A
gold nanoparticle-filled capillary electrophoresis method combined with three multiplex polymerase chain reactions (PCRs) was established for simultaneous diagnosis of five common
alpha-thalassemia deletions, including the -alpha(3.7) deletion, -alpha(4.2) deletion, Southeast Asian (--(SEA)), Filipino (--(FIL)) and Thai (--(THAI)) deletions.
Gold nanoparticles (GNPs) were used as a pseudostationary phase to improve the resolution between
DNA fragments in a low-viscosity
polymer. To achieve the best CE separation, several parameters were evaluated for optimizing the separation conditions, including the capillary coating, the concentrations of
polymer sieving matrix, the sizes and concentrations of GNPs, the
buffer concentrations, and the pH. The final CE method for separating a 200-base pair (bp)
DNA ladder and
alpha-thalassemia deletions used a DB-17 capillary, 0.6% poly(
ethylene oxide) (PEO) prepared in a mixture of GNP(32nm)
solution and
glycine buffer (25mM, pH 9.0) (80:20, v/v) as the sieving matrix with 1microM
YO-PRO-1 for fluorescence detection; the applied voltage was -10kV (detector at
anode side) and the separation temperature was 25 degrees C. Under these optimal conditions, 15
DNA fragments with sizes ranging from 0.2kb to 3.0kb were resolved within 11.5min. The RSDs of migration times were less than 2.81%. A total of 21 patients with
alpha-thalassemia deletions were analyzed using this method, and all results showed good agreement with those obtained by gel electrophoresis.