DNA integrity under alkaline conditions: An investigation of factors affecting the comet assay

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Introduction
The integrity of genomic DNA is crucial for cell function and survival.Many types of exposures induce primary DNA damages, including DNA single-and double-strand breaks.If unrepaired, these can lead to different types of mutations, which may be critical steps in tumor development [1].Even if DNA single-strand breaks (SSBs) are constantly formed in our cells, as a result of natural processes related to errors in replication, transcription and byproducts of metabolic processes [2,3], they are considered to be a genotoxic event if induced by endogenous events or exogenous agents such as irradiation, or chemicals.DNA double-strand breaks (DSBs) are less common, but also more problematic, and more difficult to repair [4].
There are many ways to identify SSBs and DSBs, but one of the more commonly used techniques is based on gel electrophoresis of DNA.The first researchers to describe the single cell gel electrophoresis technique (now known as the comet assay) were Östling and Johanson [5,6].Since they were focusing on radiation-induced DSBs, they performed the electrophoresis under what they described as neutral conditions, without any unwinding of DNA before the electrophoresis.Singh and Tice [7] modified the protocol, mainly by performing unwinding and electrophoresis under extremely alkaline conditions (pH >13).The latter protocol for the comet assay (and variants) is the most commonly used protocol, because it is generally assumed that pH > 13 is needed to detect not only actual SSBs and DSBs, but also alkali-labile sites (ALS) converted into strand breaks.
As indicated above, there are currently two preferred versions of the comet assay, the neutral [8] and alkaline assays [9].In the latter version, the agarose embedded cells are submerged and electrophoresed in a strong alkaline solution (0.3 M NaOH; pH >13) allowing the relaxation of DNA, enabling the detection of SSBs and conversion of alkali-labile sites (ALS) into strand breaks, thereby increasing the sensitivity of the assay [9].ALS is a somewhat undefined concept but is generally considered to be equal with apurinic/apyrimidinic (AP) regions, where the DNA has lost its nucleobase (adenine, thymine, cytosine, or guanine), leaving a naked DNA backbone.At physiological conditions (37 ℃ and pH 7.4), ALS exists as a balance between ring-closed (hemiacetal) and ring-opened (aldehyde) sugars, which are relatively stable.However, under alkaline conditions, β-elimination may occur between the ring-opened aldehyde and the 3´-phosphoryl group, leading to strand cleavage [10].
A common misconception is that the neutral condition is specific for detecting only double-strand breaks (DSBs) [11].In fact, both the neutral and alkaline versions of the comet assay detect both DSBs and SSBs.This is because they are driven by the same fundamental principle.Intact DNA, with its large size and supercoiled structure, does not move easily through the gel during electrophoresis.However, depending on the degree of strand breakage, whether single or double, relaxation of the supercoiled structure allows the DNA to move more easily through the matrix.The migration of DNA results in the formation of a characteristic comet-shaped bundle of DNA, where a more pronounced comet tail indicates a higher degree of DNA damage.In the alkaline version of the assay, the relaxation and unwinding process is enhanced by increasing the pH to > 13, which disrupts the Watson-Crick hydrogen bonds in the DNA, converting it into a single-stranded form and simultaneously cleaving the DNA backbone at the ALS.This combination of effects allows the DNA to migrate towards the anode even more readily.Consequently, the alkaline versions enhance DNA relaxation with the additional treatment, thereby increasing assay sensitivity [12][13][14][15].
In a recent publication, we presented a third version of the comet assay, the so-called "flash-comet".This protocol can be seen as an inbetween version of single cell gel electrophoresis under neutral conditions and the protocol used for the comet assay under very strong alkaline conditions (pH > 13).The flash-comet uses a diluted alkaline solution at slightly lower pH (12.5) with much shorter times for unwinding and electrophoresis, at substantially higher field strengths compared to its alkaline and neutral counterparts [16].The shorter run-times and higher field strengths are possible because LiOH (with lower conductivity) replaces NaOH in the comet solutions.
The introduction of the flash-comet has revealed some scientific challenges related to the ongoing debate about the type of DNA damage that is actually detected in the various comet assay protocols.In the flash-comet, the alkaline treatment during the unwinding is conducted only for 2.5 min, virtually omitting the alkaline unwinding step.In this step of the assay, the flash-comet protocol is more like the one used in the neutral version, which is performed without unwinding.As indicated in Table 1, another similarity between the neutral version and the flash-comet is that lysis of the cells is performed at pH 8.5.In the most alkaline version (pH>13), the pH during lysis is often 10.It seems to be generally believed that it is crucial to use a high alkaline pH (>13) and a rather long time for unwinding, in order to convert ALS into SSBs and relax and unwind the supercoiled DNA.It is therefore not surprising that there is ongoing debate about the types of DNA strand breaks, including those derived from ALS, that are actually detected in the different types of protocols, including the flash-comet protocol.
Here, we have investigated how the integrity of DNA is affected by three different comet assay protocols: a neutral version, the flash version, and under alkaline conditions at pH> 13.The focus was then on the importance of pH and the time for unwinding, in untreated control cells.To obtain a full picture of the effects of experimental procedures on the levels of DNA strand breaks, a multi-level approach was used, spanning the genome, gene, and DNA molecular levels (Fig. 1).Comparisons of the comet protocols were achieved by comparing the levels of DNA damage in untreated cryopreserved TK-6 cells.
To elucidate further the effect of alkaline treatment on the integrity of DNA, this was evaluated also on the gene level, by using real time quantitative PCR (RT-qPCR), measuring how amplification of an 87 base pair long commonly used house-keeping gene (human β-2-microglobulin, B2M) was affected when extracted DNA from irradiated (100 Gy) and non-irradiated TK-6 cells was exposed to solutions with different pH (ranging from pH 7 to pH>13).
How quickly does conversion of ALS into strand breaks occur at different pH values?This question was evaluated on a molecular level by monitoring the strand cleavage in two synthetic molecular beacons (hairpins), with or without an artificial 3-bp ALS-region insertion coupled to a fluorophore and a quencher (probe).One hairpin was a 27bp single-stranded DNA, the other was an equally long double-stranded DNA hairpin.To characterize further how the integrity of the hairpin was affected by the time of exposure (from 2 min to 1 h) to solutions with different pH values (from pH 7 to pH>13), the stability of the single-stranded hairpin was also evaluated using liquid chromatography with UV detection (UHPLC-UV).

