Genotyping of alpha-thalassemia deletions using multiplex polymerase chain reactions and gold nanoparticle-filled capillary electrophoresis.
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.
Chen
Yen-Ling
YL
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.
Shih
Chi-Jen
CJ
Ferrance
Jerome
J
Chang
Ya-Sian
YS
Chang
Jan-Gowth
JG
Wu
Shou-Mei
SM
D019075:Electrophoresis, Capillary / Q000379:methods*; D017353:Gene Deletion; D005838:Genotype; D005998:Glycine / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014783:Viscosity; D017085:alpha-Thalassemia / Q000235:genetics*
D011108:Polymers; D006046:Gold; D005998:Glycine