Abstract
Enzymatic ligation is essential for the synthesis of long DNA. However, the number of ligated products exponentially decays as the DNA synthesis proceeds in a random manner. The controlling of ligation randomness is of importance to suppress exponential decay and demonstrate an efficient synthesis of long DNA. Here, we report the analysis of randomness in sequential DNA ligations, named qPCR-based STatistical Analysis of Randomness (qPCR-bSTAR), by a probability distribution of ligated DNA concentration. We show that the exponential decay is suppressed in a solution of another polymer and DNA ligation is activated at an optimal crowded condition. Theoretical model of kinetic ligation explains that intermolecular attraction due to molecular crowding can be involved in the optimal balance of the ligation speed and the available ligase. Our finding indicates that crowding effects enhance the synthesis of long DNA that retains large genetic information.
Highlights
Genetic polymers, such as DNA or RNA, need to be elongated to store information in their sequences
The mechanism regarding how long DNA strands are efficiently synthesized under crowding conditions, an analytical method that can analyze ligated DNA products based on their length and concentration is needed
We report enhanced ligation of DNA in a crowded polymer solution using quantitative polymerase chain reaction (qPCR)-based statistical analysis
Summary
Genetic polymers, such as DNA or RNA, need to be elongated to store information in their sequences. Since end-to-end ligation in a test tube is a random process, new short fragments are connected from both ends to produce longer DNA, but the concentration of ligated polymers decays with the number of newly added monomers [4,5]. One scenario to drive the efficient synthesis of long DNA is the local increase of short DNA fragments using physical transport effects. A local temperature gradient or solute concentration gradient induces the directed motion of DNA as a solute, and the trapped DNA exhibits an exponential increase in the concentration [10,11]. This DNA trapping process is considered to compensate for the significant decrease in long DNA polymers
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