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 non-target polynucleotides from a mixture of non-target and target polynucleotides where differences between the target polynucleotides and the non-target polynucleotides include the extent of modified bases that are present in a greater density in the target polynucleotides than in the non-target polynucleotides. This permits the target polynucleotides to be selectively and rapidly bound to an affinity matrix such as affinity protein-coated magnetic beads providing enrichment of the non-target polynucleotides in the supernatant. One use of this enrichment is to remove human genomic DNA from a mixture of DNAs obtained from human tissue samples to enrich for polynucleotides in a microbiome so as to characterize the microbiome by DNA sequencing.
1. A composition, comprising:
(a) a mixture comprising polynucleotides from one or more bacteria and polynucleotides from a methylated genome, the polynucleotides in the mixture having a median size in the range of 10-100 Kb;
(b) an affinity matrix comprising a protein or a polypeptide domain that is capable of selectively binding to methylated polynucleotides but not non-methylated polynucleotides; and
(c) 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 methylated bases in the methylated genome occur at a frequency of at least 1 modified base in 200 bases.
4. A method for enriching polynucleotides from a mixture, comprising:
(a) combining: (i) a mixture comprising non-methylated polynucleotides from one or more bacteria and polynucleotides from a methylated genome, the polynucleotides in the mixture having a median size in the range of 10-100 Kb, with (ii) a buffer containing effective amounts of a salt and a non-ionic detergent and (iii) an affinity matrix comprising a protein or a polypeptide domain that is capable of selectively binding to methylated polynucleotides but not non-methylated polynucleotides;
(b) permitting binding of the methylated polynucleotides to the affinity matrix;
(c) collecting the unbound polynucleotides, thereby obtaining an enriched preparation of non-methylated polynucleotides; and
(d) analyzing the enriched preparation of non-methylated polynucleotides.
5. A method according to claim 4 , wherein the methylated polynucleotides that are bound to the affinity matrix are at least 90% of the polynucleotides in the mixture prior to enrichment.
6. A method according to claim 4 , wherein the non-methylated polynucleotides in the unbound fraction comprise no more than 10% of the polynucleotides in the mixture prior to enrichment.
7. A method according to claim 4 , wherein the methylated genome comprises methylated cytosines.
8. A method according to claim 4 , wherein the mixture of polynucleotides prior to enrichment is obtained from a biological sample and comprises prokaryotic and mammalian genomic DNA wherein the mixture comprises at least 50% mammalian genomic DNA.
9. A method according to claim 4 , wherein the analyzing is done by sequencing, cloning or amplification to determine a genomic identity for the non-target polynucleotides.
10. A method according to claim 4 , wherein the affinity matrix comprises a methyl-binding domain (MBD) selected from the group consisting of UHRF1(SRA), CXX1, DNMT1, MBD and methyl-binding variants thereof.
11. A method according to claim 4 , wherein the affinity matrix comprises magnetic beads.
12. A method according to claim 11 , wherein the magnetic beads are coated with protein A bound to MBD2a-Fcbu.
13. A method according to claim 4 , wherein the salt is NaCl in a concentration range of 10 mM-800 mM.
14. A method of enhancing separation of polynucleotides of a non-methylated genome from polynucleotides of a methylated genome, comprising:
(a) providing a composition according to claim 1 ;
(b) permitting the methylated polynucleotides to bind to the affinity matrix to form a product that is immobilized on the matrix; and
(c) performing amplification and/or sequencing of at least one of the bound polynucleotide or the unbound polynucleotide.
15. A method according to claim 14 , wherein the mixture of methylated and unmethylated polynucleotides comprise fragments from one or more bacterial genomes and one or more mammalian genomes.
16. A method according to claim 14 , wherein the matrix is a magnetic bead.
17. A method according to claim 14 , wherein the affinity matrix comprises a methyl-binding domain (MBD) selected from the group consisting of—UHRF1(SRA), CXX1, DNMT1, MBD and methyl-binding variants thereof.
18. A method according to claim 14 , wherein the effective amount of salt is in the range of 100 mM-800 mM NaCl.
19. The composition of claim 1 , wherein the polynucleotides from a non-methylated genome are fragments of bacterial genomic DNA and the polynucleotides from a methylated genome are fragments of mammalian genomic DNA.
20. The method of claim 4 , wherein the polynucleotides from a non-methylated genome are fragments of bacterial genomic DNA and the polynucleotides from a methylated genome are fragments of mammalian genomic DNA.