This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.
For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.
DNA polymerases perform accurate and efficient DNA synthesis and are essential to maintaining genome integrity. The ability to synthesize hundreds of nucleotides per second without making errors also makes DNA polymerases essential tools in molecular biology and biotechnology. However, these properties also limit the applications for M-DNA substrates; generally speaking, natural DNA polymerases cannot synthesize many potentially valuable M-DNA substrates, likely due to the high selective pressure against using non-standard substrates in vivo. Many groups have developed directed evolution approaches to generate mutant DNA polymerases capable of M-DNA synthesis1a,2,3,4,5; these efforts have expanded the biotechnological utility of DNA6,7,8.
To evaluate the ability of mutant DNA polymerases to synthesize M-DNA, we9,10, and others11,12,13 typically use in vitro measurements of DNA polymerase activity, which are described in this manuscript. In these experiments, DNA polymerases are co-incubated with a labeled primer/template duplex and nucleoside triphosphate substrates; the products are evaluated by gel electrophoresis. Depending on the specific experimental question, mutant DNA polymerases, modified primers, modified templates, or modified nucleoside triphosphates can be used, enabling the systematic biochemical evaluation of the mutant enzyme activity.
Historically, these assays have relied on a 5' radioactive label to track DNA synthesis; most commonly, 32P and 33P have been used; typically, labeling is achieved using T4 polynucleotide kinase11. However, due to the finite lifetime and relatively high cost of radioactive labels and their safe disposal, our group instead uses a synthetic 5' near-infrared fluorophore labeled DNA. Using a relatively low cost near-infrared gel imager, we have observed similar detection limits to prior studies using radioactive labels (unpublished results). We have successfully reproduced past observations9, and we have not observed any large quantitative difference with previously measured rate constants (unpublished results).
To analyze the size of DNA, and thus, the extent of DNA synthesis, we rely on polyacrylamide gel electrophoresis methods developed originally for Sanger sequencing14 before the advent of capillary electrophoresis15. The distance of separation or mobility can be used as a measurement of molecular weight; large format, vertical polyacrylamide gels can achieve single nucleotide resolution, enabling quantitative observation of DNA oligonucleotides of varying lengths.
Collectively, these experiments are a robust method for polymerase characterization. Due to the time sensitive nature of the reactions, preparation and care is necessary to achieve reproducible results. Further, while the acrylamide gel is a highly effective way to measure DNA synthesis, as well as numerous other DNA modifying reactions, with single nucleotide resolution, it can be technically challenging. The protocol here will hopefully enable users to perform these experiments while avoiding the most common mistakes.
1. Activity Assay
NOTE: There are two typical types of assays that are often run to characterize DNA polymerases using the methods described here. They differ in whether they qualitatively characterize overall synthesis (encompassing many steps of DNA synthesis) or whether they quantitatively focus on individual steps. We describe the necessary steps for each of these below.
NOTE: Because the assembly of materials are relatively complex, for time sensitive experiments, we recommend that all materials are assembled beforehand. The recipes of all critical components of the assay are listed below. Commercial suppliers for components are listed in the Table of Materials.
2. Assay Run
3. Gel Electrophoresis
NOTE: Because pouring the gel is time sensitive, all materials are assembled beforehand. The recipes of all critical components of the assay are listed below; suppliers are listed in the Table of Materials.
A successful polyacrylamide gel analysis of a qualitative characterization of overall activity (described in section 2.1, Figure 1) and of steady-state kinetics (described in the note at conclusion of section 2.1, Figure 2) are shown. An unsuccessful polyacrylamide gel analysis is also shown (Figure 3).
Note that no commercially available ladder or molecular marker is used. Occasionally, we will use a known polymerase-substrate combination to create a molecular marker; however, it is important to note that different modified nucleotides have very different electrophoretic mobilities, making comparison of oligonucleotides of the same length but differing nucleotide structure inappropriate.
Figure 1: Example of successful polyacrylamide gel analysis of a qualitative characterization of overall activity. Note that the individual bands are well-defined and regular. The bands represent oligonucleotides of differing length; this gel enables easy comparison between polymerases. The no enzyme control lane is indicated with a "-". Activity is judged by both the length of the products and the portion of the labeled primer that is converted to larger products. From this analysis, E6 and E5 are the most active. E3 and E2 are approximately equal in activity, but less than E6 or E5, and E4 and E1 are the least active enzymes.
Figure 2: Example of successful gel for quantitative analysis using steady-state kinetics. Note that the bands are well resolved and well defined. To measure steady-state rate constants of individual steps in oligonucleotide synthesis, an enzyme is incubated with varying quantities of nucleoside triphosphate (in this case, dCTP); note that only the n and (n+1) products are synthesized enabling quantification of the singular event.
Figure 3: Example of an unsuccessful gel for overall activity. The gel was torn during handling, resulting in visible gaps in bands. Note that the gel bands are irregularly shaped, making quantification and analysis difficult.
Here, we have described an assay to characterize the DNA polymerase-mediated synthesis of M-DNA. By using near-infrared labeled DNA primers, and using denaturing polyacrylamide gel electrophoresis to resolve differently sized oligonucleotides, we can obtain single nucleotide resolution on oligonucleotides, enabling precise measurement of synthesis. These approaches can be used to either measure the overall activity of the enzyme (section 2.1) or to measure the Michaelis-Menten parameters of individual steps (note following step 2.1). Our lab has recently used these to both characterize previously evolved enzymes9 as well as rationally engineered enzymes10.
