A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.
Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.
Although uracil is a normal base in RNA, it is a common and highly mutagenic lesion in genomic DNA. Uracil can arise from spontaneous/enzymatic hydrolytic deamination of a deoxycytidine. In each living cell, this deamination occurs 100-500 times per day under physiological conditions1,2. If these alterations are not repaired, there can be a change in the DNA sequence composition, causing mutation. As uracil in DNA prefers to pair with dATP during replication, if cytosine deaminates to uracil, in two replication events, there will be a new G:C to A:T transition mutation in half of the progeny DNA3.
Among the cellular strategies to maintain genetic stability, base excision repair (BER) is an essential mechanism that repairs damaged bases, such as uracil, in DNA4. BER is a highly evolutionarily conserved process. There are two general BER pathways: the short-patch pathway leading to a repair tract of a single nucleotide and the long-patch pathway that produces a repair tract of at least two nucleotides5. BER is a coordinated mechanism that occurs in several steps. The first step in BER is the enzymatic hydrolysis of the damaged nucleotide base by a damage-specific DNA glycosylase to generate an apurinic/apyrimidinic (AP) intermediate site6. This is followed by the cleavage of the sugar-phosphate backbone at the AP site by an endonuclease, clean-up of the DNA ends by a lyase, gap-filling by a DNA polymerase, and sealing the final nick by a ligase5.
Uracil-DNA glycosylase (UDG) hydrolyzes the uracil from uracil-containing DNA for BER in Escherichia coli. Conventional UDG assays using radiolabeled DNA involving different separation techniques6,7,8,9,10,11,12,13 are usually time-consuming, labor-intensive, with costly labeling reagents, complicated procedures, and requiring intensive training and practice to reduce risks of exposure to radioactive materials. Fluorometric oligonucleotide assays have been developed as a replacement for radioisotope labeling14, in addition to molecular beacons and Förster resonance energy transfer technology15,16,17,18,19,20. However, specific labeling is required for all the aforementioned methods. Recently label-free biosensors assays21,22,23 and colorimetric methods based on the formation of a G-quadruplex24,25,26 have been developed. However, multiple A:U pairs or specially designed sequences in the probes complicate enzyme unit definition.
MALDI-TOF MS is a technology that could be of great use in DNA analysis. Applications developed include single-nucleotide polymorphism genotyping27,28, modified nucleotide analysis29, and DNA repair intermediate identification30,31,32,33,34. MALDI-TOF MS should be readily adopted for DNA glycosylase analysis to detect AP-site-containing DNA products. However, AP-sites in DNA are prone to strand break under many experimental conditions33. A UDG assay is presented here using MALDI-TOF MS to directly measure AP site production without significant strand-break noise. This label-free method is easy to work with and has a high potential for the pharmaceutical application of DNA glycosylase inhibitor screening.
1. Substrate/template preparation
2. DNA glycosylase assay
3. Transfer UDG reaction products to a matrix chip
4. Setup the assay parameters on the mass spectrometer
5. Mass spectrometer operation
6. Viewing mass spectra and analyzing the data
Templates and substrates
Taking synthetic oligonucleotides with U in the center (U+9) paired with a G template as an example (Figure 1A), a blank control of equimolar amounts of template and uracil-containing substrate can be used for quality control of synthetic oligonucleotide purity (Figure 1B; the signals match the designated m/z and the low background noise). For the MS data analysis, the peak heights were measured (Figure 2). The 19 nt template DNA was expected to remain unchanged after glycosylase hydrolysis; therefore, the signal could serve as a reference for the quantitation of the AP-product (Figure 1C).
Reaction condition and mass spectra
This DNA glycosylase assay is simple and easy to perform using a non-labeled oligonucleotide for a standard reaction to obtain clean and reliable results (Figure 1). The range of MS spectra should cover all the signals generated from substrates, templates, and reaction products (Figure 1B). The difference of 1 nt between the substrate and the corresponding template produced a well-separated signal profile for both the template and the substrate (Figure 1B). The equimolar primers U+9 and T1 showed similar peak intensities without significant differences. Extensive digestion of UDG (0.5 units for a 30 min reaction) demonstrated complete uracil cleavage. The signal of the AP-product was also well separated from that of the uracil-containing substrate (Figure 1C d+9 AP-product m/z = 5447.6 versus Figure 1B U+9 substrate m/z = 5541.6). The relatively mild MALDI ionization technique36 used in this study minimized the signals associated with strand breaks induced by β-elimination reaction at AP sites37, which were not significant in the mass spectra. Overall, the reaction background is very clean without noticeable noise (Figure 1C).