Chemicals
Unless specifically indicated, all chemicals and reagents were purchased from Merck (Germany) of highest purity available.Primers and molecular beacons were acquired from Integrated DNA Technologies (IDT, Coralville, IA).Uracil-DNA glycosylase (UNG) was obtained from Fisher Scientific (UK).

Cell culture
Human lymphoblastoid TK6 cells were purchased from ATCC (catalogue number CRL-8015, USA) and kept frozen at − 150 ℃ until use.The cells were cultivated in Gibco™ RPMI-1640 medium (Thermofischer Scientific, UK supplemented with 10% foetal bovine serum (FBS, Biological Industries, Israel), 1% Gibco™ PenStrep (10 000 U penicillin/ml and 10 mg/ml streptomycin; Thermo-Fisher Scientific, UK).After an ampoule of frozen cells was thawed, the cells were maintained in suspension in a temperature-controlled atmosphere at 37 ℃ with 5% CO 2 until the experiments were performed on cultivation day seven.

Pre-irradiated cell cultures
Irradiation with X-rays was performed with an Elekta Versa HD linear accelerator at Uppsala University Hospital.The beam quality was 6 MV x-rays and the cells were placed at a water-equivalent depth of 10 cm by the use of water-equivalent plastic attenuators.The cells were irradiated in the dose range 0-100 Gy with 10 Gy increments and dose rate = 5 Gy/min.Directly after irradiation, the cells were washed in icecold PBS, centrifuged at 230 × g for 5 min, and resuspended in freeze medium (complete growth medium supplemented with 5% DMSO) at 1 × 10 6 cells/ml.The cryovials were placed in an isopropanol chamber and maintained at − 80 ℃ overnight.On the following day, the vials were transferred to, and kept in, a − 150 ℃ freezer until the day of analysis.

Neutral, alkaline and flash-comet assays
For analysis of irradiated cells, three different protocols were used.All have previously been described in detail elsewhere: neutral [8], alkaline [9], and flash [16,17].Each has the same general experimental protocol; the most important differences are summarized in Table 1.In brief, non-irradiated cells were brought from the − 150 ℃ freezer and thawed at 37 ℃.Cells were washed once in PBS (pH 7.4) at 4 ℃ and the pellet was then resuspended in PBS, 50 μl.The cells were embedded in 0.6% low-melting-point agarose (Thermo-Fisher Scientific, UK) and cast on microscope slides before lysis for 1 h on ice, in lysis solution.The slides were then transferred to a horizontal electrophoresis tank and immersed in electrophoresis solution.In the alkaline versions, the DNA was allowed to unwind at 4 ℃ before they were subjected to electrophoresis.The slides were then neutralized with 0.4 M Tris-HCl (pH 7.5), and fixated by EtOH (70:95:100%; using 2 min per step).After that, the slides were dried at room temperature in a hood and stored in a sealed box until day of analysis.For all protocols, the level of DNA damage in the comet tails was analysed using the software Comet Assay IV (Perspective Instruments, UK), using the tail intensities (percentage of DNA in the tail) as the indicator of DNA damage.

