DNA Fragmentation

DNA fragmentation occurs when DNA is broken at various points within the double helical structure. Spontaneous DNA fragmentation has been widely observed in apoptotic events caused by a deoxyribonuclease, known as caspase-activated DNase (CAD) or DNA fragmentation factor (DFF). In its inactive state CAD remains bound to its inhibitor (ICAD), but in the process of apoptosis, caspase 3 (a protease enzyme) cleaves these molecules and CAD becomes activated. Though many factors may contribute to spontaneous and/or programmed cell death, DNA fragmentation has proven to be a reliable marker of the apoptosis process.

DNA fragmentation may be used for the construction of a DNA library or even for forensic applications. One common assay that integrates DNA fragmentation is restriction fragment length polymorphism (RFLP) where specific recognition sites within the DNA are targeted by restriction enzymes. After gel electrophoresis and hybridization with DNA probes, variable lengths of DNA fragments can be analyzed. RFLP is particularly useful for locating genetic markers, used to follow inheritance. DNA fragments are also key for polymerase chain reaction (PCR) techniques, where it is unnecessary for an entire DNA strand to be amplified. These specific regions of DNA are used instead, and depending on the PCR process, DNA fragments usually only need to be between 1 - 40 bp long. Recombinant DNA technology also relies on DNA fragments where two or more separate fragments are conjoined and become capable of autonomous replication inside the host.

Enzymatic and Physical Fragmentation Methods

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Enzymatic Techniques

Restriction Enzymes

For DNA fragmentation methods, restriction digest is generally used to generate compatible ends on PCR products, where one or more restriction enzymes are used to fragment the sample DNA, resulting in non-directional and/or directional insertion into the compatible plasmid. These enzymes recognize roughly 6 - 8 consecutive bases, and result in larger DNA fragments. Choice of restriction enzyme(s) and digestion conditions are empirically designed based on the desired insert size for the clone library, and the enzyme may have a recognition site with multiple cloning sites. These enzymes work by cutting immediately outside the DNA target insert to produce two DNA fragments.

Transposomes

Another common method of DNA digestion is transposome mediated fragmentation that uses transposases to catalyze the random insertion of excised transposases and transposons into DNA targets. First, the transposases make random, double-stranded breaks in the DNA. They then covalently attach to the 3' end of the transposon strand and to the 5' end of the target DNA segment. This cut and paste technique is highly efficient, has high throughput capabilities, and is typically used alongside NGS.

Mechanical Techniques

Some other methods of DNA fragmentation require no expensive equipment or special instrumentation, and offer extremely quick test times.

In bead shearing, the DNA sample is vortexed in a tube containing a glass bead, a minimal volume of 5 ul may be used, and this procedure can be performed in just a couple minutes.
In needle shearing, DNA is passed through a small gauge needle, and at least 20 ul of sample is normally required, though this process may be more laborious.
In centrifugal shearing, DNA is subjected to shear forces while in a specific microcentrifuge test tube, and this process may require >150 uL of starting material, though it requires little hands-on time.
In hydrodynamic shearing, a syringe pump is used to generate shear forces within a tube or pore, and the DNA fragment size is based on the size of the constriction as well as the flow rate of the fluid. Many instruments and solution types are available, with little consumables needed. The method requires volumes of > 100 ul starting material, and many steps in the process may be automated.
Nebulization works by forcing DNA through a small hole into a chamber (of the nebulizer unit) using compressed air. In nebulization, DNA fragmentation size is determined by the input pressure used and the material may be between ~500 - 2000 bp in length. Though this method offers inexpensiveness and simplicity, microgram quantities of DNA are required as starting material and the method lacks the efficiency seen in other techniques.
Sonication may utilize a simple bench-top ultrasonic water bath, to fragment DNA samples by acoustic shearing. In this method, unfragmented DNA is placed with an appropriate amount of stock solution into a microcentrifuge tube or similar, then submerged in an ice bath and sonicated for a desired length of time. Repeat sonication may be necessary to ensure the entire working sample is fragmented, though excessive sonication should be avoided. Acoustic shearing works by focusing high-frequency energy into a small container to subject an aqueous DNA sample to cavitation, (the continuous formation and disruption of bubbles) at low-pressure regions within the sample induced by the high-velocity sound waves. Acoustic shearing methods offer no sample loss, reproducible fragment sizes, and quick benchtop procedures.

