DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Summary

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.

Abstract

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.

Introduction

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 synthesis....

Protocol

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 li....

Results

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 l.......

Discussion

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 followi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
33x42 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.