This application note details how the Agilent 2100 Bioanalyzer was used in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) fragment analysis for fish species identification. A 464bp cytochrome-b target sequence, found in all vertebrate fish, was first amplified and then digested with restriction enzymes. The fragments were resolved on the DNA 500 LabChip®, allowing simple comparison with authentic species profiles due to the accuracy of fragment size determination. Use of the Bioanalyzer offered significant benefits over traditional gel electrophoresis and DNA staining techniques for PCR-RFLP fragment analysis.
The document details an application of the Agilent 2100 Bioanalyzer for identifying fish species through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The method involves amplifying a 464bp cytochrome-b target sequence and digesting it with restriction enzymes to generate DNA profiles. These profiles are resolved on the DNA500 LabChip, allowing for accurate comparison with authentic species profiles.
The text outlines the materials used, including specific PCR primers, AmpliTaq Gold polymerase, and restriction enzymes from various suppliers. It describes the protocol for DNA extraction using a modified CTAB method and the subsequent PCR amplification conditions. The results section presents observed fragment sizes compared to expected values for four salmonid species (Red salmon, Pink salmon, Atlantic salmon, and Rainbow trout) when digested with five different enzymes.
A DNA500 LabChip was used to resolve the fragments.
The study used a 464bp cytochrome-b target sequence.
The Bioanalyzer offered significant benefits over traditional gel electrophoresis and DNA staining techniques.