This research was supported by the ISRAEL SCIENCE FOUNDATION (grant no. 1023/18).

1. Design of microarray
NOTE: DNA probes represent custom 36-nucleotide sequences, consisting of the recognition site for T7 DNA primase (GTC) located between two variable flanking regions, followed by a constant 24-nucleotide sequence tethered to a glass slide3. We used a 4 x 180,000 microarray format, which enabled spotting of each DNA sequence in six replicates, randomly distributed on the slide.
<ol>
	<li>Design of DNA library for primases with known recognition sequences 
	<ol>
		<li>Ensure that each sequence is composed of 60 nucleotides. Keep the first 24 nucleotides constant (to be attached to the glass slide). Variable regions should have the following general form: (N)16GTC(N)17; where N represents any desired nucleotide.</li>
		<li>Design the flanking region to address a specific scientific question (an example of the design is presented in the following text). The flanking regions may be designed to contain specific features such as the repeat elements or specific symmetry. 
		NOTE: We have created eight categories of different flanking sequences composed of two or three specific nucleotides: T and G (group 1); T and C (group 2); C and A (group 3); A and G (group 4); G, C and T (group 5); C, T and A (group 6); G, A and T (group 7); G, A and C (group 8). 2,000 different sequences were designed for each group. Each group had subgroups of sequences with different types and different numbers of sequence repeats.</li>
		<li>Include a set of negative control sequences (lacking the specific binding site)4. The presence of such sequences allows to validate the occurrence of specific binding in the experiment.</li>
	</ol>
	</li>
	<li>Design of DNA library for primases with unknown recognition sequences 
	<ol>
		<li>If the recognition sequence is unknown, create the DNA library by selecting the sequences (with or without specific features mentioned previously) from the genome of desired organism (bacteria, fungi, etc.).</li>
	</ol>
	</li>
	<li>Design of microarray slide 
	<ol>
		<li>Purchase custom slides (e.g., 4x180K, AMADID #78366) from a commercial supplier (for more details see Table of Materials). Order each sequence in six replicates, where each replicate should be attached to a randomly selected spot on the slide.</li>
	</ol>
	</li>
</ol>
2. Primase DNA binding experiment
NOTE: The day before (or at least 2 h before) the PBM, prepare the blocking solution [2% w/v skim milk in phosphate buffer saline (PBS)] and stir it on a magnetic stirrer. Prior to use, filter the solution with a 0.45 &#181;M filter. To detect the primase binding to the DNA strands, several steps should be performed in the following order.
<ol>
	<li>Blocking procedure 
	<ol>
		<li>Pre-wet the microarray slide in a Coplin jar with 0.01% v/v Triton X-100 in PBS (5 min at 125 rpm on a lab rotator).</li>
		<li>Briefly wash the gasket slide (also referred to as coverslip) with water and ethanol and dry using compressed air.</li>
		<li>Assemble bottom part of steel hybridization chamber (PBM chamber) with the gasket slide facing up (commercial label facing up). For more details regarding the assembly, refer to the Table of Materials.</li>
		<li>Pipet blocking solution (2% w/v skim milk in PBS) into each chamber.</li>
		<li>Remove the microarray slide from the Coplin jar, then dry the non-DNA side (the DNA should be on the same side as the company label) and edges using a fine wipe. Slowly place the microarray on the gasket slide, avoiding bubbles (company label facing down). Immediately assemble and tighten steel hybridization chamber apparatus. Incubate for 1 h at room temperature (RT).</li>
		<li>Fill one staining dish (the &#8220;PBS&#8221; dish) with 800 mL of fresh PBS. Fill a second staining dish (the &#8220;WASH&#8221; dish) with 800 mL of freshly prepared 0.5% v/v Tween-20 in PBS.</li>
		<li>Unscrew the PBM chamber and remove the microarray slide-coverslip &#8220;sandwich&#8221;, taking care not to break the seal. Disassemble it underwater in the WASH dish by placing forceps between the slide and coverslip.</li>
		<li>Shake the microarray slide underwater and quickly transfer to a Coplin jar.</li>
		<li>Wash once with 0.1% v/v Tween-20 in PBS (5 min at 125 rpm on a lab rotator).</li>
		<li>Wash once with 0.01% v/v Triton X-100 in PBS (2 min at 125 rpm on a lab rotator).</li>
		<li>Quickly transfer the slide to a Coplin jar containing PBS.</li>
	</ol>
	</li>
	<li>Protein binding 
	<ol>
		<li>Assemble the PBM chamber as previously described (steps 2.1.2&#8211;2.1.3).</li>
