Development of a new genotyping assay for detection of the BDNF Val66Met polymorphism using melting-curve analysis.
Brain-derived neurotrophic factor (BDNF) plays a critical role in the growth, differentiation and survival of neurons in the CNS. Recent research has suggested that BDNF may be implicated in the etiology of mood disorders and schizophrenia, as well as in the therapeutic action of some drugs, such as antidepressants and antipsychotics. This study aimed to develop a simple, fast and accurate new method for detecting the Val66Met polymorphism of the BDNF gene in schizophrenia patients using melting-curve analysis and a DNA-specific dye, SYBR Green I. A group of 30 schizophrenia patients were analyzed to detect the BDNF Val66Met polymorphism (rs6265) using the new genotyping method based on the analysis of fluorescence melting curves of PCR products that were labeled with SYBR Green I. The genotype results were confirmed for all 30 samples using the specific BDNF TaqMan allele discrimination assay. This new method allows the analysis of both alleles in the same reaction tube using SYBR Green I, with no need for additional steps. The addition of a GC clamp makes this method universally applicable, since the melting temperature of one allele can be adjusted as necessary to give the distinctive separation of melting curves. Therefore, this new method is simple, fast and accurate for determining the presence of the BDNF Val66Met polymorphism. It may also be useful for the analysis of other SNPs in pharmacogenetic studies.
Sánchez-Romero
María Antonia
MA
Clinical Research Centre (CICAB), Extremadura University Hospital, Servicio Extremeño de Salud (SES), 06080 Badajoz, Spain.
Dorado
Pedro
P
Guarino
Estrella
E
Llerena
Adrián
A
D000483:Alleles; D052160:Benzothiazoles; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006358:Hot Temperature; D006801:Humans; D008715:Methionine / Q000235:genetics*; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011804:Quinolines; D012559:Schizophrenia / Q000209:etiology / Q000235:genetics; D013997:Time Factors; D014633:Valine / Q000235:genetics*; D044465:White People / Q000706:statistics & numerical data
D052160:Benzothiazoles; D019208:Brain-Derived Neurotrophic Factor; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA; D008715:Methionine; D014633:Valine