Name: proofreading and dna repair assay using single nucleotide extension and maldi-tof mass spectrometry analysis
Uploaded: 2018-06-19T14:45:00
Description: Watch this Scientific Journal Video about Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis at JoVE.com

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 research grants from the Taiwan Health Foundation (L-I.L.) and the Ministry of Science and Technology, Taipei, Taiwan, ROC [MOST 105-2320-B-002-047] for Woei-horng Fang, [MOST 105-2628-B-002 -051-MY3] for Kang-Yi Su, and [MOST-105-2320-B-002-051-MY3] for Liang-In Lin.

1. Primer/template Preparation
<ol>
	<li>Design primers/templates with a balanced G+C content between 40% and 60% as in a sequencing or PCR primer design. Use primers of 18 to 21 nt for an appropriate annealing and better MS signals.</li>
	<li>Design the template by setting 50 &#176;C as the minimum melting temperature for the duplex region with at least 7 nt of 5&#39;-overhang to separate the signals between the primer and the template. 
	NOTE: For example, for the substrate P21/T28 in Table 1, t...

<ol>
	<li>Loeb, L. A., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fidelity+of+DNA+synthesis.">Fidelity of DNA synthesis.</a> Annual Review of Biochemistry. 51, 429-457 (1982).</li><li>Kornberg, A., Baker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNA replication. , (1992).</li><li>Carroll, S. S., Benkovic, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+aspects+of+DNA+polymerases:+Escherichia+coli+DNA+polymerase+I+(Klenow+fragment)+as+a+paradigm.">Mechanistic aspects of DNA polymerases: Escherichia coli DNA polymerase I (Klenow fragment) as a paradigm.</a> Chemical Reviews. 90 (7), 1291-1307 (1990).</li><li>Echols, H., Goodman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fidelity+Mechanisms+in+DNA+Replication.">Fidelity Mechanisms in DNA Replication.</a> Annual Review of Biochemistry. 60 (1), 477-511 (1991).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetic+and+chemical+mechanism+of+high-fidelity+DNA+polymerases.">The kinetic and chemical mechanism of high-fidelity DNA polymerases.</a> Biochimica et Biophysica Acta. 1804 (5), 1041-1048 (2010).</li><li>Reha-Krantz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+proofreading:+Multiple+roles+maintain+genome+stability.">DNA polymerase proofreading: Multiple roles maintain genome stability.</a> Biochimica et Biophysica Acta. 1804 (5), 1049-1063 (2010).</li><li>Fidalgo da Silva, E., Reha-Krantz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+proofreading:+active+site+switching+catalyzed+by+the+bacteriophage+T4+DNA+polymerase.">DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase.</a> Nucleic Acids Research. 35 (16), 5452-5463 (2007).</li><li>Petruska, J., Goodman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+neighboring+bases+on+DNA+polymerase+insertion+and+proofreading+fidelity.">Influence of neighboring bases on DNA polymerase insertion and proofreading fidelity.</a> The Journal of Biological Chemistry. 260 (12), 7533-7539 (1985).</li><li>Mendelman, L. V., Petruska, J., Goodman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+mispair+extension+kinetics.+Comparison+of+DNA+polymerase+&#945;+and+reverse+transcriptase.">Base mispair extension kinetics. Comparison of DNA polymerase &#945; and reverse transcriptase.</a> The Journal of Biological Chemistry. 265 (4), 2338-2346 (1990).</li><li>Lutz, S., Burgstaller, P., Benner, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+screening+technique+for+DNA+polymerases+that+can+incorporate+modified+nucleotides.+Pseudothymidine+as+a+substrate+for+thermostable+polymerases.">An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudothymidine as a substrate for thermostable polymerases.</a> Nucleic Acids Research. 27 (13), 2792-2798 (1999).</li><li>Da Silva, E. F., Mandal, S. S. S., Reha-Krantz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+2-aminopurine+fluorescence+to+measure+incorporation+of+incorrect+nucleotides+by+wild+type+and+mutant+bacteriophage+T4+DNA+polymerases.">Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA polymerases.</a> The Journal of Biological Chemistry. 277 (43), 40640-40649 (2002).</li><li>Frey, M. W., Sowers, L. C., Millar, D. P., Benkovic, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleotide+analog+2-aminopurine+as+a+spectroscopic+probe+of+nucleotide+incorporation+by+the+Klenow+fragment+of+Escherichia+coli+polymerase+I+and+bacteriophage+T4+DNA+polymerase.">The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase.</a> Biochemistry. 34 (28), 9185-9192 (1995).</li><li>Leung, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+G-quadruplex-based+luminescent+switch-on+probe+for+the+detection+of+polymerase+3'-5'+proofreading+activity.">A highly sensitive G-quadruplex-based luminescent switch-on probe for the detection of polymerase 3'-5' proofreading activity.