DNA extraction and alkaline treatment
DNA from 1 × 10 6 TK6 cells per biological replicate was extracted using the Allprep DNA/RNA mini kit (Qiagen) according to the manufacturer's instructions and stored at − 20 ℃ until use; n = 3 per exposure group.
TK6 cells were lysed and homogenized using a 27 G syringe and exposed to PBS (pH 7.4), 150 mM TBS (pH 10), 30 mM LiOH (pH 12.5) or 0.3 M NaOH (pH >13) for 1 h.Exposure was quenched by addition of 1 M Tris-HCl, pH 6.8.Exposure and neutralization buffers were eliminated from the samples by purification of DNA through a spin column, Allprep DNA/RNA mini kit (Qiagen).DNA sample concentrations were measured on a Nanodrop 2000 (Thermo-Fisher Scientific).

Real time quantitative PCR
The levels of intact DNA sequences were detected by amplification of the human β 2 -microglobulin (B2M) gene (Table 2) in the RT-qPCR.The latter technique was performed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in 10 μl reactions with 10 ng (2.5 ng/μl) DNA template and 300 nM forward and reverse primers.
Primer pair efficiency was determined from a standard curve of pooled samples.Each biological replicate was run as three technical replicates.Amplification was performed as follows: initial denaturation, 2 min 98 ℃; 39 cycles of 15 s 98 ℃ and 30 s 60 ℃.Single product amplification was ensured by generating a melt curve 65-95 ℃ with steps of 0.5 ℃.Cq values were normalized against their respective internal controls.Analyses of amplification were performed with the 2 -dCt method.

Alkaline cleavage of AP-sites in molecular beacons
Four different solutions were examined: PBS, pH 7.4; TBS, pH 10; 30 mM LiOH, pH 12.5; and 0.3 M NaOH, pH > 13.A sample (approx.800 nmol) of the AP-containing oligonucleotide was diluted (1:1) in 2 × concentrations of the solutions with different pH values.The mixtures were transferred to a 96-well plate and the fluorescence was monitored using a SpectraMax® iD3 micro plate reader (Molecular Devices, CA).Measurements were conducted after 5, 20, 40 and 60 min.After 60 min, the samples were transferred into LC-MS vials and immediately frozen and stored at − 80 • C until the UHPLC-UV analysis.

UHPLC-UV analysis of the products present after treating molecular beacons with solutions with different pH values
UHPLC analyses were conducted using a protocol similar to that used by Hadar et al., 2022 [10].In brief, analyses were conducted using a reversed-phase column at 30 • C (Waters Acquity UPLC BEH C18, 2.1 × 50 mm, 1.7 µm) eluted with a linear gradient of 5-35% acetonitrile in aqueous 0.1 M triethylammonium acetate, pH 6.8, over 20 min, at flow-rate = 0.3 ml/min.The products were monitored by absorbance at 260 nm.The focus was on the molecular beacon with an ALS, but a control experiment was also done using the molecular beacon without an ALS, but only after exposure for 60 min at pH 12.5 or pH< 13.

Statistics
Comet assay results were evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test.Differences in fluorescence were analysed using a repeated measurements two-way ANOVA followed by Tukey's multiple comparisons test.The level of statistical significance was set to P < 0.05.Comet assay data and molecular beacon experiments were analysed using GraphPad Prism, GraphPad Software, version 9.5.1.Statistical analyses of gene amplification data were performed in Statistica, TIBCO, v14.0.1.25.Initially, data were tested for normal distribution using the Lilliefors test and deemed normally distributed, p > 0.2.Amplification data were analysed with a one-way ANOVA with Bonferroni's post hoc test.

