Solid Phase DNA Amplification: A Brownian Dynamics Study of Crowding Effects

Abstract

Solid phase amplification (SPA), a new method to amplify DNA, is characterized by the use of surface-bound primers. This limits the amplification to two-dimensional surfaces and therefore allows the easy parallelization of DNA amplification in a single system. SPA leads to the formation of small but dense DNA brushes, called DNA colonies. For a molecule to successfully duplicate itself, it needs to bend so that its free end can find a matching primer, located on the surface. We used Brownian dynamics simulations (with a united-atom model) to model the basic kinetics of an SPA experiment. The simulations mimic the temperature cycles and the molecule duplication process found in SPA. Our results indicate that the steric interaction between molecules leads to a decreased duplication probability for molecules in the center of a colony and to an outward leaning for the molecules on the perimeter. These effects result in slower amplification (compared to solution PCR) and indicate that steric interaction alone can explain the loss of the exponential growth (characteristic of solution PCR) of the number of molecules in an SPA experiment. Furthermore, the growth of the colony as a function of the number of thermal cycles is found to be similar to the one obtained with a simple Monte Carlo simulation.

End-to-End Optimization of High-Throughput DNA Sequencing

“…To our knowledge, this is the first work attempting to model and analyze the cluster growth process with a view on optimizing DNA sequencing cost/yield. The detailed simulations of the surface physics associated with the bridge amplification process, [23,22], support that the disc/cluster processes we introduced earlier and will use are well suited. Work optimizing this process has taken place in industry where empirical evidence and simple rules of thumb have been used.…”

Product Length, Dye Choice, and Detection Chemistry in the Bead-Emulsion Amplification of Millions of Single DNA Molecules in Parallel

“…It is also surprising that a system using a Titanium Taq polymerase which has low processivity22 and no proofreading activity, both factors that influence normal PCR product length, produced longer products than the polymerase systems with proofreading activity (HiFi) and higher processivity (Platinum Taq). It has been suggested that in BEA6 molecular crowding on the bead’s surface might interfere with the amplification of longer products23. It is possible that the efficiency of a Titanium amplification system, or other systems with polymerases lacking 5′exonuclease activity, are less sensitive to such inhibitory conditions, an observation we already made with the extension of Alexa-coupled probes.…”

Role of Interaction on Noise-Induced Transport of Two Coupled Particles in Brownian Ratchet Devices

“…Lennard-Jones potentials (together with spring coupling) have been used to describe interaction between the monomers for a single DNA molecule in recent Brownian dynamics simulations [12,13]. In [17,18], polyelectrolyte brushes and ssDNA molecules are modelled by spring-coupled beads, with Lennard-Jones interaction terms. In this work, we concentrate on the dependence of the ratchet current on the type of interaction force between a pair of particles, in order to clarify the important similarities and differences between spring-coupling and Lennard-Jones interactions in multi-particle simulations.…”