A primer-initiated strand displacement amplification strategy for sensitive detection of 5-Hydroxymethylcytosine in genomic DNA

5-Hydroxymethylcytosine(5 hmC),an intermediate product of DNA demethylation,is important for the regulation of gene expression during development and even tumorigenesis.The challenges associated with determination of 5 hm C level include its extremely low abundance and high structural similarity with other cytosine derivatives,which resulted in sophisticated treatment with large amount of sample input.Herein,we developed a primer-initiated strand displacement amplification(PISDA)strategy to quantify the global 5 hm C in genomic DNA from mammalian tissues with high sensitivity/selectivity,low input and simple operation.This sensitive fluorescence method is based on 5 hmC-specific glucosylation,primer ligation and DNA amplification.After the primer was labeled on 5 hm C site,DNA polymerase and nicking enzyme will repeatedly act on each primer,causing a significant increase of fluorescence signal to magnify the minor difference of 5 hm C content from other cytosine derivatives.This method enables highly sensitive analysis of 5 hm C with a detection limit of 0.003%in DNA(13.6 fmol,S/N=3)from sample input of only 150 ng,which takes less than 15 min for determination.Further determination of 5 hmC in different tissues not only confirms the widespread presence of 5 hmC but also indicates its significant variation in different tissues and ages.Importantly,this PISDA strategy exhibits distinct advantages of bisulfite-free treatment,mild conditions and simple operation without the involvement of either expensive equipment or large amount of DNA sample.This method can be easily performed in almost all research and medical laboratories,and would provide a promising prospect to detect global 5 hmC in mammalian tissues.