</a> Methods. 64 (3), 224-228 (2013).</li><li>Song, C., Zhang, C., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+detection+of+DNA+polymerase+fidelity+by+singly+labeled+smart+fluorescent+probes.">Rapid and sensitive detection of DNA polymerase fidelity by singly labeled smart fluorescent probes.</a> Biosensors and Bioelectronics. 26 (5), 2699-2702 (2011).</li><li>Haff, L. A., Smirnov, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-nucleotide+polymorphism+identification+assays+using+a+thermostable+DNA+polymerase+and+delayed+extraction+MALDI-TOF+mass+spectrometry.">Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.</a> Genome Research. 7 (4), 378-388 (1997).</li><li>Haff, L. A., Smirnov, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genotyping+of+PCR+products+with+MassTag-labeled+primers.">Multiplex genotyping of PCR products with MassTag-labeled primers.</a> Nucleic Acids Research. 25 (18), 3749-3750 (1997).</li><li>Blondal, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+MALDI-TOF+based+methodology+for+genotyping+single+nucleotide+polymorphisms.">A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms.</a> Nucleic Acids Research. 31 (24), e155 (2003).</li><li>Su, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+Epidermal+Growth+Factor+Receptor+(EGFR)+T790M+mutation+predicts+shorter+EGFR+tyrosine+kinase+inhibitor+response+duration+in+patients+with+non-small-cell+lung+cancer.">Pretreatment Epidermal Growth Factor Receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer.</a> Journal of Clinical Oncology. 30 (4), 433-440 (2012).</li><li>Lee, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endonuclease+V-mediated+deoxyinosine+excision+repair+in+vitro.">Endonuclease V-mediated deoxyinosine excision repair in vitro.</a> DNA Repair. 9, 1073-1079 (2010).</li><li>Lee, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+excision+of+3'+penultimate+errors+by+DNA+polymerase+I+and+its+role+in+endonuclease+V-mediated+DNA+repair.">The excision of 3' penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair.</a> DNA Repair. 12 (11), 899-911 (2013).</li><li>Kucera, R. B., Nichols, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-Dependent+DNA+Polymerases.">DNA-Dependent DNA Polymerases.</a> Current Protocols in Molecular Biology. 84 (1), 3.5.1-3.5.19 (2008).</li><li>Tabor, S., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+residue+in+DNA+polymerases+of+the+Escherichia+coli+DNA+polymerase+I+family+is+critical+for+distinguishing+between+deoxy-+and+dideoxyribonucleotides.">A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (14), 6339-6343 (1995).</li><li>Weiss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+deoxyinosine+from+the+Escherichia+coli+chromosome+as+studied+by+oligonucleotide+transformation.">Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation.</a> DNA Repair. 7 (2), 205-212 (2008).</li><li>Cao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endonuclease+V:+an+unusual+enzyme+for+repair+of+DNA+deamination.">Endonuclease V: an unusual enzyme for repair of DNA deamination.</a> Cellular and Molecular Life Sciences. 70 (17), 3145-3156 (2013).</li><li>Su, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+single+nucleotide+extension+and+MALDI-TOF+mass+spectrometry+in+proofreading+and+DNA+repair+assay.">Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay.</a> DNA Repair. 61, 63-75 (2018).</li><li>Carver, T. E., Hochstrasser, R. A., Millar, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proofreading+DNA:+recognition+of+aberrant+DNA+termini+by+the+Klenow+fragment+of+DNA+polymerase+I.">Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (22), 10670-10674 (1994).</li><li>Santa Lucia, J., Hicks, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thermodynamics+of+DNA+structural+motifs.">The thermodynamics of DNA structural motifs.</a> Annual Review of Biophysics and Biomolecular Structure. 33, 415-440 (2004).</li><li>Su, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+I+proofreading+exonuclease+activity+is+required+for+endonuclease+V+repair+pathway+both+in+vitro+and+in+vivo.">DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo.</a> DNA Repair. 64, 59-67 (2018).</li></ol>

This study described a step-by-step proofreading activity assay analyzed by the chosen commercial instrument (see the Table of Materials) using MALDI-TOF MS. The major advantages include that the primer and template are label free and easy to perform, allowing for greater flexibility in designing experiments. A stream-lime complete processing of 30 proofreading tests would take 4 h, including 3 h for manually performing the proofreading reactions and their cleanup, while the MALDI-TOF MS analyses using t...

