Continuing Overlap and Restriction Enzyme Ligation of a Linear Plasmid for D. radiodurans

 Introduction: 

Previous work in overlap ligation has revealed problems amplifying the assembled left and tetr/a fragment in order to complete a full three part plasmid. This was deemed necessary at the time to acquire enough stock fragment to undergo multiple attempts at overlap ligation introducing the right fragment, but it was decided to go forward with what assembled fragment remained and attempt to attach the right fragment anyways. The PCR experiments this week evaluated two primary methods for overlap ligation, the Anna protocol (Behle 2019) and the Hilgarth method (Hilgarth et al 2020). The Anna protocol has been used thus far, including the successful overlap of left and tetra fragments. The Hilgarth protocol was introduced due to other successes in the lab in which assembly was achieved, with very high specificity of products (very little ghost banding). The restriction enzyme ligation team isolated all three fragments the previous week, and went forward by attempting further amplification of each fragment to create enough stock for upcoming digestion, phosphorylation, and eventual ligation steps that require 1 ug of each fragment. 

Methods: 

All gels were 1.2% agarose with 5 ul of PCR water, 5ul of samples, and 2 ul of Uview dye used for each method tested. A 1 kb ladder was also standard. 

Overlap

The first two reactions included one of each protocol, the Anna method including a total mass of 50 ng of each fragment loaded to be approximately equimolar. The "annealing" temperature was 37 degrees Celsius accommodating the homologous regions according to Snapgene. This reaction was 24 ul total volume and ran for 15 cycles. The Hilgarth protocol called for 100 ng/kb, which totaled 170 ng of the assembled left and tetr/a fragment and 100 ng of the right fragment. The "annealing" temperature was implemented for 60 seconds at 72 degrees Celsius and touchdown (-0.5 degrees Celsius/cycle) for 9 cycles followed by a secondary touchdown beginning at 67.5 degrees Celsius for 5 additional cycles with another 75 degrees stage for 60 seconds. The total volume was 50 ul. 

Four microliters from each of these reactions was used for identical amplification steps according to Hilgarth protocol, a 50 ul reaction with primers (1 and 4) diluted 1:10 from 10 uM to 1 uM. Touchdown was completed starting at 67 degrees Celsius followed by a 72 degrees step for 60 seconds, repeated for 17 cycles. A secondary cycle was done including an annealing temperature of 72 degrees for 60 seconds for another 23 cycles. A gel was ran evaluating these two samples. 

The next reactions included redoing the previous two reactions described above. Three additional reactions were also added. 2 ul of potential overlap product was used and annealed at 60 degrees Celsius for 30 cycles, no touchdown, and no diluted primers. The amount of PCR water was also adjusted to account for addition of extra primers to these reactions; 1,4, 3 and 6. Primers 1 and 4 would amplify the total plasmid, all three fragments combined. 1 and 6 would amplify left and tetr/a, and 3 and 4 would amplify the right fragment. This would indicate how much of each fragment was present, and if overlap was achieved with all three fragments, and if not, if the other input fragments could be detected. This reaction was 20 ul volume. 

A positive control was also included using Lactonase, a 20 ul reaction with an annealing temperature of 60 degrees Celsius. Genomic D. rad DNA was used for template, with forward and reverse primers loaded accordingly. These five samples were run on a gel. 

Restriction Enzyme 

Each of the three fragments was amplified in triplicates at standard PCR, 60 degrees annealing temperature for 30 cycles and undiluted primers. These triplicates were run on a large gel. 

Results: 

The first gel testing the Anna and Hilgarth protocols for overlap reactions and then amplified identically resulted in no evidence of DNA on the gel. No smears or bands were seen, and visualization of the ladder was clear. 

Figure 1 shows the gel with the five reactions, where the positive control was not seen, but the two reactions done with standard PCR amplification at 60 degrees Celsius showed DNA. There was significant smearing at low molecular weight sizes as well as a small band at approximately 1,000 bp. The two reactions using Hilgarth showed no significant evidence of DNA. 

The restriction enzyme fragments produced a gel where of the nine reactions, three resulted in a visible band. 2 reactions of the right fragment were successful and 1 reaction of tetr/a was successful, each with very minimal ghost banding and not appearing to be highly concentrated. 

Figure 1 from left to right: (1)1kb ladder, (3)Lactonase, (4)Anna at 60 degrees, (5)Anna and touchdown with diluted primers,(6) Hilgarth at 60 degrees, and (7)Hilgarth and touchdown with diluted primers.
 

Discussion: 

The results for overlap ligation seem to point to issues potentially with the Hilgarth protocol, primarily in the touchdown PCR and undiluted primer steps. The problems encountered are likely due to a misunderstanding of the literature and how each step was actually accomplished. There has been ongoing debate and review to reinterpret the initial understanding of the Hilgarth protocol, and it will be decided next week which adjustments may need to be made to see success with this method, which as been seen by fellow TRAIN students. Another problem that may be the source of this week's issues are the initial overlap reactions; if they were unsuccessful in overlap then DNA would likely not be seen on a gel because the primers would not be amplifying anything but an imaginary product. It was realized that our calculations for equimolar reactions for the overlap steps was not accurate, seeing as other fragments were also in the input samples, skewing the total  mass of the desired fragments. Future experiments will be focused on redoing the overlap steps with more accurate equimolar measurements, which may result in more successful amplification, which was incredibly unsuccessful this week. Another concern was the lack of visualization of the lactonase PCR reaction, which was supposed to be the positive control. It may be the Q5 reaction buffer, enhancer and polymerases, which haven't been used before that way, which caused this result but it is yet to be tested again. The restriction enzyme team also got odd results in their gel, but since some reactions worked and others didn't it leads us to believe human error was seen in pipetting such small volumes, or dilutions of each of the fragment samples was not done accurately due to too small of volumes used for dilutions, leading to high inaccuracy chances. They will hopefully go forward with digestion anyways, once checking fluorometer readings to confirm minimal fragment mass, as opposed to following Nanodrop data. 

Sources: 

1. Anna Behle 2019. Overlap extension PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.psndnde

2. Hilgarth RS, Lanigan TM. Optimization of overlap extension PCR for efficient transgene construction. MethodsX. 2019 Dec 4;7:100759. doi: 10.1016/j.mex.2019.12.001. PMID: 32021819; PMCID: PMC6992990. Modified GCC Biotechnology 2022.