Single-tube analysis of DNA methylation with silica superparamagnetic beads.
BACKGROUND
DNA promoter methylation is a signature for the silencing of tumor suppressor genes. Most widely used methods to detect DNA methylation involve 3 separate, independent processes: DNA extraction, bisulfite conversion, and methylation detection via a PCR method, such as methylation-specific PCR (MSP). This method includes many disconnected steps with associated losses of material, potentially reducing the analytical sensitivity required for analysis of challenging clinical samples.


METHODS
Methylation on beads (MOB) is a new technique that integrates DNA extraction, bisulfite conversion, and PCR in a single tube via the use of silica superparamagnetic beads (SSBs) as a common DNA carrier for facilitating cell debris removal and buffer exchange throughout the entire process. In addition, PCR buffer is used to directly elute bisulfite-treated DNA from SSBs for subsequent target amplifications. The diagnostic sensitivity of MOB was evaluated by methylation analysis of the CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); also known as p16(INK4a)] promoter in serum DNA of lung cancer patients and compared with that of conventional methods.


RESULTS
Methylation analysis consisting of DNA extraction followed by bisulfite conversion and MSP was successfully carried out within 9 h in a single tube. The median pre-PCR DNA yield was 6.61-fold higher with the MOB technique than with conventional techniques. Furthermore, MOB increased the diagnostic sensitivity in our analysis of the CDKN2A promoter in patient serum by successfully detecting methylation in 74% of cancer patients, vs the 45% detection rate obtained with conventional techniques.


CONCLUSIONS
The MOB technique successfully combined 3 processes into a single tube, thereby allowing ease in handling and an increased detection throughput. The increased pre-PCR yield in MOB allowed efficient, diagnostically sensitive methylation detection.
Bailey
Vasudev J
VJ
Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Zhang
Yi
Y
Keeley
Brian P
BP
Yin
Chao
C
Pelosky
Kristen L
KL
Brock
Malcolm
M
Baylin
Stephen B
SB
Herman
James G
JG
Wang
Tza-Huei
TH
D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D019175:DNA Methylation; D019942:Genes, p16; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008280:Magnetics / Q000191:economics / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011401:Promoter Regions, Genetic; D012822:Silicon Dioxide / Q000737:chemistry*
D012822:Silicon Dioxide; D004247:DNA