Under equimolar conditions, the relative signal intensity of the AP-product was comparable to that of the U-substrate using the T1 template as a reference (Figure 1B, U+9/T1 versus d+9/T1). Thus, the calculation of UDG activity is based on the exact input of the U-containing substrate (50 pmol or 20 pmol) according to Eq (1).
UDG activity = (signal of AP-product) / (Signal of U substrate + signal of AP-product) × [U] (1)
UDG kinetic parameters determined by MALDI-TOF MS assay
As shown in Figure 2, throughoutf the 3 min reaction, UDG reactions from 0.01 units to 0.1 units demonstrated both dose- and time-dependence. A concentration of 3.2 pM (0.1 units per 100 µL reaction) was used for the determination of the kinetic parameters, Km and kcat, for a G:U substrate (Table 1). The aliquots of the UDG reaction were removed and quenched for 30 s and 60 s. Kinetic data were obtained under conditions when the uracil-containing substrate was less than 50% digested, and five substrate concentrations ranging from 10 to 200 nM were subjected to analysis. The presteady-state time course showed excellent linearity for rate analysis. An example of a reaction rate plot is shown in Figure 3A, where the relative MS signal intensities of the product and substrate are converted into concentration (nM). The UDG reaction rate (v) is presented as nM of the AP site produced per second. The Km and Vmax were calculated from a Lineweaver-Burk plot (Figure 3B). The UDG kinetic parameters derived from the MALDI-TOF MS assay were thus determined to be Km = 50 nM, Vmax = 0.98 nM s-1, and Kcat = 9.31 s-1. The values are comparable to the results of previous 3H-uracil releasing assays where the Km = 40 nM and Kcat = 13.3 s-1 38.
The G:U+9 duplex was subjected to UDG sensitivity assays. The unit measured by MALDI-TOF MS assay was plotted against the manufacturer's defined unit by a conventional 3H-uracil-releasing method38. As shown in Figure 3C, the MALDI-TOF MS-measured unit was proportional to the defined unit from 0.001 units to 0.02 units with the correlation equation of y = 0.933x + 0.0003 and coefficient of determination R2 = 0.9974. The detection limit is 0.001 units as the coefficient of variation = 9.2%, ~5 times the signal-to-noise ratio. Thus, this MALDI-TOF MS assay provides sufficient sensitivity as UDG is mostly used at the unit level39.
Substrate specificity of UDG
One of the characteristic features of this UDG MALDI-TOF MS assay is that it is label-free, which renders high versatility for substrate design. This feature is very useful for analyzing the substrate specificity of UDG. As demonstrated in Table 1, uracil could be inserted at any place near the 5' or 3' end of single-stranded or double-stranded DNA for a specificity assay. For substrate specificity, it is well known that UDG is highly active in removing uracil from single-stranded DNA38. Indeed, as shown in Table 1, uracil excision from the single-stranded substrate (ssU) was decreased 3.7-fold when annealed to the complementary DNA strand, forming G:U duplex. For the commonly used A:U duplex6,7,8, the reaction rate was further lowered by nearly 10-fold relative to ssU. The UDG did not act on U at the DNA terminus (Table 1 substrates of G:U+1, G:U-1, and G:U-2), which is consistent with previous studies40,41. Interestingly, U at the 2nd and 3rd positions from the 5'-terminus exhibited a higher UDG catalytic rate than the U in the center by 2-fold and 1.5-fold, respectively (Table 1, G:U+2 and G:U+3 versus G:U). UDG excised U 3-nt from the 3'-end with 8% less activity than the U at the center (Table 1, G:U-3 versus G:U).
Uracil glycosylase inhibitor inhibition of the UDG reaction
Uracil glycosylase inhibitor (UGI) is a bacteriophage protein, which inhibits E. coli UDG by reversible protein binding with a stoichiometry of 1:142. The inhibition of UDG activity by UGI is shown in Figure 4. In the presence of 0.05 units (100 pM) of UGI, the activity of 0.05 units of UDG (8 pM) was inhibited to an undetectable level. The IC50 was 7.6 pM. Thus, the UDG MALDI-TOF MS assay can be easily modified to a mass screening assay for the discovery of inhibitors of other DNA glycosylases.
