Development of a New Genotyping Assay for Detection of the BDNF Val66Met Polymorphism Using Melting-Curve Analysis

Abstract

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.

KEYWORDS::

• BDNF
• melting curve
• schizophrenia patients
• SYBR
• Val66Met polymorphism

Acknowledgements

The collaboration of Eva Peñas-LLedó MD PhD and Alfredo de la Rubia MD PhD is gratefully acknowledged.

Financial & competing interests disclosure

This study was supported in Spain by the Ministries of Education and Science (SAF2006/13589) and Health, Instituto de Salud Carlos III-FIS and European Union (FEDER): CIBERSAM, PI06/1681 and CP/06/0030 (Pedro Dorado). The study was coordinated in the network Red Iberoamericana de Farmacogenética y Farmacogenómica (CYTED206RT0290). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This study was supported in Spain by the Ministries of Education and Science (SAF2006/13589) and Health, Instituto de Salud Carlos III-FIS and European Union (FEDER): CIBERSAM, PI06/1681 and CP/06/0030 (Pedro Dorado). The study was coordinated in the network Red Iberoamericana de Farmacogenética y Farmacogenómica (CYTED206RT0290). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed