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
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
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.