Five laboratories of the Department of Dermatology and Cutaneous Biology and the Division of Rheumatology/Department of Medicine of the Jefferson Medical School with NIH-funded laboratory projects are requesting funding for a WAVE DNA fragment analysis system (Transgenomic Inc.). Although our laboratories focus on different research areas in molecular genetics and molecular biology, we have overlapping strategies and utilize similar methodology and technology, such as mapping and cloning of disease genes, mutation detection, detection and analysis of polymorphic DNA markers, RT-PCR and purification of DNA fragments. The requested equipment is versatile, can be utilized for all of the listed applications, and can be shared to benefit all users of our departments. Most importantly, it is superior to other methods of mutation/polymorphism detection. We calculate based on our current sample volume that our 5 research laboratories combined accrue approximately 33,000 samples per annum for these applications. In addition, the requested equipment will be available to other users in our departments with an estimated volume of 1,700 analyses per annum. Therefore, we anticipate utilizing the instrument system effectively and at full capacity. The requested equipment is a fully automated, integrated nucleic acid fragment analysis system based on high-performance liquid chromatography (HPCL). The system includes the following components: HPCL analyzer with patented separation cartridge with guaranteed performance of up to 4000 injections; an autosampler formatted for 96- well microtiter plates; analytic software (WAVEMaker); an accelerator for rapid cartridge cleaning and re-equilibration; and a nucleic acid fragment collector. The WAVE system is the only instrument platform with all of the above specifications. It has 3 possible modes of operation: nondenaturing, partial denaturing, and full denaturing conditions, which allow a wide range of applications. It can be used either with UV/visible absorbance detection or high sensitivity fluorescence detection to perform rapid, automated separation and quantification of single- and double- stranded nucleic acid fragments. While the UV detection is sufficient for mutation screening and marker analysis, more sensitive fluorescence detection is required for automated genotypic and RT-PCR. The versatility of this instrument system is a major factor that allows shared usage of the system by many laboratories with different applications. It has been demonstrated that the requested equipment has several convincing advantages over other technologies currently employed in our research labs. These include high accuracy and sensitivity with a mutation detection rate of almost 100%, which is far superior to SSCP and CSGE, versatility and cost-effectiveness due to automated injection, high throughput, and low per sample costs. In summary, usage of the WAVE Nucleic Acid Fragment Analysis will substantially reduce time and cost of our research projects involving mutation screening, polymorphism analysis, competitive RT-PCR and purification of nucleic acids. The very high sensitivity of this system for detection of sequence variations makes it a superior screening method. It is highly suitable for analysis of a set of genes in a large number of individuals as well as identification of mutations and polymorphisms in many genes of interest.
| Pfendner, Ellen G; Nakano, Aoi; Pulkkinen, Leena et al. (2003) Prenatal diagnosis for epidermolysis bullosa: a study of 144 consecutive pregnancies at risk. Prenat Diagn 23:447-56 |
