Highly sensitive electrochemical methyltransferase activity assay.

A simple and highly sensitive electrochemical DNA methyltransferase (MTase) activity assay is presented in this report. The assay employs the electrocatalytic oxidation of ascorbic acid (AA) by a threading intercalator (N,N'-bis(3-propylimidazole)-1,4,5,8-naphthalene diimide (PIND) functionalized with electrocatalytic redox Os(bpy)2Cl(+) moieties (PIND-Os)). Briefly, a double-stranded DNA (ds-DNA) containing the symmetric sequence of 5'-CCGG-3' is first immobilized on a gold electrode. The electrode is then incubated with M.SssI CpG methyltransferase (M.SssI MTase) which catalyzes the methylation of the specific CpG dinucleotides, and the electrode is subsequently treated with a restriction endonuclease HpaII which recognizes the 5'-CCGG-3' sequence. Once the CpG site in the 5'-CCGG-3' is methylated, HpaII recognition is blocked. Higher M.SssI MTase activity leads to more CpG sites being methylated and consequently impedes more the restriction endonuclease HpaII digestion process. Thus, a larger amount of ds-DNA remains on the electrode surface after the HpaII treatment. Thereafter, the electrode is incubated with PIND-Os during which PIND-Os specifically inserts itself between base pairs of ds-DNA and catalyzes the electrooxidation of AA. The methylation event corresponding to the MTase activity can therefore be monitored and amplified by the electrocatalytic oxidation of AA. A linear correlation between the catalytic oxidation current of AA and the activity of M.SssI MTase ranged from 0 to 120 U/mL with a current sensitivity of 0.046 µA mL U(-1) is obtained. The inhibitor screening ability of the developed MTase activity assay is also demonstrated.

Peripheral refraction in different ethnicities.

PURPOSE: Peripheral refraction is commonly used to infer retinal shape. Because of the different prevalence of myopia in the white compared with East Asian populations, peripheral refraction along the horizontal meridian was compared in white and East Asian young adults with emmetropic, low myopic, and moderately myopic refractive errors. METHODS: Thirty-five white and 37 East Asian subjects were recruited with central refraction between +1.00 DS and -5.50 DS and ≤ -1.50 DC. Central and peripheral noncycloplegic autorefraction was measured along the horizontal meridian at 5° intervals up to ± 35°, and corneal topography maps were also quantified. RESULTS: There were no significant differences between whites and East Asians in peripheral refraction profiles in the emmetrope and low myope groups, which had peripheral myopic and emmetropic refraction, respectively. However, in the moderate myope group, there was a statistically significant difference between whites and East Asians in peripheral refraction, which was generally hyperopic. East Asian moderate myopes had more relative peripheral hyperopia than did whites of similar central refractive error. Corneal shape and power were comparable between white and East Asian subjects in the three refractive groups. CONCLUSIONS: East Asian moderate myopes have a greater degree of relative peripheral hyperopia and hence a more prolate ocular shape than do white subjects of similar central refractive error. Differences in ocular shape may play a role in the greater propensity for East Asians to develop and progress in myopia compared with whites.