial genome is determined based on a difference in the ratio of the set representing the one region compared with the ratios for one or more sets representing other regions of the mitochondrial genome.