Molecular diagnosis of alpha-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis. 
The aim of the study was to set up an automatic molecular diagnostic method for deletional alpha-thalassemia without gel electrophoresis and TaqMan probe.  Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by dissociation curve (DC) analysis were used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles (alpha alpha or alpha(T)alpha), respectively.  Positive results of the SYBR-PCRs were defined by the special shapes of the dissociation curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon > or = cutoff values.  Molecular diagnosis of alpha-thalassemia was determined by combining all four SYBR-PCR results.  The specific Tms for the SYBR-PCR1-4, which was used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles were 82.5 +/- 1 degrees Celsius, 82.8 +/- 1 degrees Celsius, 81.5 +/- 1 degrees Celsius, and 83.0 +/- 1 degrees Celsius, respectively.  The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70.  The C(T) VS log copies of a recombinant plasmid DNA showed a good linear relationship between 10(5) approximately 10(0).  Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method.  Diagnostic outcomes of the 120 alpha-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods.  The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of alpha-thalassemia.