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>
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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. 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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 ...

This method can help answer key questions in the field of DNA polymers fidelity and DNA repair such as proofreading methods and analysis and in defining the DNA repair patch. The main advantage of this technique is that it is very specific, simple, rapid, and easy to perform. Generally, individuals new to this method will struggle because of still they in DNA replication and the repair field may be not familiar with the mass spectrometry instrumentation and the data interpretation.I think the most important is the visual demonstration is very critical as we can show how our commercially developed mass spectrometry for clinical applications such as mutation and deputation can be conveniently adapted for DNA proofreading and the repair study. Demonstrating the procedure will be Rong-Syuan and Kuei-Ching graduate students from my lab. Begin this procedure with primer and template preparation as described in the text protocol.As an example, use a TG mismatch of substrate P21 with T28 for the proofreading. Dilute the P21 primer and the T28 template to 12.5 picomoles per microliter with water. Then, transfer 12 microliters of the diluted primer and 12 microliters of the diluted template to a 1.5 milliliter sterilized micro centrifuge tube.After closing the tube tightly, incubate the tube for 30 minutes in a covered 65 degree Celsius water bath. Then, incubate for 30 minutes in a 37 degree Celsius water bath. And, finally, place the tube on ice to ensure a proper annealing.To a 1.5 milliliter sterilized micro centrifuge tube, add eight microliters of primer and template mix, two microliters of 10x proofreading reaction buffer, four microliters of 4ddNTPs mix, and water up to 18 microliters. Flick the tube to mix. Dilute the DNA polymerase in ice cold 1x proofreading reaction buffer to the desired concentration.Store the diluted enzyme on ice at all times. Pre-warm the micro centrifuge tube with the substrate mix to the desired reaction temperature. Then, transfer two microliters of the diluted DNA polymerase to the pre-warmed substrate mixture and flick the tube to mix its contents.Centrifuge the tubes with enzyme and substrate for a few seconds at 3200 times gravity at ambient temperature to spin down the components of the reactions. Immediately transfer the reaction to a heating block or a water bath at the desired incubation temperature. To terminate the reaction, add an equal volume of buffered phenol and mix the reaction by vortexing for a few seconds.Then, centrifuge the reaction in a micro-centrifuge at 3200 times gravity at ambient temperature for five minutes. Transfer the aqueous phase to a clean micro-centrifuge tube and add an equal volume of chloroform. After mixing it by vortexing for a few seconds, centrifuge the sample in a microcentrifuge at 3200 times gravity at ambient temperature for three minutes.Transfer the aqueous phase to a clean micro-centrifuge tube. After incubating the reaction at 95 degrees Celsius for five minutes, place the tube on ice. Next, perform resin addition to eliminate salt contamination in a 384 well plate as detailed in the text protocol.Set the sample plates in the nanoliter dispenser. Place the matrix chip and the prepared 384 well plate in the corresponding tray. Operate the nano-dispenser to spot the reaction products to the chip.Push the run button on the touch screen of the nano-dispenser to start. Check the automated captured image of the sample spots containing saturation information on the screen to ensure the overall spotted volume on the chip is around five to 10 nanoliters. Repeat the sample spotting if the volume is insufficient.Prepare a file in Excel format containing the anticipated signal information for importing. Create and define a new assay in the application program by right-clicking import assay group in designer format and select the Excel file. Establish the target assay plate via the customer project plate option tree on the left side of the screen by right-clicking the top of the tree and giving it a file name.Select the 384 well plate type and press OK.A blank plate will appear. Select the appropriate assay on the left side of the screen. Assign the selected assay for each spotted sample position on the chip by highlighting the well and right-clicking to select add plex.Next, prepare a working list of all the tests on the chip in Excel format with no header. Import the working list via the add new sample project option. Assign the test and the working list to each position of the P21T28 assay and choose by right-clicking.Link the mass spectrometer to the chip using the application program. Select the default setting at the right side of the screen. Fill in the assay name and the chip ID at the corner of the chip.Save the settings and start the MS control program. Press the in/out button and take out the scout plate. Place the spotted chip onto the scout plate and press the in/out button for the spotted chip to enter the mass spectrometer.Click the acquire button of the application program to start the mass spectrometry and acquire the data. Open the program for the data analysis. Browse the tree of the database at the upper left corner and select the chip ID.Open the data file on the right side of the screen.Click the spectrum icon to display the mass spectrum. To crop a specific range of mass over charge, right-click to select customization dialogue. In the new window, click on x-axis and set the desired upper and lower limit and press OK to show the specified range of the spectrum.Export the spectrum for record keeping by right-clicking export. Select the JPEG file type, click on destination, select browse disc, type the file name, and then click on export. For data quantitation, use the cursor and click on the peak to show the peak height at the upper left hand corner.In MALDI-TOF MS there is an inverse relationship between peak intensity in Oligonucleotide mass. Oligonucleotides from 17 to 24 nucleotides were subjected to an MS analysis. The relative signal intensities were calculated and could be used for quantification of the proofreading or repair assay.Both template and primer signals could be found in the mass spectrum. Because of size differences, signals from templates and their non-specific partial degradation products were well separated from the mismatched primers and proofread products. Shown here is a proofreading of a three prime terminal TG.At five minutes, the corrected product was 23%versus 17%for the excision product.This difference continued to increase to 69%versus 14%at 20 minutes. Note the high resolution of MS clearly separating the sub-straight and product signals with a mass overcharge difference of only 32. Also easily resolvable were two forms of excision products for a pen ultimate TG proofreading.The one nucleotide excision intermediate and two nucleotide excision products. Proofreading of internal mismatches showed a clear trend in proofreading efficiency. As evidenced by a large proofread product speak, increased efficiency was observed when substraights had mismatches at the last, penultimate, third, and fourth nucleotides from the three prime end, as compared to mismatches at other positions.Once mastered, this technique can be done in four hours if it is performed properly. Following this procedure, other applications like polymerous proofreading heapiter screening can be performed to discover and invent new drug for pharmaceutical research. This technique also paves the way for researchers in the field of DNA replication and repair to gain more insight into the medicine of the gnome and it's faithful replication for all living organisms.After watching this video, you should have a good understanding of how to perform DNA proofreading and the repair assay using MADI-TOF mass spectrometry analysis. Don't forget that working with spheno-chloroform can be hazardous and the precautions, such as wearing personal protection gear, should always be taken while performing the procedure. The alternative protocol in the text could be considered as a safer substitute.

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