Investigation of macromolecular crowding on melting analysis.

• Main Author: Yap, Wee Min.
• Other Authors: School of Biological Sciences
• Format: Final Year Project
• Language: English
• Published: 2010
• Subjects: DRNTU::Science::Biological sciences::Biophysics
• Online Access: http://hdl.handle.net/10356/42526
• Institution: Nanyang Technological University

Biological macromolecules have evolved to function in a crowded environment. Although scientists have been able to exploit these biomolecules by recreating their biochemical environment in the laboratory, the biophysical component of macromolecular crowding (MMC) has been overlooked. Recently, MMC had been introduced into a common laboratory technique – RT-PCR and found that this resulted in a significant improvement in PCR sensitivity. In this paper, the concept of MMC was investigated in the Melting analysis. One limitation in melting analysis was the differentiation of nucleotides with high sequences similarity. It was thus hypothesized if the introduction of MMC could enhance the melting analysis. MMC was introduced into the reaction mixture by the addition of a mixture of Ficoll70 (15mg/ml) and Ficoll400 (5mg/ml). It was found that MMC resulted in better separation of melting curves of mutants from the wild type (easier diagnosis) in direct amplicon melting analysis. The effect of MMC on the separation of melting curves, though consistent, showed high variability in the extent of separation. These better curves separation was not seen when MMC was introduced into probe based melting analysis. Instead, only a general shift of melting profiles to the right was detected with no other visible improvements.