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.
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 Promega's Y-chromosome deletion detection system.
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 Promega's 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.
MagneSilTM paramagnetic particles allow the flexibility ofautomating the isolation ofDNA from as little as 20mg ofplant material to as much as 500 grains ofvegetable 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 ofsample types. The procedures used in these purification systems are similar to other methods used for the walkaway automation ofplasmid purification and DNA sequencing reaction cleanup used in genomics applications, as well as DNA purification ofDNA from PCR reactions used for genetic interogations or DNA immobilizations. These purification systems can be used with a variety ofrobotic workstations in 96 well formats.
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.
Conference Committee Involvement (5)
Molecular Probes for Biomedical Applications II
21 January 2008 | San Jose, California, United States
Microarrays, Combinatorial Techniques, and High Throughput Screening
27 January 2005 | San Jose, CA, United States
Microarrays, Combinatorial Techniques and High Throughput Screening
25 January 2004 | San Jose, CA, United States
Microarrays and Combinatorial Technologies for Biomedical Applications: Design, Fabrication, and Analysis
26 January 2003 | San Jose, CA, United States
Genomics, Proteomics, and Related Technologies for Biomedical Applications
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