Figure 1: Model system for a DNA glycosylase assay. (A) Lesion strand containing a single uridine (U+9) was annealed to a template (T1), forming a G:U mismatch (bold). Uracil-DNA glycosylase detects and removes the uracil, forming an AP site (d). When subjected to MALDI-TOF MS, the difference in m/z values between U-containing DNA and AP products can be resolved as shown in B. (B) An 18 nt DNA containing a single uridine (U+9) annealed to a 19 nt template (T1) (Table 1) was tested as an enzyme blank of 50 pmol of substrate in 40 µL of reaction buffer and subjected to MS analysis. (C) A near-complete enzyme digestion of 50 pmol of substrate in a 10 µL reaction using 0.5 units of UDG at 37 °C for 30 min. (D) An 18 nt DNA containing a single uridine (U+3) annealed to T1 enzyme blank of 50 pmol substrate in 40 µL reaction buffer was subjected to MS analysis. (E) A near-complete enzyme digestion of 50 pmol of U+3 substrate in a 10 µL reaction using 0.5 units of UDG at 37 °C for 30 min to produce d+3 and subjected to MS analysis within 30 h. (F) The reaction condition was the same as in E except the d+3 product was in 0.1 M NaOH at 95 °C for 30 min to trigger a beta-elimination reaction producing B15 fragmented product and subjected to MS analysis. (G) The reaction condition was the same as in E except the d+3 product was stored at -20 °C for more than 7 days; a fraction of d+3 converted into B15 fragmented products. This figure is modified from 43. Abbreviations: nt = nucleotide; MS = mass spectrometry; MALDI-TOF MS = Matrix-assisted Laser Desorption/Ionization time-of-flight MS; AP = apurinic/apyrimidinic; UDG = Uracil-DNA glycosylase; T1 = 19 nt G-containing template; U+9 = 18 nt U-containing substrate; d+9 = 18 nt AP site-containing product; U+3 = 18 nt U-containing substrate; d+3 = 18 nt AP site-containing product; B15 = 15 nt beta-elimination fragmented product. Please click here to view a larger version of this figure.
Figure 2: Time course analysis for UDG activity at different enzyme concentrations. Substrate DNA, 50 pmol, containing a G:U lesion was incubated with 0.01, 0.02, 0.05, and 0.1 units of UDG at 37 °C. Aliquots (10 µL) were taken from the reaction mixture at 0, 0.5, 1, 2, and 3 min and quenched with an equal volume of phenol/chloroform. (A) MALDI-TOF mass spectra illustrating the concentration dependence of processing G:U substrates by UDG. (B) The amount of product was plotted as a function of time. UDG: 0.01 (closed triangles with solid line), 0.02 (open triangle with dashed line), 0.05 (closed circle with solid line), and 0.1 units (open circles with solid line). The data are the averages of three independent determinations, and the error bars represent 1 S.D. This figure is modified from 43. Abbreviations: nt = nucleotide; MALDI-TOF = Matrix-assisted Laser Desorption/Ionization time-of-flight; AP = apurinic/apyrimidinic; UDG = Uracil-DNA glycosylase; T = 19 nt G-containing template; U = 18 nt U-containing substrate; AP = 18 nt AP site-containing product. Please click here to view a larger version of this figure.
Figure 3: Determination of kinetic parameters of UDG using MALDI-TOF analysis. Michaelis-Menten curve and Lineweaver-Burk plots to determine the Km and kcat for UDG enzyme-catalyzed excision of Uracil from DNA. DNA glycosylase MALDI-TOF MS assay was performed using 0.1 units of UDG and different concentrations (10, 30, 60, 100, 200 nM) of substrates in 100 µL reaction for 30 s and 60 s. (A) Michaelis-Menten curve generated from three independent experiments. The error bars represent 1 SD. (B) Lineweaver-Burk plot for the assay; the indicated intercepts permit calculation Km and Vmax. (C) UDG assay by MALDI-TOF MS analysis compared to the manufacturer-assigned unit. Enzyme activity was measured by uracil removal forming AP site in a 10 µL reaction containing 50 pmol (0.56 µg) of G:U within an 18/19 nt DNA duplex for 30 min at 37 °C. The UDG was diluted with buffer [50% glycerol, 20 mM Tris-HCl (pH 7.5), 30 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol] on ice. A unit was defined as the amount of enzyme that catalyzed the release of 60 pmol of uracil per minute. Error bars show the standard deviation of six experiments. This figure is modified from 43. Abbreviations: nt = nucleotide; MALDI-TOF = Matrix-assisted Laser Desorption/Ionization time-of-flight; UDG = Uracil-DNA glycosylase; AP = apurinic/apyrimidinic; V, reaction velocity nM/s. Please click here to view a larger version of this figure.
