DNA is the premier material for directing nanoscale self-assembly, having been used to produce many complex forms. Recently, DNA has been used to direct colloids and nanoparticles into novel crystalline structures, providing a potential route to fabricating meta-materials with unique optical properties. Although theory has sought the crystal phases that minimize total free energy, kinetic barriers remain essentially unstudied. Here we study interfacial equilibration in a DNA-directed microsphere self-assembly system and carry out corresponding detailed simulations. We introduce a single-nucleotide difference in the DNA strands on two mixed microsphere species, which generates a free-energy penalty for inserting 'impurity' spheres into a 'host' sphere crystal, resulting in a reproducible segregation coefficient. Comparison with simulation reveals that, under our experimental conditions, particles can equilibrate only with a few nearest neighbours before burial by the growth front, posing a potential impediment to the growth of complex structures.