Methods and compositions for enriching either target polynucleotides or non-target polynucleotides from a mixture of target and non-target polynucleotides
View Patent ↗Compositions and methods are provided for enriching mitochondrial DNA and optionally chloroplast DNA from eukaryotic cells in a simple rapid method that provides greater than 100 fold enrichment. Affinity protein-coated substrate in a buffer is used to efficiently bind chromosomal DNA and thereby remove it from the buffer. Mitochondrial sequencing reads reveal that non-biased sequence selection providing representation of a substantial proportion of mitochondrial DNA in the eukaryotic cells analyzed.
1. A composition comprising:
a mixture comprising eukaryotic chromosomal DNA and mitochondrial DNA, wherein the chromosomal DNA in the mixture has a median size of at least 10 kb,
a matrix comprising a protein that comprises a methyl binding domain, wherein the protein is capable of selectively binding to methylated polynucleotides but not non-methylated polynucleotides, and
a buffer containing effective amounts of a salt and a non-ionic detergent.
2. A composition, according to claim 1 , wherein the effective amount of salt is 10 mM-800 Mm.
3. A composition according to claim 1 , wherein the chromosomal DNA is bound to the matrix and a significant portion of the mitochondrial DNA is unbound in the buffer.
4. A composition according to claim 3 , wherein the unbound mitochondrial DNA in the buffer is enriched by at least a 100 fold compared to the mitochondrial DNA in the mixture.
5. A composition according to claim 3 , wherein less than 10% of the chromosomal DNA in the cell DNA is unbound in the buffer.
6. A composition according to claim 3 , wherein less than 10% of the mitochondrial DNA in the mixture is bound to the matrix.
7. A composition according to claim 3 , wherein at least 90% of the mitochondrial DNA and less than 10% of chromosomal DNA is unbound in the buffer.
8. A composition according to claim 1 , wherein the matrix comprises magnetic beads.
9. A composition according to claim 1 , wherein the methyl-binding domain (MBD) is selected from the group consisting of UHRF1(SRA), CXX1, DNMT1, MBD and methyl-binding variants thereof.
10. A composition according to claim 1 , further comprising chloroplast DNA.
11. A composition according to claim 10 , wherein at least 90% of the chloroplast DNA is unbound in the buffer.
12. A method for enriching cellular mitochondrial DNA from a cell sample, comprising:
(a) obtaining a composition according to claim 1 ;
(b) permitting the chromosomal DNA to bind to the matrix; and
(c) obtaining an enriched preparation of mitochondrial DNA in a unbound fraction.
13. A method according to claim 12 , further comprising determining the fraction of the mitochondrial DNA and chromosomal DNA in the unbound fraction.
14. A method according to claim 12 , further comprising, sequencing a part of the entire mitochondrial DNA.
15. A method according to claim 12 , further comprising perfoming a genetic analysis of the mitochondrial DNA.
16. A method according to claim 12 , further comprising perfoming a genetic analysis of single nucleotide polymorphisms in the mitochondrial DNA.
17. A method according to claim 15 , further comprising: analyzing the mitochondrial DNA for oxidative damage.
18. A method according to claim 12 , wherein the methyl-binding domain (MBD) is selected from the group consisting of UHRF1(SRA), CXX1, DNMT1, MBD and methyl-binding variants thereof.
19. A method according to claim 12 , wherein the matrix comprises magnetic beads.
20. A method according to claim 19 , wherein the magnetic beads are coated with protein A bound to MBD2a-Fc.
21. A method for detecting oxidative damage in mitochondrial DNA, comprising:
(a) enriching mitochondrial DNA using the method of claim 12 ;
(b) identifying a change in oxidation status of methylcytosine to hydroxymethylcytosine in chromosomal DNA bound to the MBD as an indicator; and
(c) analyzing the mitochondrial DNA in the unbound fraction for reactive oxygen damage.