Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

Summary

Staining DNA molecules for fluorescence microscopy allows a scientist to view them during an experiment. In the method presented here, DNA molecules are pre-stained with fluorescent dyes and digested with methylation and non-methylation sensitive restriction enzymes.

Abstract

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and sensitivity for DNA. In order to determine if the DNA molecules are full length after the completion of the experiment, a method is required to determine if the stained molecules are full length by digesting DNA with restriction enzymes. However, stained DNA may inhibit the enzymes, so a method is needed to determine what enzymes one could use for fluorochrome stained DNA. In this method, DNA is stained with a cyanine dye overnight to allow the dye and DNA to equilibrate. Next, stained DNA is digested with a restriction enzyme, loaded into a gel and electrophoresed. The experimental DNA digest bands are compared to an in silico digest to determine the restriction enzyme activity. If there is the same number of bands as expected, then the reaction is complete. More bands than expected indicate partial digestion and less bands indicate incomplete digestion. The advantage of this method is its simplicity and it uses equipment that a scientist would need for a restriction enzyme assay and gel electrophoresis. A limitation of this method is that the enzymes available to most scientists are commercially available enzymes; however, any restriction enzymes could be used.

Introduction

The TOTO series (TOTO-1, YOYO-1, POPO-1, BOBO-1, TOTO-3, YOYO-3, POPO-3, and BOBO-3; Table 1) is utilized in a wide variety of experiments where the visualization of DNA is required^{1,2,3,4,5,6,7,8,9,10,11,12,13....}

Protocol

1. Preparation of Dyes, Buffers and Agarose Gel

1. Prepare the following solutions for staining DNA or the digestion of stained DNA.
   1. Prepare 1x TE (Tris-HCl and ethylenediaminetetraacetic acid; EDTA) buffer using 10 mM Tris-HCl and 1 mM EDTA in a graduated cylinder or volumetric flask. Store at room temperature (18 - 25 °C).
   2. Make aliquots of each dye: TOTO-1, YOYO-1, POPO-1, BOBO-1, TOTO-3, YOYO-3, POPO-3, BOBO-3 (Table 1). Pipette 4 µL of 1 mM dye into a dark amber or black 1.5 mL tube and add 36 µL of 1x TE, mix using the pipette; this concentration makes a 100 µM dye solution (40 µL total). C....

Results

In order to determine if an intercalating dye will affect a restriction enzyme digesting DNA, the correct order of steps must be followed (Figure 1). Once DNA is stained and digested with a given enzyme, a picture of the gel can be taken to determine the number of fragments and their size (Figure 2). In order to determine the enzyme efficiency, the total number of expected visible bands divided by the number of visible bands. Enz.......

Discussion

In order to digest fluorescently labeled DNA (Figure 1), a series of steps are required. First, DNA is stained with a fluorochrome overnight. DNA can be incubated with cyanine dimers for a shorter period of time; however, Carlsson et al. found that DNA created double bands for each DNA size due to incomplete staining²⁰. To remedy this, the DNA can be stained overnight to prevent double bands from occurring. This is a critical step in the prot.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Unmethylated lambda DNA	NEB	N3013S
YOYO-1	ThermoFisher Scientific	Y3601
TOTO-1	ThermoFisher Scientific	T3600
POPO-1	ThermoFisher Scientific	P3580
BOBO-1	ThermoFisher Scientific	B3582
YOYO-3	ThermoFisher Scientific	Y3606
TOTO-3	ThermoFisher Scientific	T3604
POPO-3	ThermoFisher Scientific	P3584
BamHI	NEB	R0136S
PmlI	NEB	R0532S
ScaI	NEB	R3122S
EcoRI	NEB	R0101S
HindIII	NEB	R0104S
SmaI	NEB	R0141S
Tris	Fisher	BP152-1	Irritant
EDTA	Fisher	S316-212	Toxic
HCl	Fisher	S25358	Corrosive and irritant
Buffer 1	NEB	B7201S	NEB 1.1
Buffer 2	NEB	B7202S	NEB 2.1
Buffer 3	NEB	B7204S	NEB Cutsmart
High gelling temperature agarose gel	Lonza	50041
Acetic Acid	Fisher	S25118	Hazardous
Blue light transilluminator	Dark Reader	DR196	Wear correct eyewear
Ethidium bromide	Fisher	15585011	Carcinogen; other alternatives are Nancy 520, Gel red, etc.
Cannon EOS Rebel T3I camera	Cannon
Apogee Model H4	Biorad	1704510
Power Pac Basic Power Supply	Biorad	1645050
Ficoll	Fisher Scientific	BP525-25
Bromophenol blue	Fisher Scientific	B-392
Xylene Cyanol	Eastmen	T1579