Visualization and Analysis

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As DNA fragmentation is a marker of apoptosis, three main methods have been developed to detect and characterize cell populations that undergo apoptotic processes.

TUNEL Assay

The first method is the TUNEL assay (Terminal deoxynUcleotidyl transferase Nick-End Labeling), where cells are cultivated, harvested, and fixed in a formaldehyde solution. Cells are permeabilized using an ethanol treatment which is necessary to allow reaction reagents, including the TUNEL endonuclease terminal deoxynucleotidyl transferase (TdT), to penetrate into the nuclei. TdT then catalyzes the incorporation of labeled dUTPs onto the free -OH moieties of the fragmented DNA.

Visualization is dependent on the label types, as they may be fluorescent or enzymatic. Fluorescence detection is most commonly coupled with other techniques including flow cytometry, laser scanning cytometry or fluorescence microscopy, though light microscopy may also be used if the assay is coupled with a colorimetric stain technique.

Ladder Assay

In the DNA ladder assay cells are initially cultured and harvested under predetermined conditions. Adherent cells must be lysed, though as some cells may have previously detached a centrifugation step to assemble floating apoptotic cells might be necessary first. Lysis buffers generally contain three main components (Tris, EDTA, and NaCl) though DMSO may also be used. Fragmented DNA will then undergo isolation, which can be performed using various commercially available kits. Though kits offer a faster, safer, more sensitive, and simpler technique, this step may also be performed using an in-house procedure that utilizes a phenol-chloroform solution. Next, any contaminating RNA is digested to purify the DNA before the sample undergoes gel electrophoresis. Then negatively charged DNA fragments are separated on the agarose gel under a direct electric current, and the voltage applied depends directly on the size of the DNA fragments to be distinguished.

The fragments can be stained, most often through the use of ethidium bromide or SYBR, and then visualized using an ultraviolet transilluminator where a “ladder” pattern can then be observed.

Comet Assay

Another method consists of the comet assay, where cells are cultivated, harvested, and mixed with a ≤ 1% low melting point agarose (LMPA) liquid. The sample is placed on a microscope slide containing a normal melting point agarose (NMPA) concentration, covered, and tempered briefly at 4 °C. The slide is submerged in a lysis buffer that may contain similar components as the DNA ladder assay, DMSO, or triton X-100. Various kits and coated microscope slides are also commercially available for these steps. The slides then undergo gel electrophoresis under a direct low voltage current, and are subjected to staining via dyes like ethidium bromide, propidium iodide, DAPI, acridine orange, SYBR, or even silver.


In visualization, the comet-like image is observed on the gel where the head contains the nuclear core with macromolecules and unfragmented DNA, while the tail is predominantly the ssDNA. The tail size is indicative of the level of overall DNA damage within the sample.

Limitations and Considerations of DNA Fragmentation Assays

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The TUNEL assay can detect and quantitate apoptotic cells from cell suspension, adherent cell lines, and tissues in vitro and in situ. This assay offers higher sensitivity than other techniques, notably the DNA ladder assay. It should be noted that TUNEL assay detects DNA fragmentation in all damaged cells, including cells that underwent apoptosis, necrosis, exposure to a toxic environment, undergoing active DNA repair, undergoing autolysis post mortem, or even those undergoing degeneration. This method should be used alongside a secondary assay to confirm apoptosis, so that the amount of false positives from other sources is reduced.


The DNA ladder assay is a simple technique and does not require special equipment. This method offers sensitivity in the order of 106 cells, so is more useful for experiments on cell cultures or tissues. The DNA ladder assay however also will detect fragmentation that occurs during necrosis, which might provide a smear pattern upon visualization. The DNA ladder assay is only appropriate for proving apoptosis at later states, when internucleosomal cleavage of DNA is ongoing. Upon visualization, the absence of a DNA ladder pattern does not always mean that no apoptotic cells were occurring in the test sample, so this assay should be used alongside a secondary assay to confirm apoptosis.

Evaluation of the comet assays is tricky, and mostly is performed using software that can score the comets through various specifications. Scoring is normally performed based on the measurement of the magnitude size, and quantification of the fluorescent signal, in the core versus tail. The comet assay is useful for detecting DNA strand breaks, is inexpensive, rapid, and needs very little specialized laboratory equipment. This method, however, is incapable of distinguishing between genotoxicity and early apoptosis, so is most useful for detecting apoptosis at late stages, and may need to be multiplexed alongside another assay.