Ligation Optimization

In order to serve as the template for nanoclew synthesis, single-stranded linear DNA must be processed to form a circular template-primer complex.

In this experiment, we first closed the linear template into a loop by annealing the primer to it. To seal the nick in the template, we varied the temperature and incubation time of the ligation step to determine the most efficient method for circular complex synthesis.


Nanoclew synthesis begins with the formation of a circular template-primer complex, which is used by phi29 DNA Polymerase for the rolling circle amplification (RCA) process. During RCA, the polymerase repetitively amplifies the complex, synthesizing a length of ssDNA that folds into the nanoclew.

The process of circular template-primer complex synthesis begins by annealing the primer to the template to form a loop. This is made possible by the complementarity between the primer and the two ends of the linear template (see Image 1). Afterwards, T4 DNA ligase is used to seal the nick in the loop. According to Bullard and Bowater, nick sealing can be completed by incubating at 37C for 30 min [1]. However, NEB recommends doing ligations at room temperature for one hour [2]. Therefore, we sought to determine the optimal temperature and incubation time for creating the circular template-primer complex.

Image 1: Linear (top) and circularized (bottom) nanoclew template with the overhang binding site in yellow, palindromic sequences in orange, and primer binding site in green


To optimize the synthesis of the circular template-primer complex by varying the temperature and incubation time of the ligation reaction

Techniques Used

Agarose Gel Electrophoresis

Agarose gel electrophoresis allows for the separation of macromolecules like DNA based on size and provides semi-quantitative data regarding the amount of each fragment in the sample based on the brightness of the bands [3]. For this experiment, it was used to determine the efficiency of and confirm template circularization under each condition.

Phenol-Chloroform Extraction

Phenol-chloroform extraction is a liquid-liquid extraction technique for purifying DNA samples. While liquids partition into the lower organic phase and proteins are in the interphase, DNA can be isolated from the upper aqueous phase [4]. As a result, we used this technique to purify our circularized template-primer complexes.


Image 2: Visual abstract of ligation optimization process

Template-Primer Annealing

Template and primer (IDT) were diluted to master stocks of 100 uM using pH 7.5 IDTE Buffer. Working stocks of 10 uM of both were then made using Milli-Q water. The template and primer were then combined with T4 ligase buffer (NEB) and diluted with water to the following final concentrations:

Component Concentration
10x T4 Ligase Buffer 1x
10 uM Template Stock 0.6 uM
10 uM Primer Stock 1.2 uM

Template Ligation

Following that, the sample was heated to 95C for five minutes and cooled to room temperature over the course of approximately 2.5 hours (a decrease in one degree every two minutes). Afterwards, 10 U of T4 ligase (NEB) was added for every microliter of the reaction. The sample was then subjected to one of the treatments listed above.

After the designated incubation time, the sample was heated to 65C for 10 minutes to deactivate the T4 ligase.

Phenolchloroform Extraction

Phenochloroform extraction was performed by first adding an amount of phenol:chloroform:isoamyl alcohol (25:24:1) (Thermo Fisher) equal to that of the sample and vortexing for 20 seconds. After centrifuging for five minutes at 16,000 g, the aqueous phase was removed and transferred to another tube. 

The following reagents were then added (glycogen was acquired from Thermo Fisher):

Component Concentration Amount
Glycogen 20 ug/uL 1 uL
Ammonium acetate 7.5 M 0.5x sample volume
Ethanol 100% 2.5x sample volume

The sample was then left to incubate at -20C for an hour, before being centrifuged at 4C for 30 min at 16,000 g. The supernatant was removed and the pellet was washed twice with 70% ethanol (resuspension followed by centrifugation at 4C for 2 min at 16,000g). Finally, the pellet was left to air dry for 10 min and resuspended in Milli-Q water.

Agarose Gel Electrophoresis

A 2% agarose gel was prepared using Top Vision Agarose (Thermo Fisher) and diluted 10x TBE buffer (Sigma-Aldrich). The samples were mixed with 6x purple loading dye (NEB) and ran at 120V until the dye front was two-thirds of the way down the gel. The gel was then imaged using [MACHINE NAME].


Image 3: Photo of an agarose gel showing the banding patterns made by template-primer complexes that have been subjected to different ligation conditions

As demonstrated by Image 3, while all conditions yielded successfully ligated circular template-primer complexes, incubation for only 30 minutes at either temperature resulted in an excess of linear templates. Additionally, the band for one-hour incubation at room temperature is darker than the band for one-hour incubation at 37C, suggesting that more complexes were able to be formed.


To minimize the amount of linear template leftover and maximize the amount of circularized template-primer complexes, incubating samples at room temperature for one hour is the optimal protocol (marked by a star in Image 3). Contrary to the findings of Bullard and Bowater, incubating at 37C for 30 minutes was not sufficient to completely seal all nicks in the templates. This is supported by the band denoting excess linear templates being fainter compared to the 30-minute incubation samples, and the band for the ligated templates being darker than the one present in the sample that had been incubated at 37C.

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