Rex M. Bitner

Use of MagneSil(TM) paramagnetic particles for plasmid purification, PCR cleanup, and purification of dideoxy and big dye DNA sequencing reactions

Traditional anion exchange purification of nucleic acids requires the elution of the DNA or RNA in a salt solution, necessitating the precipitation or desalting of the nucleic acid prior to many molecular biology applications. A pH dependent anion exchange purification method is described which allows the purification of nucleic acids at one pH, followed by the elution of the nucleic acid in a low salt buffer at a second, higher pH. The benefits of this method include the avoidance of alcohol washes and the drying steps required for alcohol removal, as well as the benefits of anion exchange purification without the need for desalting of the purified DNA or RNA.

Purifying Genomic DNA from Whole Blood on Automated, High-Throughput, and Moderate-Throughput Platforms

We describe three new automated methods for purifying genomic DNA from whole blood. The MagneSil® Blood Genomic, Max Yield System uses MagneSil® paramagnetic particles (PMPs) in a 96-well format to purify the maximal amount of DNA from a 200-μL blood sample. In contrast, the MagneSil® ONE, Fixed Yield Blood Genomic System uses MagneSil® Fixed Yield PMPs to purify a normalized amount of DNA from 60 μL of blood in a 96-well format. These methods are implemented on the Beckman Coulter Biomek® FX automated workstation. The MagneSil® KF Genomic System uses MagneSil® PMPs to purify DNA from 1 to 15 samples of 200-μL blood using the moderate-throughput Thermo Electron KingFisher® mL instrument. The MagneSil® Blood Genomic System typically yields > 4 μg per 200 μL of whole blood, depending on the white blood cell content. The MagneSil® ONE System is best suited where there is a requirement for purification of a narrow concentration range of DNA. This system purifies 1 μg(±50%) of DNA from 60 μL of blood. The MagneSil® KF System purifies 2 to 6 μg of DNA from 200 μL of blood. DNA purified using all of these methods is suitable for PCR, STR, EADIT® SNP genotype analysis, and multiplexed PC analysis.

Automated high-throughput purification of genomic DNA from whole blood using Promega's MagneSilTM paramagnetic particles with either the Max Yield or MagneSilTM ONE normalized purification methods

Two different methods of automated high throughput purification of genomic DNA from human whole blood in 96 well plates are described. One method uses MagneSilTM paramagnetic particles to purify a maximal amount of the DNA present in the sample. Another method, the MagnesilTM ONE system, allows for the purification of a predetermined amount of DNA from human whole blood. Protocols for the purification of 100 ng or, alternatively 1 ug, of human genomic DNA from whole blood using MagneSilTM paramagnetic particles and a Beckman BioMekTM FX robot are described. With the maximal yield purification system, typical DNA yields fall in the range of 4-9 ug of DNA from 200ul of human whole blood, depending upon the white cell content of donor sample. For situations where DNA achiving is desired, or when the number of downstream sample applications is not clearly defined (e.g. multiple SNP analyses) the maximal yield method is usually preferred. However, in situations with a defined downstream application (e.g. criminal databasing or use of a defined set of amplifications) where purifying DNA in a narrow concentrate range streamlines the high throughput purification and analysis process, the automated MagneSilTM ONE purification system is the method of choice. DNA from either method is suitable for applications such as PCR, STR, READITTM SNP analysis, and multiplexed PCR systems such as Promegas Y-chromosome deletion detection system.

Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promegas DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs

MATERIALS Reagents Dithiothreitol (DTT), 1 M DNA IQ System (Promega; includes Resin, Lysis Buffer, 2X Wash Buffer, and Elution Buffer) Ethanol, 95-100% Isopropanol Sample (liquid blood, cotton swab, th CEP swab, 15-50 mm2 S&S paper, 3-30 mm2 FTA paper) Equipment Aerosol-resistant micropipette tips DNA IQ Spin Baskets (Promega) Heating blocks, preset to 65°C and 95oC MagneSphere Technology Magnetic Separation Stand (Promega)

Purifying Genomic DNA from Plant Tissue on Automated High-Throughput and Moderate-Throughput Platforms

We describe automated methods for purifying genomic DNA from plant tissue. The Wizard Magnetic 96 Plant System uses MagneSil paramagnetic particles (PMPs) in a 96-well format to purify sufficient DNA for polymerase chain reaction (PCR)-based plant genotyping. The system can be scaled up for high yield to provide genomic DNA for large numbers of PCR-based tests or archiving. In contrast, the MagneSil ONE Fixed Yield System uses MagneSil ONE PMPs to purify a fixed amount of ultraclean DNA for high sensitivity in Third Wave Technology Invader single nucleotide polymorphism (SNP) Genotyping assays. These methods may be implemented on Beckman Coulter Biomek automated workstations.

Automated high-throughput purification of genomic DNA from plant leaf or seed using MagneSil paramagnetic particles

Three different methods of automated high throughput purification of genomic DNA from plant materials processed in 96 well plates are described. One method uses MagneSil paramagnetic particles to purify DNA present in single leaf punch samples or small seed samples, using 320ul capacity 96 well plates which minimizes reagent and plate costs. A second method uses 2.2 ml and 1.2 ml capacity plates and allows the purification of larger amounts of DNA from 5-6 punches of materials or larger amounts of seeds. The third method uses the MagneSil ONE purification system to purify a fixed amount of DNA, thus simplifying the processing of downstream applications by normalizing the amounts of DNA so they do not require quantitation. Protocols for the purification of a fixed yield of DNA, e.g. 1 ug, from plant leaf or seed samples using MagneSil paramagnetic particles and a Beckman-Coulter BioMek FX robot are described. DNA from all three methods is suitable for applications such as PCR, RAPD, STR, READIT SNP analysis, and multiplexed PCR systems. The MagneSil ONE system is also suitable for use with SNP detection systems such as Third Wave Technology’s Invader methods.

Automation of DNA extraction from food and plants using MagneSil paramagnetic particles

MagneSil paramagnetic particles allow the flexibility of automating the isolation of DNA from as little as 20mg of plant material to as much as 500 grams of vegetable oil for use in testing for DNA sequences from genetically modified organisms (GMO), or plant breeding applications such as random amplification polymorphism detection (RAPD) or polymerase chain reaction (PCR). Given the wide variety of plant materials, foods and highly processed food ingredients that require testing, the purification system must be both scalable and flexible in its ability to purify DNA from such a wide array of sample types. The procedures used in these purification systems are similar to other methods used for the walkaway automation of plasmid purification and DNA sequencing reaction cleanup used in genomics applications, as well as DNA purification of DNA from PCR reactions used for genetic interogations or DNA immobilizations. These purification systems can be used with a variety of robotic workstations in 96 well formats.