Our objective is to provide a closed-tube system for rapid PCR, mutation scanning and genotyping that does not require fluorescently-labeled probes. For many genetic diseases, it is difficult and expensive to screen for all possible mutations that may cause the disease. We propose a relatively simple solution for rapid amplification, scanning and genotyping in a single homogeneous system. Certain DNA dyes are compatible with PCR and can detect mismatches between two copies of DNA by simple melting analysis after amplification. The dye is added before PCR, and heterozygotes are easily identified after a 1-2 min high-resolution melting curve after PCR. Specific genotype confirmation is provided by an unlabeled oligonucleotide probe.
The specific aims for Phase I of this Fast Track proposal are: 1. Develop a homogeneous, closed-tube gentotyping method that uses unlabeled oligonucleotides. 2. Prototype a rapid PCR system that integrates high-resolution melting analysis for scanning and genotyping. We will integrate the high-resolution melting capability of our HR-1 instrument into our rapid PCR machine (R.A.P.I.D./LightCycler). After PCR, each tube will be automatically analyzed by high-resolution melting. Simultaneous scanning and genotyping will be demonstrated using special DNA dyes. Advantages of our system includes speed (PCR in 15 min, analysis in 1-2 min/sample), homogeneous design (no need for sample transfer or reagent additions), closed-tube analysis (no amplicon contamination risk), both scanning and genotyping capability, and nondestructive analysis (immediately available for sequencing in the rare case when it is necessary). ? ?
| Erali, Maria; Wittwer, Carl T (2010) High resolution melting analysis for gene scanning. Methods 50:250-61 |
| 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 |
| 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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