The proofreading functions of DNA polymerases during DNA replication are essential to ensure the high fidelity of genetic information that needs to be transferred to progeny1,2,3,4,5,6,7. Being able to assess the contributions of polymerase proofreading exonucleases would clarify the mechanisms safeguarding genetic stability.
Radioisotope labeling and gel-based assays in combination with densitometric analyses of the autoradiograms or phosphor imaging8,9,10 have traditionally been used to detect proofreading activity of DNA polymerases. While functional, these assays are laborious, expensive, and not amenable to high-throughput formats. In addition, radioisotopes suffer safety issues including waste disposal. Alternatively, proofreading activities have been analyzed by fluorometric techniques. For example, 2-aminopurine (2-AP) can be incorporated into extension products during in vitro polymerase proofreading assays to produce a fluorescent signal11,12. Unfortunately, these approaches suffer from a low specificity, since 2-AP can pair with both thymine and cytosine. More recent approaches include a sensitive G-quadruplex-based luminescent switch-on probe for a polymerase 3&#39;-5&#39; exonuclease assay13 as well as a singly-labeled fluorescent probe for a polymerase proofreading assay that overcomes some of the aforementioned drawbacks14. Enthusiasm for these fluorometric methods is diminished due to the need for the specific labeling of DNA substrates.
In contrast, a MALDI-TOF MS for DNA analysis has been employed in the PinPoint assay, where the primer extension reactions with unlabeled 4 ddNTPs can be used to identify polymorphisms at a given locus15,16,17 and has been widely adopted in clinical applications for mutation detections and cancer diagnoses18. Using these basic principles, we have created a label-free assay for the in vitro determination of DNA polymerase proofreading activity exploiting the high resolution, high specificity, and high-throughput potential of MALDI-TOF MS. Using the E. coli DNA polymerase I Klenow fragment as a model enzyme, dideoxyribonucleotide triphosphates (ddNTPs) as substrates can take a &#34;snapshot&#34; of proofreading products after a single nucleotide extension via MALDI-TOF MS (Figure 1).
Likewise, this method was also developed for a DNA repair assay where primers containing 3&#39; penultimate dI lesions are subjected to a pol I repair assay which mimics endo V nicked repair intermediates. While not fully understood, the endo V repair pathway is the only repair system known to employ the pol I proofreading exonuclease activity for lesion excision19,20. Using MALDI-TOF MS, we show a clearly defined repair patch where dI can be excised by pol I when occurring in the last 2 nt of the primer before adding the correct complemented nucleotide.
For the study of proofreading and DNA repair, this method is faster and less laborious than previous methods and provides additional information towards mechanism and function.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1125">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:331px;">Oligonucleotides</td>
			<td style="width:283px;">Mission Biotech (Taiwan)&#160;</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">phosphorothioate modified oligonucleotides</td>
			<td>&#160;Integrated DNA Technologies (Taiwan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNA polymerase I, Large (Klenow) Fragment</td>
			<td>New England Biolabs, MA</td>
			<td>M0210L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Klenow fragment (3&#39;&#8594;5&#39; exo-)</td>
			<td>New England Biolabs, MA</td>
			<td>M0212L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NEBuffer 2.1 (10X)</td>
			<td>New England Biolabs, MA</td>
			<td>B7202S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddATP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddGTP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddTTP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddCTP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dATP, dGTP, dCTP, dTTP set</td>
			<td>Clubio, Taiwan</td>
			<td>CB-R0315</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SpectroCHIP array</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#01509</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MassARRAY&#160;</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Typer 4.0 software</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#10145</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Clean Resin Tool Kit&#160;</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#08040</td>
			<td></td>
		</tr>
	</tbody>
</table>

proofreading and dna repair assay using single nucleotide extension and maldi-tof mass spectrometry analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

Templates and primers:
Using the procedure presented here, equal molar synthetic oligonucleotide templates and primers of relevant sequences obtained from commercial sources were checked for their purity and quality (Figure&#160;3A; note the signals matched the designated mass and the low background) as well as for the relationship between the peak intensity and the analyte mass (<s...

Watch this Scientific Journal Video about Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis at JoVE.com

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

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