Our assays described here differ from prior methods in their use of a non-radioactive DNA label. Historically, radioactivity has been used to track DNA synthesis due to the high sensitivity that can be obtained using radioactive phosphorous labels11,18. Unfortunately, the high cost of disposal as well as the limited shelf life of radioactive labels can make the use of radioactive labels prohibitive. Here, we describe the use of near-infrared fluorophore labeled DNA, which does not suffer from these drawbacks. Notably, with near-infrared fluorescent dyes we see similar detection limits to radioactively labeled DNA (unpublished results). However, with fluorescent dyes in the visible range, we were not able to observe similar detection limits (unpublished results). While our group typically uses commercially prepared DNA bearing near-infrared fluorophores, these dyes are compatible with a number of established post-synthesis 5' modification chemistries that can be used to install these labels.
Near-infrared fluorescent dyes are also advantageous in that there are multiple commercially available colors, which can enable more complex experiments that monitor synthesis of two labeled oligonucleotides in a single experiment. With radioactive labels, these types of multicomponent experiments are not easily executed. This will likely enable a number of more complex experiments, particularly for orthogonal replication experiments.
This assay, and any assay using denaturing polyacrylamide gel electrophoresis, is primarily limited by the technical challenge of executing the electrophoresis, the low throughput of these experiments, as well as the limited size ranges that are observable at single nucleotide resolution. We hope that this detailed protocol allows groups to overcome the technical challenges of these assays. Notably, while the throughput of the assay is limiting, the use of near-infrared fluorescent labels does increase the throughput, as it does not require the user to develop an autoradiograph. However, the gel still takes approximately 4-6 h to set up and run, limiting the number of experiments that can be run per day. The limited range is caused by the electrophoretic capabilities of polyacrylamide. Practically speaking, these limitations mean that these assays are best for focused research questions that require single nucleotide resolution.
Recently, high throughput DNA sequencing19 has been used increasingly as a method of characterizing DNA polymerases20,21. These assays are noteworthy for their dramatically increased throughput, which enables broader questions focused on sequence biases and error spectra. Importantly, while high throughput sequencing can enable many parallel experiments, it can be challenging to interpret on a single nucleotide basis. This provides an exciting opportunity for enzyme assays employing polyacrylamide gel electrophoresis to fill in the gaps in high throughput sequencing, ensuring that the methods described in this article are relevant for many years to come.
The authors have nothing to disclose.
This work was supported by the Research Corporation for Scientific Advancement (Cottrell College Scholar Award #22548) and by TriLink Biotechnologies (ResearchReward Grant #G139).
Tris HCl | Promega | H5123 | |
Tris Base | Promega | H5131 | |
MgCl2 | Fisher Scientific | BP214-500 | |
Acetylated BSA | Promega | PR-R3961 | |
KCl | Sigma | P4504 | |
Dithiothreitol (i.e. DTT) | Research Products International | D11000 | |
Ethylenediaminetetraacetic acid (i.e. EDTA) (0.5M solution) | Sigma | 03690-100mL | |
Glycerol | Sigma | G5516 | |
Formamide | Acros | AC42374-5000 | |
Orange G | Sigma Aldrich | O3756 | |
Bromophenol blue | Fisher Scientific | 50-701-6973 | |
dNTPs | Fisher Scientific | FERR0191 | |
M-dNTPs (riboNTPs) | Fisher Scientific | 45-001-341 (343, 345, 347) | |
M-dNTPs (all other modified NTPs) | TriLink Biotechnologies | assorted | |
primer 1 | IDT DNA / TriLink Biotechnologies | Custom Syntheses | We use the IR700 dye which can be purchased as a custom synthesis. We typically purchase the oligonucleotides HPLC purified. Sequence is 5’-dTAATACGACTCACTATAGGGAGA |
template 1 | IDT DNA / TriLink Biotechnologies | Custom Syntheses | We typically purchase the oligonucleotides HPLC purified. Sequence is 5’-dCGCTAGGACGGCATTGGATCAGTCTCCCTATAGTGAGTCGTATTA |
Acrylamide | Research Products International | A11405 | 38.67% acrylamide and 1.33% bis-acrylamide |
Tris/Borate/EDTA (TBE) solid | Research Products International | T22020 | |
Urea ultrapure | Research Products International | U20200 | |
Gel tape | CBS Scientific | GT-72-10 | |
Large white spring clamp polypropylene | CBS Scientific | GPC-0001 | |
Ammonium persulfate (APS) | Fisher Scientific | BP179 | |
Tetramethylethylenediamine (TEMED) | Fisher Scientific | BP15020 | |
0.75 mm spacers | CBS Scientific | SGS-20-0740A | |
33×42 Notched Glass Plate Set | CBS Scientific | SGP33-040A | |
Wedge plate separator | CBS Scientific | WPS-100 | |
Comb for gel electrophoresis | CBS Scientific | SG33-0734 | |
Gel electrophoresis rig | CBS Scientific | SG-400-33 | |
ultrapure water | we use a Milli-Q system from Millipore | ||
DNA polymerases | we prepare these in our laboratory using published protocols. |