		<li>Pipette protein binding mixture containing 5 &#181;M T7 DNA primase, 30 mM Tris-HCl pH 7.5, 6.5 mM MgCl2, 30 mM K-glutamate, 6 mM dithiothreitol (DTT), 65 &#956;M ribonucleoside triphosphate (rNTP), 2% w/v skim milk, 100 ng/&#956;L bovine serum albumin (BSA), 50 ng/&#956;L salmon testes DNA, and 0.02% v/v Triton X-100 (TX-100) into each chamber of the gasket slide (without touching the rubber sides and without introducing bubbles).</li>
		<li>Rinse the microarray slide briefly by dipping it in the PBS dish, which removes excess detergent from the surface of the slides. Wipe the non-DNA surfaces of the slide with a fine wipe.</li>
		<li>Slowly lower the microarray slide (facing down) onto gasket slide, being careful to prevent leakage from one chamber to another. Immediately assemble and tighten PBM chamber apparatus without introducing bubbles. If bubbles do form, they can be removed by gently tapping the steel chamber against a hard surface.</li>
		<li>Incubate for 30 min at room temperature (RT).</li>
	</ol>
	</li>
	<li>Fluorescent antibody attachment
	<ol>
		<li>Unscrew the PBM chamber and remove the microarray slide-coverslip &#8220;sandwich&#8221;, taking care not to break the seal. Disassemble underwater in the WASH dish using forceps. Shake the slide underwater and quickly transfer to a Coplin jar containing PBS.</li>
		<li>Assemble the PBM chamber with gasket slide as previously described (steps 2.1.2&#8211;2.1.3).</li>
		<li>Add Alexa 488-conjugated anti-his antibody (10 ng/&#956;L in binding buffer) to the gasket slide.</li>
		<li>Rinse the slide briefly by dipping it in PBS dish as before, then remove the slide from PBS slowly, wipe the non-DNA surface and place it facing down onto gasket slide.</li>
		<li>Incubate 30 min at RT in the dark (fluorescence probe is light- sensitive) to reduce photo-bleaching.</li>
		<li>Disassemble the PBM chamber and coverslip as before, removing the coverslip underwater in the WASH dish.</li>
		<li>Rinse the slides briefly by dipping them in the PBS dish. Dry the slides with compressed air. Store in the dark in a slide box until ready to scan.</li>
	</ol>
	</li>
	<li>Scanning the microarray slide 
	<ol>
		<li>Scan the chip by using microarray scanner (refer to Table of Materials) with excitation of 495 nm and emission of 519 nm and collect the median fluorescence intensity.</li>
	</ol>
	</li>
</ol>
3. Microarray data analysis
NOTE: All data processing was performed using custom written scripts in MATLAB.
<ol>
	<li>Perform the initial PBM data processing using Wilcoxon rank sum test p-value as described previously4.</li>
	<li>Use the median value of the binding intensity for each DNA sequence for further analysis.</li>
	<li>Next, using the one-way ANOVA p-values, compare statistical significance of the observed differences in primase-DNA binding intensities obtained for different groups of DNA probes, as explained above in the DNA library design section3.</li>
</ol>
4. Template-directed RNA synthesis catalyzed by T7 DNA primase
<ol>
	<li>Preparation of denaturing polyacrylamide gel
	<ol>
		<li>To prepare 100 mL of gel mixture, combine 62.5 mL of 40% acrylamide-bisacrylamide (19:1), 42 g of urea, 1.1 g of Tris base (2-Amino-2-(hydroxymethyl)-1,3-propanediol), 0.55 g of boric acid, and 0.4 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA) solution.</li>
		<li>Divide the mixture into 14 mL aliquots (the amount needed for one gel of 16.5 cm x 26 cm x 0.03 cm) and keep them protected from light at 4 &#176;C for up to 1 month.</li>
		<li>To prepare one denaturing polyacrylamide gel, add 4 &#181;L of TEMED and 40 &#181;L of 10% w/v ammonium persulfate to 14 mL of the previously prepared mixture, cast it, and leave it to polymerize for at least 2 h at RT. The gel can be kept for one day at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Sample preparation 
	NOTE: Keep the reagents, reaction mixture, and samples on ice.
	<ol>
		<li>To prepare the reaction mixture required for ten reactions combine 11 &#181;L of 5x activity buffer (200 mM Tris-HCl, pH = 7.5; 50 mM dithiothreitol, 250 mM potassium glutamate, 50 mM MgCl2), 5.5 &#181;L of 2.5 mM mixture of nucleotide tri-phosphate (NTPs), and 5.5 &#181;L of radiolabeled ribonucleotide [e.g., ATP, (&#945;-32P) 3000 Ci/mmol]. 
		NOTE: All amounts have been increased by 10% due to possible pipetting errors. Radiolabeled ribonucleotides should be diluted according to the strength of radioactive signal (initial dilution is usually 1:10 or more).</li>
		<li>Transfer 2 &#181;L of the reaction mixture to ten PCR tubes.</li>
		<li>Add 1 &#181;L of 100 &#181;M DNA template to the corresponding PCR tube and spin down.</li>
		<li>Add 2 &#181;L of 16 &#181;M T7 DNA primase, spin down, mix gently, and spin down again.</li>
