Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue

Area of Science:

• Molecular Biology
• Plant Genomics
• Biotechnology

Background:

• High-quality genomic DNA (gDNA) is essential for next-generation sequencing (NGS) applications, particularly in plant breeding.
• Existing protocols for plant gDNA extraction, including the nuclear pellet (NP) method for maize, often yield DNA insufficient for NGS requirements.
• Reliable and accessible methods for high-quality plant gDNA isolation are needed for large-scale genomic studies.

Purpose of the Study:

• To optimize the nuclear pellet (NP) method for extracting high-quality genomic DNA (gDNA) from rice (Oryza sativa L.) suitable for next-generation sequencing (NGS).
• To develop a robust and accessible protocol for researchers with limited resources.
• To ensure the extracted gDNA meets the stringent quality standards for Illumina sequencing platforms.

Main Methods:

• Optimization of the nuclear pellet (NP) method involving nucleus enrichment, cell lysis, and extraction.
• Integration of purification steps including proteinase K and RNase A treatments.
• Application of phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol for removal of proteins, RNA, and phenol.

Main Results:

• The optimized NP method consistently yielded high-quality, undegraded genomic DNA (gDNA) from rice leaves.
• The extracted gDNA was free from protein and RNA contamination.
• The gDNA met the quality metrics required for NGS library preparation using the Illumina HiSeq X Ten platform and TruSeq DNA PCR-Free Library Prep Kit.

Conclusions:

• The optimized NP method provides a reliable and accessible protocol for high-yield, high-quality gDNA extraction from rice.
• This method facilitates NGS library preparation for rice whole-genome sequencing.
• The protocol is valuable for molecular biologists, especially those with limited laboratory resources.