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Extraction of high purity genomic DNA from pine for use in a high-throughput Genotyping Platform

Emily Telfer1*, Natalie Graham1, Lisa Stanbra1, Tim Manley2 and Phillip Wilcox1

Author Affiliations

1 Scion, 49 Sala St, Private Bag 3020, Rotorua, New Zealand

2 Formerly of AgResearch, Invermay Agricultural Centre, Mosgiel, New Zealand

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New Zealand Journal of Forestry Science 2013, 43:3  doi:10.1186/1179-5395-43-3

Published: 18 March 2013

Abstract

Standard protocols for extracting genomic DNA from Pinus radiata D. Don needles, such as CTAB-based methods, can yield large quantities of DNA. However, final DNA purity can be an issue due to carry over of contaminants that can impede accurate high throughput genotyping. This study evaluated eight DNA extraction and purification protocols to determine which method provided the greatest improvement in call rates and accuracy when using the Sequenom iPLEX® Gold MassARRAY® genotyping technology. Of the methods tested, genomic DNA extracted using the Machery-Nagel NucleoSpin®-96 Plant II kit performed the best overall, and was more efficiently and accurately genotyped than genomic DNA extracted using the standard CTAB method. This study also demonstrated that the quality and assay performance of CTAB-extracted genomic DNA is greatly improved by further purification with the Qiagen® QIAquick 96 PCR Purification kit. Using these improvements, the Sequenom iPLEX® Gold MassARRAY® genotyping technology is now a viable option for genotyping plant genomes such as Pinus radiata.

Keywords:
Genomic DNA; Genotyping; Pine; Sequenom