DNA Extraction Method Selection for Insect Specimens

Main Findings:

• Compared E.Z.N.A kit, Monarch HMW DNA extraction kit, Qiagen Blood & Cell Culture DNA kit, and phenol-chloroform extraction for beetle specimens
• All tested commercial kits and phenol-chloroform extraction had limitations primarily due to insoluble DNA pellets with impurities and poor absorbance ratios
• Isolation of nuclei followed by anion-exchange chromatography resulted in optimal quality HMW DNA suitable for Nanopore sequencing
• Method optimized across different developmental stages (larvae, pupae, adults)

Relevance to Course: Demonstrates that different extraction methods perform differently depending on specimen type and downstream application requirements, justifying the need for method selection guidance.

Main Findings:

• Compared four HMW DNA extraction kits: Nanobind CBB Big DNA, Fire Monkey, Gentra Puregene Cell, and Qiagen Blood & Cell Culture DNA kit with Genomic-tip
• Significant variation in DNA yield, purity, and fragment length across methods
• Method selection depends on tissue type, downstream application, and required DNA fragment length
• HMW DNA between 50-250 kb needed for long-read methods; commercial kits may recover DNA up to 1Mb but often insufficient amounts of very HMW DNA >250kb

Relevance to Course: Provides evidence that extraction method choice significantly impacts DNA quality and downstream success, particularly important when planning for sequencing applications.

Main Findings:

• Developed method for isolating HMW DNA from small insects with hard exoskeletons
• Originally developed for BAC library construction and proven reliable for long-range optical mapping
• For insects, the least complex tissue (e.g., legs vs. whole body) works best
• Protocol emphasizes gentle lysis and minimal mechanical disruption to preserve DNA integrity

Relevance to Course: Provides foundational understanding of how specimen selection and tissue choice impact DNA quality, relevant for student sample preparation decisions.

Main Findings:

• Magnesium-based buffer systems preserve HMW DNA during extraction
• Single specimen extraction possible with optimized protocols
• Method suitable for small invertebrate specimens
• Achieved DNA fragments >100 kb suitable for long-read sequencing

Relevance to Course: Demonstrates that buffer composition (particularly Mg²⁺ concentration) is critical for HMW DNA preservation, relevant to understanding extraction chemistry principles.