We thank Jorge Cebrian and Anastasia Rastokina who started developing this approach in our lab, Massimo Lopes for providing us with pML113 plasmid and invaluable advice, Ylli Doksani for insightful discussions, and members of the Mirkin lab for their support. The work in the Mirkin lab is supported by the National Institute of General Medical Sciences [R35GM130322] and NSF-BSF [2153071].

NOTE: The plasmid designed for our outlined 2DGE analysis in mammalian cells should contain an SV40 origin of replication several kb upstream of structure-prone repeats (Figure 2). Leading and lagging synthesis should be kept in mind when choosing what orientation relative to the origin the repeats should be cloned into the plasmid.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67229/67229fig02.jpg" /> 
Figure 2: Digestion of repeat-containing plasmid for 2DGE analysis. Structure-prone repeats are depicted several kb downstream from the right-moving replication fork. Digestion with unique cutters 1 and 2 will place the repeat sequence on the descending arm of the Y-arc, given the sequence beyond the halfway point of the digested fragment. Abbreviation: 2DGE = Two-dimensional neutral/neutral gel-electrophoresis. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Plasmid transfection into mammalian cells
<ol>
	<li>Seed 600,000 HEK293T cells in a 10 cm tissue culture plate prior to transfection. Allow the cells to recover at 37 &#176;C overnight. 
	NOTE: Many cell lines may be used for this experiment, although cells containing the SV40 Tag are recommended for optimal results. CAUTION: HEK293T cells are considered BSL-2 and all culturing work should be carried out in a biosafety cabinet using appropriate aseptic technique and proper PPE.</li>
	<li>When the cells reach 60% confluency, transfect 8 &#956;g of repeat-containing plasmid DNA into the seeded cells using appropriate transfection reagents in accordance with the manufacturer&#39;s protocol.</li>
	<li>If not isolating intermediates at this time point, aspirate old media and replace with 10 mL of fresh media after 24 h.</li>
	<li>Begin collecting cells 24-48 h following transfection.
	<ol>
		<li>Aspirate media and carefully wash with 10 mL of phosphate-buffered saline (PBS). Detach and collect cells using 0.5 mL of trypsin and spin down at 340 &#215; g for 4 min.</li>
		<li>Aspirate the supernatant and wash the cell pellets with PBS. Spin again at 340 &#215; g for 4 min and aspirate the supernatant. 
		NOTE: The experiment may be paused here by freezing cell pellets at -80 &#176;C. We have found the best resolution isolating replication intermediates 48 h following transfection; however, 24 h has yielded viable results.</li>
	</ol>
	</li>
</ol>
2. Isolation of replication intermediates
<ol>
	<li>Resuspend the cells in 1.5 mL of modified Hirt lysis buffer [10 mM tris-HCl (pH 7.5), 10 mM ethylenediaminetetraacetic acid (EDTA)] in 50 mL conical tubes and begin cell lysis.
	<ol>
		<li>Add sodium dodecyl sulfate (SDS) to a final concentration of 0.6% (approximately 650 &#956;L of stock 2% SDS) and proteinase K to a final concentration of 100 &#956;g/mL (approximately 10 &#956;L of stock 20 mg/mL proteinase K) to remove nucleases.</li>
		<li>Mix gently by pipetting until homogeneous and incubate the mixture at 37 &#176;C for at least 90 min.</li>
	</ol>
	</li>
	<li>Increase NaCl concentration to 1 M (approximately 540 &#956;L of stock 5 M NaCl) and mix gently until homogeneous. Incubate overnight (18-24 h) at 4 &#176;C to allow for precipitation of cell debris, RNA, and protein by salting out. 
	NOTE: The mixture will be highly viscous, so take care and be patient while mixing well.</li>
	<li>The following day, separate the DNA from cell debris, RNA, and protein.
	<ol>
		<li>Centrifuge the mixture at 29,500 &#215; g for 45 min at 4 &#176;C.</li>
		<li>Transfer the supernatant containing DNA, add one volume of phenol:chloroform:isoamyl alcohol 25:24:1 (v/v), and mix briefly until homogeneous. 
		CAUTION: Phenol:chloroform:isoamyl alcohol is a hazardous material and should be handled with appropriate PPE in a chemical fume hood.</li>
		<li>Centrifuge again at 15,000 &#215; g for 5 min at room temperature. Transfer the aqueous layer to a new conical tube.</li>
	</ol>
	</li>
	<li>Precipitate and wash the isolated DNA.
	<ol>
		<li>Add one volume of pure isopropanol and incubate at room temperature for at least 5 min. Spin down the DNA at 15,000 &#215; g for 30 min at 4 &#176;C.</li>
		<li>Decant the supernatant and wash the pellet with cold 70% ethanol to remove excess salt.</li>
		<li>Spin once more at 15,000 &#215; g for 30 min at 4 &#176;C, air dry, and gently resuspend the pellet in Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA). 
