Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

Table 1 Sequences of oligonucleotides used in the assay

Met-p16	cgc cac cac cct cca acc t
Dem-p16	tgc cac cac cct cca acc t
Probe1	SH-(CH₂)₃-aaa aaa aaa aaa tta ttt agg ttg gag ggt ggt ggc g
Probe2	SH-(CH₂)₃-aaa aaa tta ttt agg ttg gag ggt ggt ggc g
Probe3	SH-(CH₂)₃-tta ttt agg ttg gag ggt ggt ggc g
MPs p16	gtt ttt tag aat gtt ggg att ata ga
MPa p16	ctc aaa aaa cta aaa caa aaa aat c
NPs	ttg tta ttt agg ttg gag ggt ggt
PC p16	tta ttt agg ttg gag ggt ggt ggc gcg att tcg gtt tat tgt aat ttt tgt ttt tcg gg
NC p16	tta ttt agg ttg gag ggt ggt ggt gtg att tcg gtt tat tgt aat ttt tgt ttt tcg gg
Probe E-cad	SH-(CH₂)₃-aaa aaa tta ggt tag agg gtt atc g
MPs E-cad	ttt agt aat ttt agg tta gag ggt tat
MPa E-cad	aaa ctc aca aat act tta caa ttc c
PC E-cad	taa ttt tag gtt aga ggg tta tcg cgt tta tgc gag gtc ggg tgg gcg ggt cgt tag
NC E-cad	taa ttt tag gtt aga ggg tta ttg tgt tta tgc gag gtc ggg tgg gcg ggt cgt tag
Probe p15	SH-(CH₂)₃-aaa aaa gat tat tcg ggt cgt tgc g
MPs p15	agg aga ata agg gta tgt tta gtg g
MPa p15	ccc taa aac ccc aac tac cta aat
PC p15	acg gtg gat tat tcg ggt cgt tgc gcg ttt ggg ggt tgc gga atg cgc
NC p15	acg gtg gat tat tcg ggt cgt tgt gtg ttt ggg ggt tgc gga atg cgc

Note: Met-p16 and Dem-p16 correspond to a partial methylated and demethylated DNA sequence of p16, respectively. A partial E-cadherin, p15, and p16 sequences were amplified with MPs E-cad and MPa E-cad, MPs p15 and MPa p15, MPs p16 and MPa p16 primers, respectively, that were identified from Methprimer. NPs and P16MPa primers were used to perform nested PCR. PC and NC sequences were all dsDNA. The special cutting site for BstUI could be found in the PC group but not in the NC group. Thus, PC E-cad, NC E-cad, PC p15, NC p15, PC p16, and NC p16 sequences were used as positive and negative controls during the DNA methylation detection of E-cadherin, p15 and p16, respectively