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

Vi takker NCFPB integreret Core facilitet for funktionel genomforskning (Taipei, Taiwan) og NRPB farmakogenomforskning Lab (Taipei, Taiwan) for deres tekniske support. Dette arbejde blev støttet af forskningstilskud fra Taiwan Health Foundation (L-I.L.) og ministeriet for videnskab og teknologi, Taipei, Taiwan, ROC [mest 105-2320-B-002-047] af Wei-Yao Hu, [mest 105-2628-B-002-051-MY3] for Kang-Yi Su og [ Most-105-2320-B-002-051-MY3] for Liang-i Lin.

1. primer/skabelon forberedelse
<ol><li>Design primere/skabeloner med en afbalanceret G + C indhold mellem 40% og 60% som en sekvensering eller PCR primer design. Bruge primere 18 til 21 nt for en passende udglødning og bedre MS signaler.</li>
	<li>Designskabelon ved 50 ° C som minimum smeltende temperatur for regionen duplex med mindst 7 nt af 5'-overhæng at adskille signaler mellem primeren og skabelonen. Bemærk: For eksempel, til substratet P21/T28 i tabel 1, 21-nt primer er parret med en 28-...

<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>

Denne undersøgelse beskrev en trinvis korrekturlæsning aktivitet assay analyseres ved den valgte kommercielle instrument (Se Tabel af materialer) ved hjælp af MALDI-TOF MS. De store fordele omfatter er primer og skabelon etiket gratis og nem at udføre, giver mulighed for større fleksibilitet i at designe eksperimenter. En stream-lime komplet behandling af 30 korrekturlæsning tests ville tage 4 h, herunder 3 h for manuelt udfører korrekturlæsning reaktioner og deres oprydning, mens MALDI-TOF MS an...

DNA polymeraser under DNA replikation korrekturlæsning funktioner er afgørende for at sikre high fidelity af genetiske oplysninger, der skal overføres til afkom1,2,3,4, 5,6,7. At være i stand til at vurdere bidragene fra polymerase korrekturlæsning exonucleases ville afklare mekanismerne sikre genetisk stabilitet.
Radioisotop mærkning og gel-baserede assays i kombination med densitometric analyser af autoradiograms eller fosfor imaging8,9,10 har traditionelt været brugt til at registrere korrekturlæsning aktivitet af DNA polymeraser. Mens funktionelle, er disse assays besværligt, dyrt og ikke indstillet til høj overførselshastighed formater. Derudover lider radioisotoper sikkerhedsspørgsmål, herunder bortskaffelse af affald. Alternativt, korrekturlæsning aktiviteter er blevet analyseret af fluorometriske teknikker. For eksempel, kan 2-aminopurine (2-AP) indarbejdes i forlængelse produkter under in vitro- polymerase korrekturlæsning assays for at producere en fluorescerende signal11,12. Desværre, disse tilgange lider af en lav specificitet, da 2-AP kan parre med både thymin og cytosin. Nyere tilgange omfatter en følsom G-quadruplex-baserede selvlysende Tænd sonden for en polymerase 3' - 5' exonuclease assay13 samt en enkeltvis mærket fluorescerende sonden for en polymerase korrekturlæsning assay, der overvinder nogle af de ovennævnte ulemper14. Begejstring for disse fluorometriske metoder er formindsket på grund af behovet for den specifikke mærkning af DNA substrater.
Derimod en MALDI-TOF MS for DNA-analyse har været ansat i PinPoint assay, hvor primer udvidelse reaktioner med umærkede 4 ddNTPs kan bruges til at identificere polymorfier på en given locus15,16,17 og har været udbredt i kliniske applikationer for mutation opdagelser og cancer diagnoser18. Brug disse grundlæggende principper, har vi skabt en etiket-fri assay til in vitro- bestemmelse af DNA polymerase korrekturlæsning aktivitet at udnytte høj opløsning, høj specificitet og høj overførselshastighed potentiale af MALDI-TOF MS. med E. coli DNA polymerase jeg Klenow fragment som en model enzym, dideoxyribonucleotide triphosphates (ddNTPs) som substrater kan tage et "snapshot" af korrekturlæsning produkter efter en enkelt nukleotid udvidelse via MALDI-TOF MS (figur 1).
Ligeledes, denne metode blev også udviklet til en DNA reparation assay hvor primere indeholdende 3' næstsidste dI læsioner er udsat for en pol jeg reparere analyse som efterligner endo V hakker reparation mellemprodukter. Mens ikke fuldt forstået, er endo V reparation vej den eneste reparation system kendt at ansætte pol jeg korrekturlæsning exonuclease aktivitet for læsion excision19,20. Bruger MALDI-TOF MS, vi vise et klart defineret reparation patch hvor dI kan være skåret ud af pol jeg når forekommer i de sidste 2 nt primer før du tilføjer den rigtige suppleret nukleotid.
Til undersøgelse af korrekturlæsning og DNA reparation, denne metode er hurtigere og mindre besværligt end tidligere metoder og giver yderligere oplysninger mod mekanisme og funktion.