		<li>Incubate the reaction at RT for 20 min.</li>
		<li>Stop the reaction by adding 5 &#181;L of quenching solution (95% formamide, 20 mM EDTA, 0.05% w/v xylene cyanol and bromophenol blue), mix, and spin down.</li>
	</ol>
	</li>
	<li>Separation of RNA products by electrophoresis through denaturing polyacrylamide gel and subsequent signal detection
	<ol>
		<li>Pre-run the gel for 1 h (10 mA, 600 V) in 1x TBE buffer (90 mM Tris-borate, 2 mM EDTA).</li>
		<li>Load up to 2 &#181;L of each sample and perform electrophoresis (20 mA, 1100 V) in 1x TBE buffer (90 mM Tris-borate, 2 mM EDTA).</li>
		<li>Dry the gel for 2 h at 80 &#176;C under a vacuum.</li>
		<li>Expose the phosphor imaging plate to radioactive gel for a few hours to overnight, depending on the strength of radioactive signal.</li>
		<li>Record the signal (photostimulated luminescence) by using phosphorimager (see Table of Materials).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Berger, M. F., Bulyk, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+binding+microarrays+(PBMs)+for+rapid,+high-throughput+characterization+of+the+sequence+specificities+of+DNA+binding+proteins.">Protein binding microarrays (PBMs) for rapid, high-throughput characterization of the sequence specificities of DNA binding proteins.</a> Methods in Molecular Biology. 338, 245-260 (2006).</li><li>Berger, M. F., Bulyk, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+protein-binding+microarrays+for+the+comprehensive+characterization+of+the+DNA-binding+specificities+of+transcription+factors.">Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors.</a> Nature Protocols. 4 (3), 393-411 (2009).</li><li>Afek, A., 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+Sequence+Context+Controls+the+Binding+and+Processivity+of+the+T7+DNA+Primase.">DNA Sequence Context Controls the Binding and Processivity of the T7 DNA Primase.</a> iScience. 2, 141-147 (2018).</li><li>Afek, A., Schipper, J. L., Horton, J., Gordan, R., Lukatsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-DNA+binding+in+the+absence+of+specific+base-pair+recognition.">Protein-DNA binding in the absence of specific base-pair recognition.</a> Proceedings of the Nationaly Academy of Sciences of the Unitet States of America. 111 (48), 17140-17145 (2014).</li><li>Frick, D. N., 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=Interaction+of+bacteriophage+T7+gene+4+primase+with+its+template+recognition+site.">Interaction of bacteriophage T7 gene 4 primase with its template recognition site.</a> Journal of Biological Chemistry. 274 (50), 35889-35898 (1999).</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=Template+recognition+sequence+for+RNA+primer+synthesis+by+gene+4+protein+of+bacteriophage+T7.">Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7.</a> Proceedings of the Nationaly Academy of Sciences of the Unitet States of America. 78 (1), 205-209 (1981).</li><li>Fried, M., Crothers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibria+and+kinetics+of+lac+repressor-operator+interactions+by+polyacrylamide+gel+electrophoresis.">Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis.</a> Nucleic Acids Research. 9 (23), 6505-6525 (1981).</li><li>Galas, D. J., Schmitz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAse+footprinting:+a+simple+method+for+the+detection+of+protein-DNA+binding+specificity.">DNAse footprinting: a simple method for the detection of protein-DNA binding specificity.</a> Nucleic Acids Research. 5 (9), 3157-3170 (1978).</li><li>Jost, J. P., Munch, O., Andersson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+protein-DNA+interactions+by+surface+plasmon+resonance+(real+time+kinetics).">Study of protein-DNA interactions by surface plasmon resonance (real time kinetics).</a> Nucleic Acids Research. 19 (10), 2788 (1991).</li><li>Bowen, B., Steinberg, J., Laemmli, U. K., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+detection+of+DNA-binding+proteins+by+protein+blotting.">The detection of DNA-binding proteins by protein blotting.</a> Nucleic Acids Research. 8 (1), 1-20 (1980).</li><li>Oliphant, A. R., Brandl, C. J., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+sequence+specificity+of+DNA-binding+proteins+by+selecting+binding+sites+from+random-sequence+oligonucleotides:+analysis+of+yeast+GCN4+protein.">Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein.</a> Molecular Cell Biology. 9 (7), 2944-2949 (1989).</li><li>Marmorstein, R., Fitzgerald, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+DNA-binding+domains+for+sequence-specific+DNA+recognition.">Modulation of DNA-binding domains for sequence-specific DNA recognition.</a> Gene. 304, 1-12 (2003).</li></ol>

