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High‐resolution melt analysis without DNA extraction affords rapid genotype resolution and species identification
Author(s) -
RugmanJones Paul F.,
Stouthamer Richard
Publication year - 2017
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12599
Subject(s) - amplicon , biology , genotyping , dna extraction , high resolution melt , real time polymerase chain reaction , polymerase chain reaction , genotype , fluorescence , dna , primer dimer , dna sequencing , computational biology , microbiology and biotechnology , genetics , gene , physics , quantum mechanics , multiplex polymerase chain reaction
Extracting and sequencing DNA from specimens can impose major time and monetary costs to studies requiring genotyping, or identification to species, of large numbers of individuals. As such, so‐called direct PCR methods have been developed enabling significant savings at the DNA extraction step. Similarly, real‐time quantitative PCR techniques ( qPCR ) offer very cost‐effective alternatives to sequencing. High‐resolution melt analysis ( HRM ) is a qPCR method that incorporates an intercalating dye into a double‐stranded PCR amplicon. The dye fluoresces brightly, but only when it is bound. Thus, after PCR , raising the temperature of the amplicon while measuring the fluorescence of the reaction results in the generation of a sequence‐specific melt curve, allowing discrimination of genotypes. Methods combining HRM (or other qPCR methods) and direct PCR have not previously been reported, most likely due to concerns that any tissue in the reaction tube would interfere with detection of the fluorescent signal. Here, we couple direct PCR with HRM and, by way of three examples, demonstrate a very quick and cost‐effective method for genotyping large numbers of specimens, using Rotor‐Gene HRM instruments ( QIAGEN ). In contrast to the heated‐block design of most qPCR / HRM instruments, the Rotor‐Gene's centrifugal rotor and air‐based temperature‐regulation system facilitate our method by depositing tissues away from the pathway of the machine's fluorescence detection optics.