Figure 4: UDG enzyme inhibition curve by addition of UGI. The inhibition curve was determined using reactions of 20 µL with 0.05 units of UDG (8 pM) and 50 pmol of substrate for 15 min in the presence of UGI from 0.001 units (5 pM) to 0.1 units (200 pM). Data are the averages of three independent determinations, and the error bars represent 1 S.D. Data fit with an online IC50 calculator (see the Table of Materials). This figure is modified from 43. Abbreviations: UDG = Uracil-DNA glycosylase; UGI = uracil glycosylase inhibitor. Please click here to view a larger version of this figure.
DNA substrates | Oligonucleotidesa | DNA Sequenceb | Initial ratec | k cat | |
(pmol/sec) | (s-1) | ||||
ssU+9 | U+9 | 5’-GACCAGTCUGGACGTTGG-3’ | 0.560 ± 0.098 | 35 | |
ssU+3 | U+3 | 5’-GAUCAGTCCGGACGTTGG-3’ | 0.756 ± 0.083 | 47.3 | |
ssU-3 | U-3 | 5’-GACCAGTCCGGACGTUGG-3’ | 0.448 ± 0.087 | 28 | |
G:U | T1 | 3’-CTGGTCAGGCCTGCAACCG-5’ | 0.149 ± 0.046 | 9.31 | |
U+9 | 5’-GACCAGTCUGGACGTTGG-3’ | ||||
A:U | T2 | 3’-CTGGTCAGACCTGCAACCG-5’ | 0.053 ± 0.018 | 3.31 | |
U+9 | 5’-GACCAGTCUGGACGTTGG-3’ | ||||
G:U5’+3 | T1 | 3’-CTGGTCAGGCCTGCAACCG-5’ | 0.256 ± 0.044 | 16 | |
U+3 | 5’-GAUCAGTCCGGACGTTGG-3’ | ||||
G:U5’+2 | T3 | 3’-CGGGTCAGGCCTGCAACCG-5’ | 0.567 ± 0.016 | 35.4 | |
U+2 | 5’-GUCCAGTCCGGACGTTGG-3’ | ||||
G:U5’+1 | T4 | 3’-GTGGTCAGGCCTGCAACCG-5’ | NDd | ND | |
U+1 | 5’-UACCAGTCCGGACGTTGG-3’ | ||||
G:U3’-3 | T5 | 3’-CTGGTCAGGCCTGCAGCCG-5’ | 0.138 ± 0.015 | 8.63 | |
U-3 | 5’-GACCAGTCCGGACGTUGG-3’ | ||||
G:U3’-2 | T6 | 3’-CTGGTCAGGCCTGCAAGC-5’ | ND | ND | |
U-2 | 5’-GACCAGTCCGGACGTTUG-3’ | ||||
G:U3’-1 | T7 | 3’-CTGGTCAGGCCTGCAACGG-5’ | ND | ND | |
U-1 | 5’-GACCAGTCCGGACGTTGU-3’ |
Table 1: Substrate compositions and UDG specificities. This table shows the 18 nt U-containing DNA designated by U±N. The '+' is indicates counting the uracil position from the 5' terminus, and the '-' indicates counting the uracil position from the 3' terminus. For example, U+9 is the 9th position from the 5' terminus, U-3 is the 3rd position from the 3' terminus. The complementary 19 nt template of T1 to T7 would be paired with the U-containing DNA forming G:U or A:U mismatches as indicated. Uracil bases are in bold. Reactions were using 50 pmol of U substrates, 0.05 units of UDG, in 10 µL as described in protocol section 2. This Table is modified from 43. Abbreviations: UDG = uracil DNA glycosylase; nt = nucleotide; ND, not detectable.
