High-resolution melting analysis for bird sexing: a successful
Data
2013
Autores
Cabral, João Alexandre
Título da revista
ISSN da revista
Título do Volume
Editora
Blackwell Publishing Ltd
Resumo
High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/
specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of
PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable
the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the
length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research
areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations.
Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sexspecific
CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed
(plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced
the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination
of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp.
The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity)
of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and
limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing
of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an
excellent approach for improving advanced molecular methods for bird sexing.
Descrição
Palavras-chave
Birds , CHD1 gene , High-resolution melting analysis , Molecular sex identification , Real-time PCR
Citação
Morinha F. el al, 2014