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Feature Articles : Apr 15, 2008 ( )
Increasing Yield in Nucleic Acid Sample Prep
Researchers Discuss Ways to Combat Sample Diversity and Degradation Issues
Extraction and purification of nucleic acids from biological materials, whether for research use such as genomic analysis, gene-expression studies, and genotyping or clinical applications like pharmacogenomics, diagnostic and prognostic use, or forensics, involve many of the same challenges. These include the diversity of sample types, the susceptibility of DNA and RNA to nucleases present in cell lysates, and the demand for high-quality, pure nucleic acid suitable for amplification and quantification, use in microarrays, and storage/sample archiving.
Tepnel’s nucleic acid prep kits are based on the company’s Nucleon™ extraction chemistry. The Nucleon HT DNA extraction system can be used to extract DNA from paraffin-embedded tissues or other hard tissues such as mouse tail that require proteinase K digestion for cell lysis. The protocol takes less than 40 minutes and yields more than 40 µg of high purity (A260/280=1.8–1.9) and high molecular weight (>50 kb) DNA per cm of mouse tail, according to Dr. Dodsworth.
BioServe recently moved into its new headquarters in Beltsville, MD, and also maintains a facility in Hyderabad, India. This new, larger home has the capacity to store the Global Repository®, which is composed of more than 600,000 DNA, tissue, and serum samples together with associated clinical data, which BioServe gained as part of its acquisition of Genomics Collaborative one year ago.
“Extraction of nucleic acids is a core competency of the company,” says Dr. Krenitzky. “The first step in mining this data is extraction, purification, and QC of the nucleic acid. We have already completed this on about 100,000 samples. Using our multianalyte purification system, we can extract DNA, RNA, and protein.”
Dr. Krenitzky has seen a shift in the market over the past 12 months or so from DNA extraction to isolating RNA from biological samples, which yields information about differential gene expression.
In the area of breast cancer diagnostics, for example, while genetic analysis provides a picture of the DNA one inherits, which is basically static except for mutation events, identification and quantification of RNA transcripts enables a comparison of differences in gene expression between normal and tumor tissue.
Dr. Krenitzky describes a trend in which, by comparing the RNA present in tumor cells with the DNA from the peripheral blood of the same person, researchers can begin to understand how an individual’s genetic profile combined with environmental risk factors affect changes in gene expression.
BioServe has developed processes and reagents for nucleic acid extraction and optimizes those for each type of sample. For example, it has developed a protocol for isolating DNA from breast tissue, which is a particularly challenging sample to work with because of its high fat content. The company has also optimized an extraction process for purifying DNA from blood clots.
All processes are done manually. “When you use a robot, you lose some control over the process, and we want to maximize yield at each step,” explains Mike Seddon, laboratory director at BioServe. Plans are also under way to commercialize the DNA Qwik™ line of DNA extraction kits, which will be followed with an RNA extraction kit.
Strategies to maximize target recovery for nucleic acid detection in clinical specimens will be the topic of a “GOT Summit” talk to be given by Bernhard Kaltenboeck, D.V.M., Ph.D., professor and director of the molecular diagnostics lab, department of pathobiology, at Auburn University.
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