Ultrasensitive Amplification-Free Quantification of a Methyl CpG-Rich Cancer Biomarker by Single-Molecule Kinetic Fingerprinting

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR i...

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Published inAnalytical chemistry (Washington) Vol. 96; no. 43; pp. 17209 - 17216
Main Authors Dai, Liuhan, Johnson-Buck, Alexander, Laird, Peter W., Tewari, Muneesh, Walter, Nils G.
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 29.10.2024
Subjects
Amino acids
analytical chemistry
Biomarkers
Biomarkers, Tumor - blood
Biomarkers, Tumor - genetics
Bisulfite
bisulfites
blood
Cancer
Chain branching
CpG Islands
Cytosine
Deoxyribonucleic acid
detection limit
DNA
DNA fingerprinting
DNA Methylation
DNA microarrays
Epigenetics
Gene sequencing
Genetic fingerprinting
Humans
Kinetics
Limit of Detection
microarray technology
Neoplasms - genetics
Nucleotide sequence
Polymerase chain reaction
Promoter Regions, Genetic
Sulfites - chemistry
Transaminase
Transaminases - genetics
Transaminases - metabolism
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Summary:The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish <5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.
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ISSN:0003-2700
1520-6882
1520-6882
DOI:10.1021/acs.analchem.4c03002