Cancer diagnostic method based upon DNA methylation differences
AssigneeUSC/Norris Comprehensive Cancer Ctr.
InventorsMark L. Gonzalgo, Peter A. Jones, Gangning Liang
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Abstract
There is disclosed a cancer diagnostic method based upon DNA methylation differences at specific CpG sites. Specifically, the inventive method provides for a bisulfite treatment of DNA, followed by methylation-sensitive single nucleotide primer extension (Ms-SNuPE), for determination of strand-specific methylation status at cytosine residues.
Claims11
A method for determining DNA methylation status at a cytosine residue of a CpG sequence, comprising the steps of:
- (a) obtaining genomic DNA from a DNA sample to be assayed;
- (b) reacting the genomic DNA with sodium bisulfite to convert unmethylated cytosine residues to uracil residues while leaving any 5-methylcytosine residues unchanged to create an exposed bisulfite-converted DNA sample having binding sites for primers specific for the bisulfite-converted DNA sample;
- (c) performing a PCR amplification procedure using top strand or bottom strand specific primers;
- (d) isolating the PCR amplification products;
- (e) performing a primer extension reaction using a Ms-SNuPE primer, (32P) dNTPs and Taq polymerase, wherein the Ms-SNuPE primer comprises from about a 15 mer to about a 22 mer length primer sequence that is complementary to the bisulfite-converted DNA sample and terminates immediately 5′ of the cytosine residue of the CpG sequence to be assayed;
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- (f) determining the methylation status at the cytosine residue of the CpG sequence by measuring the incorporation of different 32P-labeled dNTPs.
A Ms-SNuPE primer that terminates immediately 5′ upstream of a cytosine residue in a CpG sequence of a CpG island that is frequently hypermethylated in promoter regions of somatic genes in malignant tissue, wherein said Ms-SNuPE primer comprises an oligonucleotide consisting of at least 15 contiguous nucleotides of a gene sequence located immediately 5′ upstream from the CpG sequence.
A method for obtaining a Ms-SNuPE primer sequence that terminates immediately 5′ of a cytosine residue in a CpG sequence of a CpG island, comprising finding a hypermethylated CpG sequence in a CpG island in a somatic gene from a malignant tissue or cell culture, determining the sequence located immediately 5′ upstream from the hypermethylated CpG sequence, and synthesizing, based at least in part on using the sequence located immediately 5′ upstream from the hypermethylated CpG sequence as a template, a Ms-SNuPE primer comprising a 15 to 25 nucleotide sequence immediately 5′ upstream from the hypermethylated CpG sequence.
A Ms-SNuPE primer comprising a 15 to 25 mer oligonucleotide sequence obtained by the process comprising finding a hypermethylated CpG sequence in a CpG island in a somatic gene from a malignant tissue or cell culture, determining the sequence located immediately 5′ upstream from the hypermethylated CpG sequence, and synthesizing, based at least in part on using the sequence located immediately 5′ upstream from the hypermethylated CpG sequence as a template, a Ms-SNuPE primer comprising a 15 to 25 nucleotide sequence immediately 5′ upstream from the hypermethylated CpG sequence, wherein said primer terminates immediately 5′ upstream from the hypermethylated CpG sequence.
Dependencies are inferred from the claim text.