Method for using probe based PCR detection to measure the levels of circulating demethylated β cell derived DNA as a measure of β cell loss in diabetes

摘要

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.

主权项

1. A method for monitoring beta cell death, comprising:
extracting and purifying DNA from a body fluid of an animal or human; treating the extracted purified DNA with bisulfite to convert demethylated cytosine to uracil while sparing the methylated cytosines; amplifying the bisulfite-treated DNA using polymerase chain reaction; purifying the amplified bisulfite-treated DNA; providing methylation-status specific probes configured to measure, in a quantitative methylation-status sensitive probe hybridization reaction, demethylated insulin DNA of β cell origin and methylated insulin DNA of non-β cell origin, comprising: a) at least one probe for detection of methylated insulin DNA of non-β cell origin selected from the group consisting of ACCTCCCGACGAATCT SEQ ID NO: 003 and TACCTCTCGTCGAATCT SEQ ID NO: 004; and b) at least one probe for detection of demethylated insulin DNA of β cell origin selected from the group consisting of ACCTCCCAACAAATCT SEQ ID NO: 005 and TACCTCCCATCAAATCT SEQ ID NO: 006; performing the quantitative methylation sensitive probe hybridization reaction on the purified bisulfite-treated DNA using the methylation-status specific probes; and quantitatively analyzing relative amounts of methylated insulin DNA and demethylated insulin DNA.