DNA strand breaks in nucleoids from single cells
Three different protocols were used for the comet assay: one under neutral conditions without unwinding (the original protocol), one using 2.5 min for DNA-unwinding at pH 12.5 (the flash-comet protocol), and one using 40 min for DNA-unwinding at pH > 13 (the most commonly used protocol).The lowest background level of DNA damage was seen using the flash protocol (Fig. 2).

Integrity of the reference gene B2M
The main purpose for the RT-qPCR experiments was to study more specifically the effect of pH on DNA isolated from cells that had been exposed to pH 7, pH 10, pH 12.5, or pH> 13 for 1 h, both in control cells and in cells irradiated with 100 Gy.The efficiency of gene amplification of the BM2-gene containing 87 bp decreases with increasing pH, indi-cating degradation of the gene, especially at pH> 13 (Fig. 3).However, a radiation effect alone could not be detected in this experimental set up (data not shown).

Integrity of molecular beacons, with or without ALS
In the fluorescence experiments using hairpins, the major aim was to study the effect of pH (7, 10, 12.5, or >13), in combination with the effect of time (5, 20, 40 or 60 min).Almost no increase in fluorescence could be observed at pH 7 and pH 10 in the time interval 5-60 min, neither in the hairpin of single-stranded DNA with an ALS (Fig. 4B), nor in the one with double-stranded DNA with an ALS (Fig. 4D).In contrast, fluorescence started to increase after 30 min at pH 12.5, but already after 5 min at pH > 13, both in the single-stranded (Fig. 4B) and in the double-stranded DNA (Fig 4D ) with ALS, indicating an increase in DNA strand breaks.Fluorescence from the hairpins of single-stranded DNA without ALS was slightly higher at pH 12.5 and at pH > 13 than at pH 7, but the fluorescence remained at the same level from 5 to 60 min (Fig. 4C).No effects of pH or time were seen in the hairpin of doublestranded DNA without ALS (Fig. 4E).
To characterize further how the hairpins were affected by time of exposure (2 min-1 h) to solutions with different pH values (7 to >13), the stability of the single-stranded hairpin with ALS was also evaluated using liquid chromatography with UV-detection (UHPLC-UV).The hairpin with 25 nucleotides was almost intact after 60 min exposure to solutions at pH 7 and pH 10, but was almost completely degraded at pH 12.5 and pH > 13 (Fig. 5).When the effect of incubation time was evaluated at pH 12.5 (Fig. 6A) and pH > 13 (Fig. 6B), the hairpin was almost intact after 30 min exposure to pH 12.5, but almost completely degraded at pH > 13.The original peak for the hairpin had disappeared after 60 min exposure to pH 12.5, but there were peaks with shorter retention times at pH 12.5.These peaks could not be seen in the chromatograms after 60 min exposure to pH > 13 (Fig. 6A and B).Supporting the experiments measuring fluorescence, there was no evidence of degradation of the molecular beacon without ALS, not even after 60 min exposure to pH> 13 (see Fig. 7).