<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

Vedligeholdelse af genomet og dens trofaste replikering er altafgørende for at bevare genetisk information. For at vurdere high fidelity replikering, vi har udviklet en simpel, ikke-mærket og ikke-radio-isotopiske metode ved hjælp af en matrix assisted laser desorption ionisering med time-of-flight (MALDI-TOF) masse massespektrometri (MS) analyse for en korrekturlæsning undersøgelse. Her, en DNA-polymerase [fx Klenow fragment (KF) af Escherichia coli DNA polymerase jeg (pol jeg) i denne undersøgelse] i overværelse af alle fire dideoxyribonucleotide triphosphates bruges til at behandle en uoverensstemmelse mellem primer-skabelon duplex. Den forkerte primer er derefter læse korrektur/forlænget og underkastes MALDI-TOF MS. Produkterne er kendetegnet ved en masse ændring af primer ned til enkelt nucleotid variationer. Vigtigere er, kan en korrekturlæsning også bestemmes for interne indre uoverensstemmelser, omend på forskellig virkningsgrader. Mismatchproblemer beliggende på 2-4-nukleotider (nt) fra 3' enden var effektivt korrekturlæst af pol, og en uoverensstemmelse på 5 nt fra primer terminus viste kun en delvis korrektion. Ingen korrekturlæsning opstod for interne uoverensstemmelser ligger på 6-9 nt fra primer 3' enden. Denne metode kan også anvendes til DNA reparation assays (f.eks. vurdering af en base-læsion reparation af substrater for endo V reparation pathway). Primere som indeholder 3' næstsidste deoxyinosine (dI) læsioner kunne korrigeres ved pol jeg. Faktisk, næstsidste T-jeg, G-jeg, og A-jeg substrater havde deres sidste 2 dI-holdige nukleotider skåret ud af pol jeg før du tilføjer en korrekt ddN 5'-monophosphate (ddNMP) mens næstsidste C-jeg uoverensstemmelser blev tolereret af pol jeg tillader primer udvides uden reparation, demonstrerer følsomhed og opløsning af MS analyse for at måle DNA reparation.

Skabeloner og primere:
Ved hjælp af den procedure, der er præsenteret her, lige kindtand syntetiske oligonukleotid skabeloner og primere relevante sekvenser fra kommercielle kilder blev kontrolleret for deres renhed og kvalitet (figur 3A, Bemærk signalerne, der matchede den udpegede masse og lave baggrunden) samt for forholdet mellem den maksimale intensitet og analysanden massen...

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

Korrekturlæsning og DNA reparation analyse ved hjælp af Single Nucleotide udvidelse og MALDI-TOF-massespektrometri analyse

En ikke-mærket, ikke-radio-isotopiske metode for DNA polymerase korrekturlæsning og en DNA reparation assay blev udviklet ved hjælp af høj opløsning MALDI-TOF-massespektrometri og en enkelt nukleotid udvidelse strategi. Analysen viste sig at være meget specifikke, enkel, hurtig og let at udføre for korrekturlæsning og reparation patches kortere end 9-nukleotider.

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

Research

Education

JoVE Journal

JoVE Core

Biochemistry

Cell Biology

Molecular Biology

Unit 1

DNA Replication

DNA Replication and Repair

SCIENTISTS IN ACTION

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Løsning affinitetsrensede proteinkomplekser ved Blå Native PAGE og protein Korrelation Profilering

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid Droplet Isolation for Kvantitativ massespektrometri Analyse

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Bestemmelse af Protease specificitet i mus væv ekstrakter af MALDI-TOF-massespektrometri: manipulere PH for at forårsage specificitet ændringer

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

Brug af open source-MALDI TOF-MS IDBac-rørledningen til analyse af mikrobiel protein og specialiserede metabolit data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Nitropeptid profilering og identifikation illustreret af angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

Dual DNA-linealer til at studere mekanismen af Ribosome translokation med enkelt-nukleotid opløsning

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

En integreret Raman spektroskopi og massespektrometri platform til at studere enkelt-celle stof optagelse, metabolisme, og effekter

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Kvantitativ analyse af cellulære lipidom af Saccharomyces Cerevisiae Ved hjælp af flydende kromatografi kombineret med tandem massespektrometri

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Umålrettet flydende kromatografi-massespektrometri-baserede metabolomiske analyse af hvedekorn

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...