2nd-PCRP | 1st-PCRP | AMP_LEN | UP_CONF | MP_CONF | Tm | PcGC | PWARN | UEP_DIR | UEP_MASS | UEP_SEQ | EXT1_ CALL | EXT1_ MASS |
NA | NA | NA | NA | NA | NA | NA | F | 5789.8 | GCCAACGTCCGGACTGGTC | |||
NA | NA | NA | NA | NA | NA | NA | F | 5541.6 | GACCAGTCUGGACGTTGG |
Table 2: FILE I.xlsx file. The settings were used for Figure 1B, C, and Figure 2A; 0 min entries. The MALDI-TOF MS system used in this study was designed specifically to analyze single-nucleotide polymorphism by a single nucleotide extension of the extended primer into the region of amplified gene sequence variation. For UDG assay, only six fields were used when preparing the assay group FILE I. Abbreviations: WELL = the well number assigned to the assay; TERM = termination mix; SNP_ID = name of the input sequence of the single nucleotide polymorphism; 2nd-PCRP = forward amplicon primer; 1st-PCRP = reverse amplicon primer; AMP_LEN = amplicon length; UP_CONF = uniplex amplification score; MP_CONF = multiplex amplification score; Tm = melting temperature; PcGC = percent GC content;PWARN = assay design warning codes; UEP_DIR = direction of primer extension; UEP_MASS = unextended-primer mass; UEP_SEQ = unextended-primer sequence; EXT1_CALL = name given to the analyte 1 mass peak; EXT1_MASS = mass of analyte 1.
0620_1-2 |
0620_1-3 |
0620_1-4 |
0620_1-5 |
0620_1-6 |
0620_1-7 |
0620_1-8 |
Table 3: 0620.xlsx file. These settings were used for the UGI inhibition reactions in Figure 4A. Abbreviation: UGI = uracil glycosylase inhibitor.
This paper provides a detailed procedure for using a UDG MALDI-TOF MS assay method to directly detect AP-containing DNA products. The main advantages of this method are that uracil-containing substrates are label-free, scalable, easy to work with, and afford greater flexibility in substrate design.
The UDG supplier-recommended phenol/chloroform extraction enables inactivation of the enzyme to prevent degradation of product DNA. However, the phenol extraction protocol involves tedious phase-separation of hazardous chemicals. An alternative acid termination method using HCl to lower the reaction pH to 2 ± 0.5 also effectively inactivated UDG. Subsequent neutralization with Tris base could prevent DNA damage. When the AP product was subjected to MS analysis within 30 h, there were no signs of base loss or modification in the mass spectra (Figure 1E). The Tris buffer in the reactions was provided with the commercial UDG. However, various amines, including Tris, can incise AP sites via beta-elimination, albeit at high reagent concentrations44. Some alternatives, such as HEPES and phosphate buffer, can be considered to avoid the risk of cleavage of the AP site by beta-elimination.
As a potential UDG reference method, a duplex of centered G:U substrate (Table 1, T1/U+9) is recommended as the standard substrate for the following reasons: 1. for physiological relevance, G:U is superior to A:U as deamination of cytosine in G:C pairs results in G:U lesions. 2. While E. coli UDG demonstrates higher specificity to hydrolyze U from single-stranded DNA, repair of a U lesion in single-stranded DNA is uncommon. 3. For all the G:U substrate tested, G:U+2 and G:U+3 demonstrated higher reaction rates than G:U+9. However, a DNA deamination lesion adjacent to a strand break is uncommon. For mimicking native lesions, placing a G:U at the center of the DNA duplex would be more appropriate.
Interestingly, the MALDI-TOF MS method generated UDG kinetic parameters, and the enzyme units of the G:U reaction (Figure 3A–C) were very similar to conventionally derived results using 3H-uracil38. However, the single G:U substrate in this study is very different from PBS1 bacteriophage DNA previously described with multiple A:U from DNA synthesis errors38. Moreover, UDG showed 3 times higher activity with G:U than with the A:U substrate (Table 1, Km and Kcat of G:U versus A:U). This seemingly contradictory result can be attributed to the DNA sequence of PBS1 substrate containing 36% of U in the form of multiple A:U lesions45 compared to only 3% of U in the G:U substrate (Table 1). Meanwhile, UDG is a very 'processive' enzyme, i.e., a single protein-DNA binding event elicits multiple uracil cleavages12. The finding of a near-perfect one-to-one correlation between these two UDG methods makes this MS method an attractive option instead of the traditional radioisotope assay.
