Discovery and diagnostic methods using 5-methylcytosine DNA...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase

Reexamination Certificate

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C435S004000, C435S006120, C435S015000

Reexamination Certificate

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10389853

ABSTRACT:
The present invention provides methods for identification of methylated, and/or potentially methylatable CpG dinucleotides in genomic DNA sequences, and methods for isolating genomic DNA sequences comprising methylated CpG dinucleotide sequences. The present invention further provides methods for comparison of the methylation status of specific CpG dinucleotides, and patterns thereof between normal and diseased genomic DNA sequences, along with methods for determining all potentially methylatable CpG dinucleotides in a genomic DNA sample. Specifically, the present invention discloses a novel use of 5-methylcytosine DNA glycosylase (5-MCDG) in combination with art-recognized DNA base excision repair (BER) enzymes, and in particular embodiments, in combination with DNA methyltransferase to specifically label methylated CpG dinucleotide sequences in genomic DNA sequences.

REFERENCES:
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Yan et al. CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer. Clin Cancer Res. Apr. 2000;6(4):1432-8.
Toyota et al. Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Res. May 15, 1999;59(10):2307-12.
Zhu et al. 5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence. Nucleic Acids Res. Nov. 1, 2000;28(21):4157-65.
Costello et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet. Feb. 2000;25(2):132-8.
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Fortini et al., “The Type of DNA Glycosylase Determines the Base Excision Repair Pathway in Mammalian Cells,” J. Biol. Chem. 274(21): 15230-15236, 1999.
Ye et al., “Heterogeneous repair of N-Methylpurines at the Nucleotide Level in Normal Human Cells,” J. Mol. Biol. 284: 269-285, 1998.

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