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CE-LIFcapillary electrophoresis with laser induced fluorescence detection
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We used this CE-LIF technique to analyze serum samples from 55 fasting subjects with episodes of deep-vein thrombosis or arterial occlusive disease, and we compared the results with those obtained from an ion-exchange amino acid analyzer (Beckman 6300 analyzer).
The same calibrator, i.e., homocystine (Sigma), was used in both methods: in the ion-exchange chromatography method, homocystine was diluted in lithium citrate buffer (0.2 mol/L, pH 7.0), and the calibrator curve in aqueous solution was determined using the ninhydrin reaction (specific for amine groups); in the CE-LIF method, homocystine was diluted in carbonate buffer (pH 9.5), and the calibrator curve in plasma solution was determined using IAF, which reacts specifically with thiol groups.
This purification step was included to remove fluorescent material and fluorescent primers, which interfered with the analysis of the CMC cleavage products by CE-LIF. The final volume of the purified DNA sample was 50 [micro]L.
In the present work, we demonstrate the use of CMC coupled to CE-LIF for the rapid detection of the T833C and G919A substitutions that are the most prevalent pathogenic mutations in the CBS gene.
Here, we demonstrate a method for analyzing the C677T mutation in the MTHFR gene, in which the technique based on PCR amplification and HinfI restriction cleavage [3] has been modified and adapted to a CE-LIF format.