Methods for detection of methyl-CpG dinucleotides
The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5′-C Me CpGG-3′. Methods for making and using the rMcrA protein, and derivatives thereof are provided.
1. A bacterial expression vector for expressing a recombinant, full length McrA protein (rMcrA), or fusion derivative thereof encoded in said vector, which full length protein or fusion derivative binds 5′-C Me CpGG-3′ sequences in vitro and wherein said fusion derivative is a fusion between full length rMcrA and an affinity tag.
2. A bacterial cell comprising the expression vector of claim 1 .
3. The cell according to claim 2 which further comprises a pRIL tRNA plasmid.
4. A method for isolating substantially pure, active, full length rMcrA, or active fusion derivative thereof comprising:
a) culturing the bacterial cells of claim 2 under conditions appropriate for expression;
b) allowing expression of rMcrA to occur in said cells;
c) harvesting said cells following expression;
d) lysing the harvested cells of c);
e) separating particulate matter from soluble matter in the lysed cells of d) to form a soluble fraction and an insoluble fraction;
f) contacting the soluble fraction from e) with a Ni-chelate resin so as to bind the expressed rMcrA;
g) washing the resin of f) to remove non-specifically bound matter; and
h) eluting the bound rMcrA, or fusion derivative thereof, by washing the resin with a buffer containing imidazole, thereby isolating substantially pure, active, full length rMcrA or active fusion derivative thereof.
5. The method according to claim 4 wherein the fusion derivative of rMcrA is a fusion between full length rMcrA and an affinity tag.
6. The method according to claim 5 wherein the affinity tag is selected from the group consisting of GST, S Tag, T7 tag, Strep tag, FLAG, chitin binding domain, and calmodulin binding protein.
7. The method according to claim 6 wherein the substantially purified fusion derivative is further purified by binding to a specific binding partner of GST, S Tag, T7 tag, Strep tag, FLAG, chitin binding domain, or calmodulin binding protein.
8. A method for isolating substantially pure active, full length rMcrA, or active fusion derivative thereof comprising: culturing the bacterial cells of either claim 2 or claim 3 under conditions appropriate for expression; allowing expression of rMcrA to occur; harvesting said cells following expression; lysing the harvested cells to release a soluble fraction; contacting the soluble fraction with a Ni-chelate resin so as to bind the expressed rMcrA; eluting the bound rMcrA, or derivative thereof, by washing the resin with a buffer containing imidazole, thereby isolating substantially pure active full length rMcrA or active fusion derivative thereof.
9. A method for isolating substantially pure, active, full length rMcrA, or active fusion derivative thereof comprising:
a) culturing the bacterial cells of claim 3 under conditions appropriate for expression;
b) allowing expression of rMcrA to occur in said cells;
c) harvesting said cells following expression;
d) lysing the harvested cells of c);
e) separating particulate matter from soluble matter in the lysed cells of d) to form a soluble fraction and an insoluble fraction;
f) contacting the soluble fraction from e) with a Ni-chelate resin so as to bind the expressed rMcrA;
g) washing the resin of f) to remove non-specifically bound matter; and
h) eluting the bound rMcrA, or fusion derivative thereof, by washing the resin with a buffer containing imidazole, thereby isolating substantially pure, active, full length rMcrA or active fusion derivative thereof.
10. The expression vector according claim 1 wherein the affinity tag is selected from the group consisting of GST, S Tag, T7 tag, Strep tag, FLAG, chitin binding domain, and calmodulin binding protein.