The authors declare no conflict of interest.

The PBM method has been widely used to investigate binding properties of transcription factors and can also be applied to DNA processing enzymes, such as DNA primase, that bind to DNA with low affinity. However, certain modifications of experimental procedures are required. The microarray experiment involves several steps: design of the DNA library, preparation of the chip, binding of the protein target, fluorescent labeling, and scanning. Mild washing steps are critical, since the long washes with solutions containing d...

HTPP makes use of DNA binding microarray technology combined with biochemical analysis (Figure 1) to statistically identify specific features of DNA templates that affect the enzymatic activity of DNA primase. Therefore, HTPP provides a technological platform that facilitates a knowledge leap in the field. The classical tools used to determine primase recognition sites do not have the ability to yield massive amount of data, whereas HTPP does.
PBM is a technique routinely used to determine the binding preferences of transcription factors to DNA1,2; however, it is not suitable for detection of weak/transient binding of proteins to DNA. Unlike the universal PBM that provides information about average protein binding specificity to all possible sequences consisting of eight base pairs, HTPP is based on the library of single-stranded DNA templates comprising unique sequence elements. Such DNA sequence elements involve tens of thousands short (few tens of bp) genomic sequences, as well as computationally designed DNA sequences enriched in certain DNA repetitive sequence elements present in the genome, which possess different average GC content. Such a high-throughput approach allows determination of, in a systematic, quantitative, and hypothesis-driven way, the sequence-related properties that are important for primase binding and its enzymatic activity3. In particular, the important link between primase-DNA binding preferences, (modulated by DNA sequences flanking specific tri-nucleotide binding sites) and primase processivity has been identified for this enzymatic system4.
The new technology was applied to revisit our understanding of primase recognition sites even for the T7 DNA primase that has been extensively studied5. Specifically, re-examination of classical concepts, such as DNA recognition sites of T7 DNA primase (which were determined almost four decades ago 6) using protein-DNA binding microarray (PBM) has led to unprecedented insight into features related to the flanking sequence of these recognition sites3. It was expected that the sequences flanking tri-nucleotide recognition site of T7 DNA primase (5&#39;-GTC-3&#39;) will be random. Instead, we found that TG-rich flanking sequences increase the chances of T7 DNA primase to synthesize longer RNA primers indicating an increase in processivity.
Other methods that can be used to study DNA-binding properties of proteins in vitro include the electrophoretic mobility shift assay (EMSA)7, DNase I footprinting8, surface-plasmon resonance (SPR)9, and Southwestern blotting10. These are, however, low-throughput methods only applicable to investigating a small number of DNA sequences. In addition, the precision and sensitivity of some of these techniques (e.g., EMSA) is low. On the other hand, in vitro selection11 is a technique that, similarly to PBM, can be used for the identification of numerous binding sequences. However, low affinity sequences are usually excluded in most applications of in vitro selection; therefore, this approach is not suitable for obtaining comparative binding data for all available sequences. The universal PBM1,2 is mainly used to characterize the binding specificities of transcription factors from prokaryotes and eukaryotes as well as specific factors (e.g., presence of certain ligands, cofactors, etc.) that may affect this interaction12.
HTPP expands the PBM application to DNA processing enzymes by combining unprecedented high-throughput statistical power with high precision to provide information on binding sequence context. Such data has not yet been obtained for primases and related enzymes (that have weak/transient binding to DNA) due to aforementioned technical limitations of other available techniques.

