Development of a Genotyping Method by Novel Melting Curve Analysis
Date Issued
2009
Date
2009
Author(s)
Su, Hsiao-Han
Abstract
This work proposes a new method of melting curve analysis. The fluorescence would change in temperature as a function of distance. The samples in glass capillary were melted by a heating surface with temperature gradient. The fluorescence intensity was decreased along with the distance. The melting temperature can be calculated by analyzing temperature versus position with the change in sample fluorescence. The first main part of experiment is system testing, including the temperature distribution of heating surface, the exposure time and gain of the optical device, the thermal-induced reaction time of fluorescence, and the variation analysis within different channels. The experimental results indicate that the characteristic of new MCA platform are described as follow. (1) By using two ends of heating/cooling system, a steady temperature gradient can be induced. (2) The value of exposure time and gain will affect the noise signal. The image intensity and sensitivity can be enhanced by increasing exposure time appropriately. But gain should be kept short. (3) The transient effects associated with the material and wall thickness of the sample container should be considered. (4) The variation in analytic results of different channels can be neglect. The second main part is the experiment of new melting curve analysis. For proof-of-concept testing, several DNA sample has been investigated. The results from new melting curve analysis were compared to the commercial instrument. Further, we also successfully distinguished the genotyping of 1:1 mixing sample. This work demonstrated the platform of the distance-dependent melting curve analysis by simple two-ends heating/cooling devices. The cost of system can be reduced significantly. The time-independent method has the potential advantages in automation and parallel detection. One application could be in the field of SNP detection. Combine the new melting curve analysis with PCR systems has a promising future and it offers much the same functionality.
Subjects
Linear Temperature Gradient
Genotyping
Melting Curve Analysis
Single Nucleotide Polymorphisms
Type
thesis
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