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A Thermal Trap for DNA Replication

Life first appeared on Earth more than three billion years ago. Around that time the first complex chemical compounds formed, presumably in the oceans, of which subsequently the first monads consisted. However, for this to happen, the molecules probably dissolved in seawater at low concentration had to find each other. Even at this early point a form of selection should have started, which according to Darwin is the basis of evolution. We try to find conditions where first living creatures can evolve. In my experiment a simple temperature gradient managed to selectively accumulate and replicate genetic information in a solution. The dimensions of the measurement-chamber as well as the type of the temperature gradient applied were similar to the ones found in rock pores near hot undersea springs.

To show that the main ingredients of evolution, exponential replication and selection can be found under such conditions, I filled an ultra-thin glass capillary with buffer, DNA molecules, polymerase enzymes and nucleotides that are the building blocks of DNA. An infrared laser beam was then focused inside the capillary, which resulted in an elongated temperature gradient within the measurement chamber. By moving the laser beam quickly along the capillary, simultaneously a convection flow was created, which transported the DNA into the cold and warm areas of the capillary.

 
Picture: © Physical Review Letters, May 7, 2010

(a) In the chamber center, the convection flow was tracked with 1 μm particles. The chamber averaged flow speed is 45 μm/s. (b) The corresponding flow profile for the finite element simulation. (c) The temperature profile was measured by the temperature dependent fluorescence of the dye BCECF. (d) Molecular trajectories by the superposition of fluid flow and thermophoresis. (e) Accumulation of PCR product by convection and thermophoresis, visualized by SYBR Green fluorescence. (c) Center concentration over time fits with a convolution of the exponential time characteristics of the trap with the sigmoidal concentration increase of DNA by PCR. Delays of replication due to different template concentrations allows to infer the DNA replication doubling time with τ = 50s.

 

 

 

The principle of this molecule trap is based on the fact that the double-stranded DNA molecules migrate without any difficulty from warm to colder areas. However, their diffusion back into the warmer area occurs at a significantly lower diffusion rate. Some molecules even stay behind and accumulate in spots in the colder region while simultaneously shifted to the capillary center by the convective fluid flow. The former effect is mainly due to the principle of thermophoresis, which is the motion of particles, such as bio-molecules, along a temperature gradient. The temperature gradient-induced change in the migration rate of molecules is different for each compound and a typical  - selective - feature thereof. The solvent and also the single-stranded DNA molecules, for example, flow back to the laser-heated area far more easily than the double-stranded DNA molecules.

The temperature gradient in addition to the accumulation also permits the replication of the double-stranded DNA. In order to duplicate, a DNA molecule first needs to split into two strands. This is achieved by melting the DNA at approximately 90°C in the warmer zone of the test capillary. However, the replication of the two halves into two new double-stranded DNA molecules can only take place after they have been transported into the colder area by the convection current. Moreover, the enzyme polymerase, which is essential for the replication of the genetic molecules, must be added to the test setting. However, by adding these enzymes to our solution, the first steps on the way to life were not quite realistically re-enacted, since, in order to build the enzyme, long DNA molecules and a translation mechanism including proteins would have had to be present at those times. We will therefore switch to RNA instead of DNA and polymerases in future experiments. This compound is chemically very similar to DNA, and does not necessarily need an enzyme to support its replication.

The principle of the molecule trap can also be used to very efficiently and selectively replicate individual molecule types coming from a mixture of different molecules. Each compound in a temperature gradient migrates at a different diffusion rate, and via different test parameters we can influence which compounds accumulate.

We therefore have demonstrated a first step towards a minimal Darwinian process: While the DNA is replicating, the molecule trap catches the new double-stranded molecules and protects them from diffusing out into the surrounding environment. Thus we have shown that the trap can host both continuous replication and selection in a single chamber.

Publication:
“Thermal Trap for DNA Replication”,
Christof B. Mast and Dieter Braun,
Physical Review Letters, May 7, 2010
Article ID: LH11815

Christof Mast
Education

Since 2009
PhD student in the group of
Prof. Dieter Braun, LMU Munich

2008 – 2009
Diploma Thesis in the group of
Prof. Dieter Braun, LMU Munich

Selected Publication

Christof B. Mast and Dieter Braun:
“Thermal Trap for DNA Replication”
Physical Review Letters, May 7, 2010
Article ID: LH11815