Douglas H. White

Automated Purification of Transfection-Grade Plasmid DNA Using Wizard MagneSil Tfx System

We describe a reagent system and robotic protocol for the isolation of highly purified plasmid DNA from cultured cells. The method is based on the Wizard® MagneSil™ Plasmid Purification System, which purifies sequencing-grade plasmid DNA. Two modifications to the sequencing-grade system were made to create the Wizard MagneSil Tfx™ System. This system provides lower endotoxin and other contaminant levels, giving higher quality plasmid for transfection applications. The Wizard MagneSil Tfx™ System uses MagneSil™ Paramagnetic Particles (PMPs) to clear lysate and bind plasmid, eliminating the need for filtration devices. The endotoxin removal step uses MagneSil™ (PMPs) and a guanidine/isopropanol wash to remove RNA and protein. One 96-well plate may be processed in 45 minutes on the Beckman Biomek® FX robotic workstation. We provide data showing DNA yield, contaminant levels, and transfection efficiency for 5 commonly used cell lines. Comparisons with other systems are also shown.

Abstract 492: Automated circulating cell-free DNA purification from large volume draws

Circulating, cell-free DNA (ccfDNA) has emerged as an important tool in molecular oncology research. Because it is highly fragmented, present in small quantities, and highly susceptible to degradation, purification of ccfDNA poses unique challenges to researchers. While plasma is the main focus of many researchers for ccfDNA, recent work has shown that it is present in other biological fluids. Promega has developed a unique chemistry to selectively purify ccfDNA from plasma. The Maxwell RSC ccfDNA Plasma system is a completely automated system that allows purification of ccfDNA from 1ml of plasma. To add flexibility, we adapted the chemistry to automated platforms in 24-well configurations. These platforms include the Hamilton Microlab STAR series and the KingFisher Flex Processor. Because the biomarkers in ccfDNA can be of very low frequency, many researchers prefer to process volumes of samples >1ml which can exceed the capacity of many purification systems. Using a sequential bind strategy, we demonstrate that ccf DNA can be purified from at least 8ml sample draw using a fully automated method. Liquid draws tested include plasma and urine. DNA quantity was assessed by qPCR using an autosomal target, and quality was assessed using an internal PCR control. This study shows the flexibility and robustness of the Maxwell RSC ccfDNA chemistry. We successfully adapted it for purification from relatively large volume liquids in a fully automated manner making this a convenient option for early biomarker research. Citation Format: Robert Ray, Mark Bratz, Doug Wieczorek, Douglas White, Douglas Horejsh, Eric Vincent, Trista Schagat. Automated circulating cell-free DNA purification from large volume draws. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 492.

Abstract 1872: A novel method for efficient and hands-free purification of circulating DNA from human plasma

Circulating or Cell-Free DNA in plasma can be used to detect biomarkers that show great promise for diagnosis and monitoring of cancer, and is already being used as a non-invasive method to detect trisomy in fetuses. There is currently great interest in the discovery of new cancer biomarkers and their potential clinical application. However, reproducible and efficient purification of these highly fragmented and low-concentration species represents a major challenge. Here we will present a novel method which is completely automated and allows parallel purification of circulating nucleic acid from plasma and serum using a medium-throughput robot. Sixteen samples can be processed simultaneously. The method was optimized to produce high-quality DNA that is suitable for use in quantitative PCR and next generation sequencing. In addition, the absence of any pre-processing steps improves reproducibility and lowers the risk of contamination.Initial characterization on plasma from pregnant women showed that fetal DNA could be detected as early as 4 weeks into gestation and could be tracked throughout pregnancy. Further characterization showed that the system was able to reliably detect less than 25 copies/ml plasma of fragmented DNA that was spiked into the sample. Subsequent work on plasma from patients with colorectal cancer showed that the system was able to detect DNA containing both wild-type and mutated EGFR, suggesting that this method can be a useful tool when screening plasma samples for biomarkers of interest. Citation Format: Douglas White, Douglas Horejsh, Zhiyang Zeng, Tetsuo Uyeda, Poncho Meisenheimer, Marjeta Urh. A novel method for efficient and hands-free purification of circulating DNA from human plasma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1872. doi:10.1158/1538-7445.AM2014-1872