Discussion
We have investigated the effect of alkaline treatment on the structural integrity of DNA, with a three-step approach.At the first level, DNA integrity of whole genome was evaluated using three different protocols ´-TCTCTGCTCCC CACCTCTAAGT-3T able 3 Description of the molecular beacon sequences.for the comet assay: neutral, flash, and alkaline using pH > 13.Although the comet assay is a well-established method for the evaluation of DNA damage in single cells, much work has been, and still is, done in order to evaluate different parameters of the protocol [8,9,[18][19][20][21][22]. One of the steps considered to be most important, with respect to assay sensitivity, was the introduction of alkaline treatment at pH> 13, first suggested by Singh et al. [7].For more than 30 years, this step has remained practically unchanged: 20-40 min incubation of the agarose-embedded, protein-depleted DNA in 0.3 M NaOH, with a minor addition of EDTA.
A substantial background level of DNA damage was observed for all three protocols, with the most pronounced effect seen in the alkaline protocol (Fig. 2).The increase in background damage is most likely due to damages arising from cryopreservation and thawing processes.The flash protocol had the lowest increase in background damage, but it was still considerably higher than our historical controls.When comparing the flash-comet with the other two protocols, a significant factor be considered is the much shorter overall time spent in unwinding/electrophoresis solutions, affecting DNA diffusion and migration.The shortened exposure time limits DNA dispersion during electrophoresis and may also affects ALS cleavage.
An additional issue regarding the alkaline step of the comet assay is its striking similarity to the alkaline-lysis plasmid preparation method [23].In the latter protocol, a nearly equally strong alkaline solution is used to remove linear DNA from the plasmids.The principle behind this step is that a plasmid is much more stable in an alkaline environment, due to its circular conformation.However, alkaline treatment actually damages the DNA, resulting in reduced recovery of the plasmids [24].This issue, combined with the ongoing debate about the types of strand breaks detected in different comet protocols, including those resulting from ALS, led us to investigate the effect of alkali treatment on DNA integrity on the gene level.This was done by monitoring how the amplification performance of the B2M gene by RT-qPCR was affected after 60 min of alkaline treatment of extracted DNA from either irradiated (100 Gy) or non-irradiated TK6 cells.The rationale for using such an extreme exposure was to provoke a strong difference and to be able to discern a potential effect on the level of a single gene.
Our data (Fig. 3) show that there is a significant reduction of amplification quality directly correlated with the increase in alkaline treatment; pH> 13 showed the strongest effect.This indicates that extremely high pH, by itself, induces DNA degradation.Since the results of the qPCR experiment showed that there are differences between pH 7, pH 12.5, and pH > 13, we designed a molecular beacon experiment to investigate to what extent and how rapidly an ALS is converted into strand breaks at different pH conditions (Figure 4A).Two different molecular beacons were used: one single-stranded (ssDNA) and one double-stranded (dsDNA).Both were constructed with a threenucleotide uridine insertion with an internal fluorophore at the 3′-end of the uridine triplet and a quencher at the 5′-end of the beacon (Table 3).ALS regions were then created by treating the beacons with uracil-DNA glycosylase (UNG).If a strand break occurs at the ALS, this would lead to a release of the quencher, allowing the fluorophore to emit light.
After the creation of ALS, the molecular beacons were exposed to different alkaline solutions and fluorescence was monitored.There was no apparent increase in fluorescence at pH 7 and 10 (Fig. 4 B and D), indicating that no conversion of ALS to DNA strand breaks had occurred.However, at pH 12.5 there was a rapid increase in fluorescence already after 5 min, continuing to increase over time.At the highest pH level (pH>13), a maximal signal was observed already after 5 min, indicating immediate conversion of the ALS into strand breaks.However, when no ALS were present in the probes, there was no increase in fluorescence over time, and only minor increase as a result of pH (Fig. 4C and E).
One might argue that UNG treatment affects the fluorophore or the quencher, leading to an increase in fluorescence in comparison to the non-UNG treated beacons.To confirm that the increase in fluorescence represents actual conversion of the ALS and not a random stand cleavage or destruction of either the fluorophore or quencher, the single-stranded molecular beacons with ALS were also analyzed using liquid chromatography coupled to UV detection, following the DNA at 260 nm.These   data suggested that there was no conversion of the ALS, neither at pH 7 nor at pH 10.However, at the higher pH levels (pH 12.5 or pH >13) no parent probe could be detected any longer.Instead, triplets of daughter fragments became visible in the chromatograms, indicating full conversion of the ALS in those samples (Fig. 5).These data confirmed the results from the comet and qPCR experiments, namely, that by increasing the pH, the stability of the DNA is rather dramatically decreased.However, since no increase in fluorescence occurred in the non-UNG treated samples (i.e., in the molecular beacons without ALS) these beacons seemed to remain intact.Hence, our data suggest that the alkaline treatment seems to be rather selective towards AP-sites.
How rapidly the conversion of the ALS into DNA strand breaks occurs at the different pH conditions could not be answered by the molecular beacon experiment, but at pH > 13, it was obviously at a maximum already after 5 min (Fig. 4B and D).The rate of ALS conversion was therefore monitored by UHPLC-UV, by repeated injections of the single stranded molecular beacon into a reversed-phase column.Conversion of the ALS was clearly time-dependent at pH 12.5, with almost no conversion after 2-30 min (Fig. 6A).After 60 min at pH 12.5, the parent probe was fully fragmented into three daughter fragments.No such fragments could be seen after 30 min at pH> 13, leaving only minute levels of the parent probe.
An additional finding was that no apparent DNA fragmentation was observed in the non-UNG treated beacon (i.e., the one without ALS) after 60 min, not even at the highest pH (Fig. 7).This is noteworthy, since the beacons used in the present study contained rather large modifications, including mismatched non-DNA-specific bases (uracil) and covalently bound fluorophores and quenchers.It can be argued that all these modifications are examples of adducts -different types of primary DNA damage that could cause changes in the structural stability of the DNA.However, neither of the modifications, except for the ALS, resulted in fragmentation of the probes (Fig. 7).This further strengthen our hypothesis that alkaline treatment is selective towards ALS in the form of apurinic and apyrimidinic sites.
Thus, in the present study, we have shown that the flash protocol of the comet assay detects DNA single-and double-strand breaks after 2.5 min unwinding at pH 12.5, but also ALS, if the time for unwinding is increased to 60 min.Unwinding of DNA at pH> 13, using a relatively long unwinding time, i.e., conditions most commonly used in the alkaline version of the comet assay, will jeopardize the integrity of DNA, inducing higher background levels of DNA damage than under less alkaline conditions.

