Abstract
High-resolution melting (HRM) is a post-PCR method that allows to discriminate genotypes based on fluorescence changes during the melting phase. HRM is used to detect mutations or polymorphisms (e.g. microsatellites, SNPs, indels). Here, the (TTTAT)3-5 microsatellite polymorphism within intron 6 of the LDHA gene in pigeons was analysed using the HRM method. Individuals (123 homing pigeons) were genotyped using conventional PCR. Birds were classified into groups based on genotype type and the results were tested by qPCR-HRM and verified using sequencing. Based on the evaluated protocol, five genotypes were identified that vary in the number of TTTAT repeat units (3/3, 4/4, 3/4, 4/5, and 5/5). Sequencing have confirmed the results obtained with qPCR-HRM and verified that HRM is a suitable method for identification of three-allele microsatellite polymorphisms. It can be concluded that the high-resolution melting (HRM) method can be effectively used for rapid (one-step) discrimination of the (TTTAT)3-5 microsatellite polymorphism in the pigeon’s LDHA gene.
Highlights
LDHA–lactate dehydrogenase isoform A is a member of a larger LDH gene family encoding L-lactate dehydrogenase (LDH, EC.1.1.1.27) [1,2]
After polymerase chain reaction (PCR) reaction, the obtained amplicons spanning the (TTTAT)3-5 microsatellite polymorphism within intron 6 of the LDHA gene were separated on a high-resolution agarose gel and the presence of five genotypes was confirmed in the analysed population of homing pigeons (Fig 1A and 1B)
High-resolution melting (HRM) genotyping of microsatellite polymorphism identified in agarose electrophoresis demonstrated two amplicons corresponded to alleles of different length and additional amplicon above PCR product (Fig 1A–heterozygote samples–C and D, additional amplicons–heteroduplexes were marked by stars)
Summary
LDHA–lactate dehydrogenase isoform A (specific for muscle) is a member of a larger LDH gene family encoding L-lactate dehydrogenase (LDH, EC.1.1.1.27) [1,2]. Lactate dehydrogenase regulates aerobic and anaerobic metabolism, which has an significant impact on the physiological condition of the body, e.g. muscle endurance, recovery, aerobic capacity, survivability during racing competitions as well as overall physiological performance [1,2,3,4]. The latest research suggest that lactate is a specific “cellular fuel”, including hippocampal nerve cells that are responsible for consolidating information from shortterm to long-term memory, and spatial memory that determines navigation ability [3,7,8,9]. Subsequent studies demonstrated that allele A of the LDHA gene
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