Methods and compositions for the quantitation of mitochondrial nucleic acid
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Publication Number
US-10774375-B2
Publication Date
2020-09-15
Expiration Date
Abstract
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.
Core Innovation
The invention provides a multiplex method for determining dosage of mitochondrial nucleic acid relative to nuclear nucleic acid in a sample from a subject. The method contacts nucleic acid of the sample with nucleic acid from a first species and a second species, where copy number information for the mitochondrial genome and nuclear genome is known and paralogous regions are used across species in both nuclear and mitochondrial genomes.
Sets of nuclear polynucleotides of paralogous regions and sets of mitochondrial polynucleotides of paralogous regions are amplified under amplification conditions. Each set comprises a polynucleotide from the first species and a polynucleotide from the second species, with mitochondrial polynucleotides and nuclear polynucleotides being native and different sets using different mitochondrial and nuclear polynucleotides.
The mitochondrial polynucleotides and the nuclear polynucleotides are defined by a 5′-J-V-K-3′ relationship with a contiguous sequence 5′J-V-K-3′, identical J and K across the species within a set, and one or more nucleotide positions V where the polynucleotides differ between the first and second species. The method compares amplicons across species within each set and determines the relative dosage of mitochondrial nucleic acid to nuclear nucleic acid in the sample based on the comparisons for all sets.
Claims Coverage
The input identifies one independent claim, directed to a multiplex method for determining relative dosage of mitochondrial nucleic acid to nuclear nucleic acid. The claim includes amplification of paralogous native mitochondrial and nuclear regions defined by a 5′-J-V-K-3′ segment, cross-species amplicon comparisons within each set, and multi-set determination of relative dosage.
Multiplex relative dosage of mitochondrial versus nuclear nucleic acid using paralogous regions
A multiplex method that contacts sample nucleic acids with nucleic acid of a first species and nucleic acid of a second species with known mitochondrial and nuclear genome copy numbers, and determines relative dosage of mitochondrial nucleic acid to nuclear nucleic acid using paralogous regions between species.
Native mitochondrial and nuclear paralog amplification defined by a 5′-J-V-K-3′ segment
Amplifying sets of paralogous nuclear polynucleotides and paralogous mitochondrial polynucleotides, where each set comprises native mitochondrial polynucleotides and native nuclear polynucleotides defined by 5′-J-V-K-3′ with identical J and K within a set across species and one or more nucleotide positions V that differ between the first and second species.
Cross-species amplicon comparisons and multi-set relative dosage determination
Comparing amplicons across species for mitochondrial polynucleotides and for nuclear polynucleotides within each set, and determining the relative dosage of mitochondrial nucleic acid to nuclear nucleic acid in the sample based on the comparisons for all sets.
The independent claim centers on multiplex amplification of native paralogous mitochondrial and nuclear regions defined by a 5′-J-V-K-3′ structure, followed by cross-species amplicon comparisons at discriminating positions V and determination of mitochondrial-to-nuclear relative dosage based on comparisons across multiple sets.
Stated Advantages
Documented Applications
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