This approach is easily scalable. All UDG reactions in this study were performed manually using micropipettes and microcentrifuge tubes, with ~30 tests performed on a given day. Thus, less than one-tenth the capacity of the 384-well plate format chip array for the MALDI-TOF MS system (see the Table of Materials) described in sections 3-5 was used. In contrast, the adaptation of an automated pipetting system for a 384-well microplate could easily increase the daily output of this approach by using the acid termination method. Thus, 300 UDG reactions would take ~1 h. The MALDI-TOF MS system (see the Table of Materials) could output mass spectra data for up to 300 reactions in 1 h. Hence, a streamlined process would require 2 h to complete 300 UDG MALDI-TOF MS assays.
The authors have nothing to disclose.
We thank the NCFPB Integrated Core Facility for Functional Genomics (Taipei, Taiwan) and the NRPB Pharmacogenomics Lab (Taipei, Taiwan) for their technical support. This work was supported by the Ministry of Science and Technology, Taiwan, R.O.C. [grant number MOST109-2314-B-002 -186 to K.-Y.S., MOST 107-2320-B-002-016-MY3 to S.-Y.C., MOST 110-2320-B-002-043 to W.-h.F.]. H.-L. C. is a recipient of a doctoral fellowship from National Taiwan University. Funding for open access charge: Ministry of Science and Technology, R.O.C.
2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris base) | J.T baker | Protocol 1,2 | |
Autoclaved deionized water | MILLIPORE | Protocol 1,2 | |
EDTA | J.T Baker | Protocol 1,2 | |
Gloves | AQUAGLOVE | Protocol 1,2,3 | |
Hydrochloric acid (HCl) | SIGMA | Protocol 1,2 | |
Ice bucket | Taiwan.Inc | Protocol 2 | |
Low retention pipette tips(0.5-10 µL) | extra gene | Protocol 1,2 | |
Low retention pipette tips(1,250 µL) | national scientific supply co, Inc. | Protocol 1,2 | |
Low retention pipette tips(200 µL) | national scientific supply co, Inc. | Protocol 1,2 | |
MassARRAY | Agena Bioscience, CA | Protocol 4, 5 Mass spectrometry control programs include Typer Chip Linker, SpectroACQUIRE, and Start RT Process. |
|
MassARRAY Nanodispenser | AAT Bioquest, Inc. | RS1000 | Protocol 3 |
Microcentrifuge | Kubota | Protocol 2 | |
Microcentrifuge | Clubio | Protocol 2 | |
Microcentrifuge tube (1.5 mL) | National scientific supply co, Inc. | Protocol 2 | |
Microcentrifuge tube rack | Taiwan.Inc | Protocol 1,2 | |
Micropipette (P1000) | Gilson | Protocol 1,2 | |
Micropipette (P2) | Gilson | Protocol 1,2 | |
Micropipette (P10) | Gilson | Protocol 1,2 | |
Micropipette (P100) | Gilson | Protocol 1,2 | |
Micropipette (P200) | Gilson | Protocol 1,2 | |
Micropipette (SL2) | Rainin | Protocol 1,2 | |
Oligonucleotides | Integrated DNA Technologies (Singapore) | Protocol 1,2 | |
Quest Graph IC50 Calculator (v.1) | AAT Bioquest, Inc. | Fig. 4 https://www.aatbio.com/tools/ic50-calculator-v1 |
|
Sodium hydroxide (NaOH) | WAKO | Protocol 2 | |
SpectroCHIP array | Agena Bioscience, CA | #01509 | Protocol 3, 5 |
Timer | Taiwan.Inc | Protocol 2 | |
Typer 4.0 software | Agena Bioscience, CA | #10145 | Protocol 6 Typer 4.0 consists four programs including Assay Designer, Assay Editor, Plate Editor, and Typer Analyzer. |
UDG Reaction Buffer (10x) | New England Biolabs, MA | B0280S | Protocol 2 |
Uracil Glycosylase Inhibitor | New England Biolabs, MA | M0281S | Protocol 2 |
Uracil-DNA Glycosylase | New England Biolabs, MA | M0280L | Protocol 2 |
UV-VISBLE spectrophotometer UV-1601 | SHIMADZU | Protocol 1 | |
Water bath | ZETA ZC-4000 (Taiwan.Inc) | Protocol 2 |