An investigation into the application of microfluidics to the analysis of chromosome conformation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular BioScience at Massey University, Albany, New Zealand

Abstract

Ever since the discovery of DNA, biologists have been striving to unravel its mysteries. Many efforts have been made over the years to further our understanding of genes, what they do and how they function. Genomes exist as a 3D structure inside the nucleus and they are not randomly arranged. However, there are still many gaps in the knowledge of how the structure fills this 3D space. Using chromosome conformation capture (3C) and other methods based on proximity ligation, interactions between different sections on the chromosome can be captured. A computer simulated 3D chromosome model can then be created based on the interaction data. Currently, global interaction maps can only be created for populations of cells. The overall goal of this research is to develop a protocol that will enable the capture of chromosome interactions within a single cell. This requires the use of microfluidic chips due to the minute quantity of DNA within a single cell. Therefore the main objectives of this research are to: 1) build and test a microfluidic system (lab-on-a-chip or LOC) that will aid in the capture of interand intra- chromosomal interactions of a single cell; and 2) characterize the restriction and ligation of DNA that will be performed in a microfluidic system. In order to assess the efficiency of DNA digestion within microfluidic chips, EcoRI and MspI digestion kinetics within microtubes is first characterized to establish a base line for comparison with digestion kinetics within microfluidic chips. The Km, Vmax and Kcat for EcoRI within microtubes are 32 nM, 0.14 nM s-1 and 1.4 fmol s-1 U-1 respectively. The Km, Vmax and Kcat for MspI within microtubes are 125 nM, 1.46 nM s-1 and 29.2 fmol s-1 U-1 respectively. On the other hand, the digestion kinetics within microfluidic chips is undetermined, because both restriction enzymes exhibit non-specific nuclease activity within microfluidic chips under the conditions tested. The exhibition of non-specific nuclease activity is unexpected and causes ligation of DNA performed in microfluidic chips to fail. The non-specific nuclease activity of EcoRI and MspI within microfluidic chips is also problematic for the overall goal of developing a protocol that will enable the capture of chromosome interactions within a single cell, because the non-specific nuclease activity would cause loss of template and random variations in results obtained.