Fig. 1 .
Fig. 1.An overview of the multifactorial approach used when evaluating the integrity of DNA at three different levels:(i) the genome, (ii) the gene, and (iii) the DNA molecule.

Fig. 2 .
Fig. 2. Using the percentage of DNA in the tail (% TDNA) as the indicator of DNA damage, the level of damage was measured in TK-6 cells.Three different protocols for the Comet assay were used: A neutral version (pH 10), the Flash version (pH 12.5) and an alkaline version using pH> 13.The data are presented as means ( ± SEM) after pooling the data from three independent gels (n = 150 analyzed comets per protocol).Statistical significance was evaluated using the Kruskal-Wallis test followed by Dunn's multiple comparisons test.* ** p < 0.001.

Fig. 3 .
Fig. 3. Transcript levels of the reporter gene β 2 -microglobuline (B2M) in TK-6 cells at pH 7.4 (used as controls), and under different alkaline conditions: pH 10; 12.5 or > 13.The cells were exposed to the solutions with different pH during 60 min.Data are presented as means fold change ( ± SEM) relative to the controls.The number of observations was 9 for each treatment.Statistical significance was evaluated with a one-way ANOVA followed by Bonferroni's post hoc test.* * p < 0.01; * ** p < 0.001.

Fig. 4 .
Fig. 4. The effect on the stabilities of self-quenched molecular beacons subjected to increasing strengths of alkaline treatment was measured over time.(A) General overview over the principle behind the molecular beacon experiment.Self-quenched uracil containing molecular probes either single-(upper panel) or double stranded DNA (lower panel) are treated with Uracil-DNA glycosylase to create an ALS region.Alkaline solutions are then added to the probes and monitored over time.At pH > 11 double stranded DNA is converted to single stranded DNA due disruption of the hydrogen bonds (unwinding).If alkaline treatment incudes strand cleavage it results in the release of the quencher and activation of the fluorophore.(B) single-stranded ALS containing DNA, (C) non-ALS containing single stranded DNA, (D) double-stranded ALS containing DNA, and non-ALS containing double stranded DNA (E).The probes where incubated in either PBS pH 7.4, TBS pH 10, LiOH pH 12.5 or NaOH > 13.The data is presented as means ( ± SEM).Statistical significance was evaluated using a two-way repeated measures ANOVA followed by Tukey post hoc test.Different from preceding time point: * p < 0.05; * * p < 0.01; * ** p < 0.001.Different from: a) pH 7.4 (p < 0.01); b) pH 10 (p < 0.01); c) pH 12.5 (p < 0.01).

Fig. 5 .
Fig. 5. UHPLC-UV analysis of the products generated by strand cleavage of an alkaline-labile site (ALS) in a single stranded molecular beacon containing ALS after 60 min exposure to the following solutions: (a) PBS at pH 7.4; (b), TBS at pH 10; (c), LiOH at pH 12.5, or (d) NaOH at pH > 13.The peak at a retention time between 12 and 14 min represent the intact molecular beacon.The number of observations for each treatment was three.

Fig. 6 .
Fig. 6.UHPLC-UV analysis of the products generated by strand cleavage in a single stranded molecular beacon containing an alkali-labile site (ALS) after exposure during 2 -60 min to either (A) 30 mM LiOH at pH 12.5) or (B) 0.3 M NaOH at pH > 13.The peak at a retention time between 12 and 14 min represent the intact molecular beacon.

Fig. 7 .
Fig. 7. UHPLC-UV analysis of the products generated by strand cleavage in a single stranded molecular beacon without an alkali-labile site (ALS) after 60 min exposure to either (A) PBS at pH 7.4, or (B) NaOH at pH > 13.The peak at a retention time between 12 and 14 min represent the intact molecular beacon.

Table 1
Important differences between the three different protocols for the comet assay used in the present study.

Table 2
Description of the gene-specific primer sequences.