Abstract Detail

Phylogenomics

Kang, Myeongbo [1], Chanderbali, Andre [2], Lee, Seungyeon [3], Soltis, Pamela [4], Soltis, Douglas [4], Kim, Sangtae [3].

High-molecular-weight DNA extraction for sequencing plant genomes – an optimization of standard methods.

Developing an effective and easy-to-use high-molecular-weight (HMW) DNA extraction method is essential for genomic research, especially in the era of 3rd generation sequencing. However, even if a technology capable of generating long-read sequence is used, this technology cannot efficiently be applicable if the length of the extracted DNA is short. We suggest a standard HMW DNA extraction method for various plant groups, which combines 1) a nuclei extraction method followed by 2) a traditional CTAB DNA extraction method for plants (http://amborella.net/Kim_Lab_Protocols/Kim_Lab_HMW_DNA_protocol_v20220403.pdf). To evaluate the effectiveness of this method for genomic studies, we compared the products of this method with those of a commercial DNA extraction kit (Exgene™ Plant SV, GeneAll Biotechnology) based on DNA binding filters for 18 taxa from different plant orders. We checked 1) the highest peak and percentage of DNA fragments >20 kbp in the electropherogram from the Femto Pulse System and 2) the purity of extracted DNA using the Nanodrop. Although results varied depending on the taxon, our protocol produced DNA fragments (percentage of >20 kbp) that were, on average, ca. five times longer than those from a commercial kit. The absorbance ratio of 260/280, which indicates protein contamination, in both methods showed similar results with low contamination (average 1.82 each). However, our protocol more effectively removed carbohydrates and organic solvents (average 260/230=1.77) than a commercial kit (average 260/230=1.32). We also discuss factors influencing the results: pipetting number, grinding time in liquid nitrogen, and g value in centrifugation. The efficacy of our HMW DNA extraction method was confirmed in diverse plant taxa, and the protocol will contribute to plant genome studies as a standard protocol for unstudied taxa.

1 - Sungshin Women's University, Dept. of Biotechnology, Seoul, 01133, Republic of Korea
2 - University Of Florida, Florida Museum Of Natural History, Gainesville, FL, 32611, USA
3 - Sungshin University, Department of Biology, Seoul, 01133, Republic of Korea
4 - University Of Florida, Dept. of Biology, Gainesville, FL, 32611, USA

Keywords:
DNA extraction
genomic DNA
high molecular weight DNA
plant DNA.

Presentation Type: Poster
Number: PPL003
Abstract ID:305
Candidate for Awards:None