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.
Liu
Jingzhong
J
Basic Medical Research Center, Beijing Chaoyang Hospital affiliate of Capital University of Medical Sciences, Beijing, China. liujingzhong@hotmail.com
Yan
Mei
M
Wang
Zhangyong
Z
Wang
Lirong
L
Zhou
Yan
Y
Xiao
Bai
B
D000483:Alleles; D001331:Automation; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D017353:Gene Deletion; D005838:Genotype; D006801:Humans; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics*
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I