Our objective is to provide a homogeneous, closed-tube system for DNA mutation scanning on a high-throughput platform. For many complex genetic diseases, it is very difficult and expensive to screen for all possible mutations that may cause the disease. We propose a relatively simple solution. Certain dyes are compatible with PCR and can detect sequence alterations present in one copy of DNA by simple melting analysis after amplification. The dye is added before PCR, and optionally followed in real-time during amplification. Heterozygotes are easily identified after a 5 min. high-resolution melting curve after PCR.
Specific aims for Phase I of this Fast Track proposal include: 1. Identify and/or synthesize scanning dyes that can be used with standard fluorescein optics. 2. Demonstrate that heterozyotes can be identified by high-resolution melting on a 384-well platform. 3. Achieve 90% sensitivity and specificity for single base change heterozygote detection in PCR products up to 300 bps in length. Our currently manufactured 384-well, low-resolution genotyping instrument will be modified for high-resolution scanning. A robust DNA scanning dye for use with standard fluorescein optics will be identified and/or synthesized for this instrument and potentially others. A comprehensive screen of single base mismatches in engineered plasmids will be used to establish the sensitivity and specificity of the system, and provide the criteria for Phase II funding. Advantages of high-resolution melting analysis for mutation scanning include speed (< 5 min), homogeneous design (no need for automation), closed-tube analysis (no amplicon contamination risk), and sample disposition (immediately available for sequencing if necessary).
| Wittwer, Carl T (2010) Making DNA melting useful. Clin Chem 56:1500-1 |
| Hill, Harry R; Augustine, Nancy H; Pryor, Robert J et al. (2010) Rapid genetic analysis of x-linked chronic granulomatous disease by high-resolution melting. J Mol Diagn 12:368-76 |
| Erali, Maria; Wittwer, Carl T (2010) High resolution melting analysis for gene scanning. Methods 50:250-61 |
| Wittwer, Carl T (2009) High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30:857-9 |
| Lyon, Elaine; Wittwer, Carl T (2009) LightCycler technology in molecular diagnostics. J Mol Diagn 11:93-101 |
| Erali, Maria; Voelkerding, Karl V; Wittwer, Carl T (2008) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85:50-8 |
| Elenitoba-Johnson, Oluwole; David, Derek; Crews, Niel et al. (2008) Plastic versus glass capillaries for rapid-cycle PCR. Biotechniques 44:487-8, 490, 492 |
| Montgomery, Jesse; Wittwer, Carl T; Kent, Jana O et al. (2007) Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis. Clin Chem 53:1891-8 |
| Vandersteen, Joshua G; Bayrak-Toydemir, Pinar; Palais, Robert A et al. (2007) Identifying common genetic variants by high-resolution melting. Clin Chem 53:1191-8 |
| Poulson, Matthew Dean; Wittwer, Carl T (2007) Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis. Biotechniques 43:87-91 |
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