Cancer diagnostic method based upon DNA methylation differences
AssigneeUniversity of Southern California
InventorsMark L. Gonzalgo, Peter A. Jones, Gangning Liang
Life of this patent
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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.
Claims10
A method for determining DNA methylation patterns at cytosine sites, 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 provide primers specific for the bisulfite-converted genomic sample for top strand or bottom strand methylation analysis;
- (c) performing a PCR amplification procedure using the top strand or bottom strand specific primers;
- (d) isolating the PCR amplification products;
- (e) performing a primer extension reaction using Ms-SNuPE primers, [32P]dNTPs and Taq polymerase, wherein the Ms-SNuPE primers comprise a from about a 15 mer to about a 22 mer length primer that terminates immediately 5′ of a single nucleotide to be assayed;
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- (f) determining the relative amount of allelic expression of CpG methylated sites by measuring the incorporation of different 32P-labeled dNTPs.
A Ms-SNuPE primer sequence designed to anneal to and terminate immediately 5′ of a desired cytosine codon in a CpG target site, comprising an oligonucleotide sequence of at least 15 base pairs and corresponding to a gene sequence located immediately 5′ upstream from the CpG island that is frequently hypermethylated in promoter regions of somatic genes in malignant tissue.
The Ms-SNuPE primer sequence wherein the primer sequence is from about 15 to about 25 base pairs in length and selected from the group consisting of GaL1 [SEQ ID NO. 1], GaL2 [SEQ ID NO. 2], GaL4 [SEQ ID NO. 3], HuN1 [SEQ ID NO. 5], HuN2 [SEQ ID NO. 6], HuN3 [SEQ ID NO. 7], HuN4 [SEQ ID NO. 8], HuN5 [SEQ ID NO. 8], HuN6 [SEQ ID NO. 9], CaS1 [SEQ ID NO. 10], CaS2 [SEQ ID NO. 11], CaS4 [SEQ ID NO. 12], and combinations thereof.
A method for obtaining a Ms-SNuPE primer sequence designed to anneal to and terminate immediately 5′ of a desired cytosine codon in the CpG target site, comprising finding a hypermethylated CpG island in a somatic gene from a malignant tissue or cell culture, determining the sequence located immediately 5′ upstream from the hypermethylated CpG island, and isolating a 15 to 25 mer sequence 5′ upstream from the hypermethylated CpG island for use as a Ms-SNuPE primer.
A Ms-SNuPE primer comprising a 15 to 25 mer oligonucleotide sequence obtained by the process comprising, finding a hypermethylated CpG island in a somatic gene from a malignant tissue or cell culture, determining the sequence located immediately 5′ upstream from the hypermethylated CpG island, and isolating a 15 to 25 mer sequence 5′ upstream from the hypermethylated CpG island for use as a Ms-SNuPE primer.
Dependencies are inferred from the claim text.