Highly Sensitive Detection Of DNA Methylation Based On Ligase Chain Reaction(LCR)

DNA methylation is an indispensable epigenetic modification of eukaryotic genomes,and occurs exclusively at cytosine of Cp G dinucleotides. Aberrant methylation of tumor suppressor genes has been reported in a wide variety of human cancers and is associated with transcriptional repression. DNA methylation has been increasingly utilized as a biomarker for cancer detection and differential diagnosis. Therefore, it is necessary to establish a fast and sensitive method to detect DNA methylation. Generally, one type of cancer is associated with several Cp G methylation and detection of multiplexed Cp G methylation can greatly improve the accuracy of cancer diagnosis.In this paper, we have developed two ligase chain reaction(LCR)-based methods for highly sensitive detection of Cp G methylation in genomic DNA at single-base resolution. The first method: the LCR products hybridize with the adjacent two probes modified on gold nanoparticles(Au NPs), which brings the Au NPs close to each other and forms the network aggregation because every Au NP bears many DNA strands. The aggregation generates a red-to-blue color change, allowing the visual detection of DNA methylation with naked eye and quantitative determination with the absorption spectral measurements. This method can detect as low as 0.01 fmol/L methylated DNA fragments and 1 ng methylated genomic DNA.0.1 % methylated DNA can be detected in the presence of large excess of normal DNA. The second method: one of the probes is modified with fluorescent group, the LCR products can be separated and detected by ABI 310 Genetic Analyzer. Due to the capacity of the LCR to discriminate one base mismatch, the proposed method can achieve high sensitivity and specificity. As low as 0.01 fmol/L methylated DNA fragments and 10 ng methylated genomic DNA can be detected by this method. And 0.1% methylated DNA can be detected in the presence of large excess of unmethylated DNA. Moreover, by simply encoding one of the LCR probes with different length of poly(A) for detection of methylation at different Cp G sites, the Cp G methylation at different sites can produce LCR products with different length,and thus, can be simultaneously detected with one-tube LCR amplification.