<table>
	<tbody>
		<tr>
			<td>40% acrylamide-bisacrylamide (19:1) solution</td>
			<td>Merck</td>
			<td>1006401000</td>
			<td></td>
		</tr>
		<tr>
			<td>95% formamide</td>
			<td>Sigma-Aldrich</td>
			<td>F9037-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated anti-his antibody</td>
			<td>Qiagen</td>
			<td>35310</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonuium persulfate (APS)</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, [&#945;-32P] &#8211; 3000 Ci/mmol</td>
			<td>Perkin Elmer</td>
			<td>NEG003H250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid, granular</td>
			<td>Glentham Life Sciences</td>
			<td>GE4425</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Roche</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA microarray</td>
			<td>Agilent</td>
			<td></td>
			<td>4x180K (AMADID #78366) 
			https://www.agilent.com</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Acros Organics</td>
			<td>AC118430010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fujifilm FLA-5100 phosphorimager</td>
			<td>FUJIFILM Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide staining rack</td>
			<td>Thermo Scientific</td>
			<td>12869995</td>
			<td>If several slides are used</td>
		</tr>
		<tr>
			<td>Lab rotator</td>
			<td>Thermo Scientific</td>
			<td>88880025</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>63064-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microarray Hybridization Chamber</td>
			<td>Agilent</td>
			<td>G2534A</td>
			<td>https://www.agilent.com/cs/library/usermanuals/Public/G2534-90004_HybridizationChamber_User.pdf</td>
		</tr>
		<tr>
			<td>Microarray scanner (GenePix 4400A)</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium glutamate</td>
			<td>Alfa Aesar</td>
			<td>A172232</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonucleotide Solution Mix (rNTPs)</td>
			<td>New England BioLabs</td>
			<td>N0466S</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon testes DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D1626-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Sigma-Aldrich</td>
			<td>70166-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining dish</td>
			<td>Thermo Scientific</td>
			<td>12657696</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad</td>
			<td>1610800</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base (2-Amino-2-(hydroxymethyl)-1,3-propanediol)</td>
			<td>Sigma-Aldrich</td>
			<td>93362-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U6504-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol</td>
			<td>Alfa Aesar</td>
			<td>B21530</td>
			<td></td>
		</tr>
	</tbody>
</table>

dna sequence recognition by dna primase using high-throughput primase profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

This technological advance for mapping the primase binding sites allows the obtaining of DNA binding properties that are difficult, if not impossible, to observe using classical tools. More importantly, HTPP enables the revisiting of the traditional understanding of primase binding sites. Specifically, HTPP reveals binding specificities in addition to known 5&#39;-GTC-3&#39; recognition sequences, which leads to changes in functional activities of T7 DNA primase. Namely, two groups of seq...

Watch this Scientific Journal Video about DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling at JoVE.com

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

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Research

JoVE Journal

Biochemistry

8.6K Views.  Ben-Gurion University of the Negev. Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

Video: DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...