Thermal Trap for DNA Replication

The hallmark of living matter is the replication of genetic molecules and their active storage against diffusion. We implement both in the simple nonequilibrium environment of a temperature gradient. Convective flow both drives the DNA replicating polymerase chain reaction while concurrent thermophoresis accumulates the replicated 143 base pair DNA in bulk solution. The time constant for accumulation is 92 s while DNA is doubled every 50 s. The experiments explore conditions in pores of hydrothermal rock which can serve as a model environment for the origin of life.

Figure 1

Trapping and replicating DNA by thermal convection. (a) As molecules are directed to the right by thermophoresis, they are transported downward by the convection and accumulate at the bottom of the chamber. (b) Convection provides a temperature oscillation to periodically denature DNA with subsequent binding of short primers and replication by a polymerase protein. As a result, DNA is duplicated in each convection cycle. (c) A borosilicate capillary with rectangular cross section is heated symmetrically with an infrared laser. Directed scanning provides a convectionlike flow pattern due to light-driven microfluidics [20, 21].

Figure 2

Accumulation of DNA. (a) In the chamber center, the convection flow was tracked with $1\mu \mathrm{m}$ particles. The chamber averaged flow speed is $45\mu \mathrm{m}/\mathrm{s}$. (b) The corresponding flow profile for the finite element simulation. (c) The temperature profile was measured using a temperature dependent dye [8]. (d) Molecular trajectories by the superposition of fluid flow and thermophoresis. (e) Fluorescence images show the DNA accumulation over time. (f) The concentration pattern over time is reasonably described theoretically. (g) The kinetics of central accumulation is well described theoretically. (h) Optimal convection speed for accumulation is around $2.5\mu \mathrm{m}/\mathrm{s}$. The experimental conditions favor faster PCR replication under reduced accumulation.

Figure 3

Replication Trap. (a) Accumulation of PCR product by convection and thermophoresis, visualized by SYBR Green fluorescence. (b) No DNA is found without template DNA. (c) Center concentration over time and fits with Eq. (6). Delays of replication due to different template concentrations allow us to infer the replication doubling time with ${\tau }_{\mathrm{doubling}}=50\mathrm{s}$.

Figure 4

Verification of the product. (a) Gel electrophoresis of product from a standard PCR cycler. Different primers yield product lengths of 86, 143, and 1530 base pairs. (b) In the capillary, the product is identified using thermophoresis [27, 28]. Upon local heating with a laser spot, DNA is locally depleted. The amplified product matches the 143 base pair product and is well bracketed by 86 mer and 1530 mer product.