Method for using probe based PCR detection to measure the levels of circulating demethylated beta cell derived DNA as a measure of beta cell loss in diabetes
A method for measuring blood levels of β cell DNA that is released upon β cell death by using a quantitative probe technology to detect amplified methylated and demethylated forms of the insulin gene DNA, representing normal tissue and β cell specific origin, respectively. Using probes permits the sensitive and specific identification of demethylated insulin DNA patterns that are present only in β cells. The method offers a bioassay for detecting β cell loss in diabetes, useful for screening of prediabetes, monitoring of disease progression, and selection and monitoring of therapies. The technique finds potential use in both Type I and Type II diabetes, as well as gestational diabetes.
1. A kit for detecting beta-cell-derived demethylated genomic DNA in a biological sample, wherein the kit comprises: a first oligonucleotide probe selected from the group consisting of ACCTCCCGACGAATCT (SEQ ID NO: 003) and (TACCTCTCGTCGAATCT SEQ ID NO: 004); and a second oligonucleotide probe selected from the group consisting of ACCTCCCAACAAATCT (SEQ ID NO: 005) and TACCTCCCATCAAATCT (SEQ ID NO: 006), where the first and/or second oligonucleotide probe comprises a fluorophore.
2. The kit according to claim 1 , wherein the fluorophore is 6-carboxyfluorescein or tetrachlorofluorescein.
3. The kit according to claim 1 , wherein the first and second oligonucleotide probes each further comprise a quencher, and wherein in a binding interaction of the first oligonucleotide probe with a bisulfite-converted CpG methylation site of a target sequence, and the second oligonucleotide probe with a bisulfite non-converted CpG methylation site of the target sequence, a change from a first conformation to a second conformation occurs, thereby changing an interaction between the fluorophore and quencher of the respective probe.
4. The kit according to claim 3 , wherein the quencher is tetramethylrhodamine.
5. The kit according to claim 1 , further comprising a pair of forward and reverse PCR primers consisting of the sequences set forth in SEQ ID NOS: 001 and 002, SEQ ID NOS: 007 and 008; or SEQ ID NOS: 009 and 010.