Abstract
The cheap and easy identification of species is necessary within multiple fields of molecular biology. The use of high-resolution melting (HRM) of DNA provides a fast closed-tube method for analysis of the sequence composition of the mitochondrial genes 12S rRNA and cytochrome b. We investigated the potential use of HRM for species identification within eleven different animal groups commonly found in Europe by animal-group-specific DNA amplification followed by DNA melting. Influence factors as DNA amount, additional single base alterations, and the existence of mixed samples were taken into consideration. Visual inspection combined with mathematical evaluation of the curve shapes did resolve nearly all species within an animal group. The assay can therefore not only be used for identification of animal groups and mixture analysis but also for species identification within the respective groups. The use of a universal 12S rRNA system additionally revealed a possible approach for species discrimination, mostly by exclusion. The use of the HRM assay showed to be a reliable, fast, and cheap method for species discrimination within a broad range of different animal species and can be used in a flexible “modular” manner depending on the question to be solved.
Highlights
During the last years, analysis of non-human DNA got more important especially in the fields of food analysis, forensic science, and ecological studies [1,2,3,4]
high-resolution melting (HRM) DNA Analysis for Species Differentiation identification is widely performed by using standard methods as Sanger sequencing and Restriction fragment length polymorphism (RFLP) methods [5] analyzing mostly mitochondrial genes for cytochrome b, 12S ribosomal ribonucleic acid (12S rRNA) or cytochrome oxidase I (COXI) [5,6,7,8,9,10,11]
Not all the known species contained in an animal group were considered for analysis due to limitations in sampling and to reliable and successful amplification (e. g. too many differences in the primer sequence led to the exclusion of water buffalo from the Bovini tribe assay)
Summary
Analysis of non-human DNA got more important especially in the fields of food analysis, forensic science, and ecological studies [1,2,3,4]. HRM DNA Analysis for Species Differentiation identification is widely performed by using standard methods as Sanger sequencing and Restriction fragment length polymorphism (RFLP) methods [5] analyzing mostly mitochondrial (mt) genes for cytochrome b (cytb), 12S ribosomal ribonucleic acid (12S rRNA) or cytochrome oxidase I (COXI) [5,6,7,8,9,10,11]. Both methods are time-consuming and susceptible to contamination due to a multi-step process.
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