Detection of methylation of human p16(Ink4a) gene 5'-CpG islands by electrochemical method coupled with linker-PCR

Abstract

Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. p16, an inhibitor of the cyclin D-dependent protein kinases, is a classical tumor suppressor gene, and its inactivation is closely associated with carcinogenesis. p16 hypermethylation could be detected in each stage, which is consistent with the finding that aberrant methylation of p16 is a very early event in carcinogenesis. We have developed an electrochemical procedure for detecting DNA methylation of the human p16(Ink4a) gene. The procedure is based on the coupling of DNA electrochemical sensors with linker-PCR- amplified DNA from human gastric tumor tissue and whole blood cells of healthy human. The synthesized oligonucleotide was immobilized on the modified gold electrode to fabricate a DNA biosensor. The hybridization reaction on the electrode surface was monitored by cyclic voltammogram (CV) and square wave voltammogram (SWV), using [Co(phen)(3)](ClO(4))(3) as a redox indicator. Methylation status of human p16(Ink4a) gene was detected and the results were validated by bisulfite DNA sequencing. A good reproducibility was observed in several parallel experiments. The coupling of DNA electrochemical sensors with PCR allowed quick detection and have the potential of the quantitative evaluation of the methylation status of the human p16(Ink4a) gene.

Figure 1

A schematic outline of the procedure for the preparation of the DNA targets. M, B and B^m represent MseI, unmethylated and methylated BstUI recognition sites, respectively. Genomic DNA comes from whole blood cells of healthy human (normal) and gastric tumor tissue (tumor).

Figure 2

CV at the scan rate of 50 mV/S (a) and anodic peak currents versus root of the scan rate of CV v^1/2 (b) for negative DNA/AET/Au (1), sample DNA/AET/Au (2) and positive DNA/AET/Au (3) in pH 7.1 Tris–HCl buffer containing 0.12 mM [Co(phen)₃](ClO₄)₃.

Figure 3

SWV for positive hybrid-DNA/AET/Au (1), sample hybrid-DNA/AET/Au (2) and negative hybrid-DNA/AET/Au (3) with an amplitude of 25 mV, a pulse frequency of 30 Hz and step potential of 4 mV, in pH 7.1 Tris–HCl buffer containing 0.12 mM [Co(phen)₃](ClO₄)₃.

Figure 4

The plot of current intensity ratios of CV and SWV for differently hybridized electrodes. P/N, positive hybrid-DNA/AET/Au/negative hybrid-DNA/AET/Au; S/N, sample hybrid-DNA/AET/Au/negative hybrid-DNA/AET/Au.

Figure 5

(a) MSP of p16 gene. After treatment with bisulfate, amplification of DNA from gastric tumor tissue (A) and whole blood cells of healthy human (B) using unmethylated primer; amplification of DNA from gastric tumor tissue (C) and whole blood cells of healthy human (D) using methylated primer. Marker (M): from top to bottom, the bands are 2000, 1000, 750, 500, 250 and 100 bp, respectively. (b) Sequencing result of the PCR product with an ABI377A. The box indicates that the CpG sites in BstUI recognition sequence are methylated.