		NOTE: The experiment can be paused here, and samples can be frozen at -20 &#176;C; however, freeze/thaw cycling should be avoided as this can decrease the quality of the DNA replication intermediates.</li>
	</ol>
	</li>
</ol>
3. Sample preparation and 2-dimensional gel electrophoresis
<ol>
	<li>Digest the isolated intermediates of plasmid replication.
	<ol>
		<li>Add 100 units of the appropriate restriction enzymes to the sample to digest the plasmid DNA, specifically placing the repeat-containing sequence on the origin-distal half of the linear fragment (Figure 2). In addition, add DpnI to cut the methylated DNA, thus removing any plasmid DNA that was not fully replicated in cultured human cells. 
		NOTE: For best results, restriction enzymes should be unique cutters that yield a 3-5 kb fragment that places the structure-prone sequence on the descending arm of the Y-arc.</li>
		<li>Incubate the samples at 37 &#176;C for 6-10 h to allow full plasmid digestion.</li>
		<li>Precipitate the DNA with either 2.5 volumes of cold pure ethanol and incubate at -20 &#176;C overnight, or add one volume of isopropanol and incubate at room temperature for 5 min.</li>
		<li>Centrifuge the digested and precipitated samples at 15,000 &#215; g for 30 min at 4 &#176;C.</li>
		<li>Decant the supernatant and wash the sample with cold 70% ethanol. Spin again at 15,000 &#215; g for 30 min at 4 &#176;C.</li>
		<li>Decant the supernatant, air dry for 10 min, and resuspend the samples in 15 &#956;L of TE buffer.</li>
	</ol>
	</li>
	<li>Prepare the first-dimension agarose gel at 0.4-0.5% in 1x Tris-borate-EDTA (TBE) (89 mM tris base, 89 mM boric acid, 2 mM EDTA). Allow the solution to solidify for at least 1 h.</li>
	<li>Begin loading the samples into the first dimension.
	<ol>
		<li>Load the ladder within the first 3 cm relative to the gel&#39;s leftmost edge. Then, load the entirety of the prepared samples, ensuring 3 cm between each pair.</li>
		<li>Run the gel in 1x TBE for 19-24 h at 0.85 V/cm to separate the intermediates with respect to their size. Ensure the chamber is covered to protect the samples from light, which may cause damage to the DNA.</li>
	</ol>
	</li>
	<li>The following day, remove the gel from the buffer and estimate the location of the digested linear fragment using a ruler.
	<ol>
		<li>Excise the first 3 cm of the gel containing the ladder and stain the gel segment in 1x TBE containing 0.3 &#956;g/mL ethidium bromide for 10-15 min. Visualize the ladder using a gel documentation system.</li>
		<li>Add 1.3 cm to the estimated location, yielding value a. Then, subtract 7.5 cm from value a, yielding value b. Align the ruler against the first-dimension gel and cut horizontally across at values a and b. Then, cut vertically down the 3 cm space reserved for each sample. Refer to Figure 3 for a visual schematic.</li>
		<li>In a new casting tray, rotate the segments clockwise and place them in the position of the sample wells (Figure 3).</li>
	</ol>
	</li>
	<li>Prepare the second-dimension agarose gel at a concentration of 1-1.3% in 1x TBE at 0.3 &#956;g/mL ethidium bromide.
	<ol>
		<li>Upon cooling to approximately 55 &#176;C, pour the second-dimension gel over the rotated first-dimension segments and allow it to solidify for at least 1 h.</li>
		<li>Transfer the second dimension to a chamber with 1x TBE at 0.3 &#956;g/mL ethidium bromide, and allow the gel to equilibrate for at least 30 min.</li>
		<li>Run the gel, again covered, for 9-10 h at 4.23 V/cm at 4 &#176;C to separate the intermediates with respect to their shape.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67229/67229fig03.jpg" /> 
Figure 3: Excision of first-dimension intermediates prior to second-dimension separation. Following visualization of the ladder, the mobility of unreplicated fragments can be estimated. (I)&#160;This value can then be used to determine appropriate cut sites (a and b) to excise it and its replicated counterparts (II). The section of the gel should then be rotated and placed in the position of wells for the second-dimension separation. Abbreviation: CW = clockwise. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Southern blotting and hybridization with radiolabeled probe
<ol>
	<li>Remove the second-dimension gel from the chamber and depurinate the DNA fragments for 10 min in a 0.24 M HCl solution with gentle rocking. Rinse the gel with deionized water and soak it in 0.4 M NaOH for 10-15 min. 
	CAUTION: HCl and NaOH are corrosive and should be handled with appropriate PPE in a chemical fume hood.</li>
	<li>Begin assembling the Southern blot to facilitate the transfer of the separated intermediates from the gel to the membrane. Refer to Figure 4 for a comprehensive schematic.
	<ol>
		<li>Fill a sufficiently sized container with 1 L of 0.4 M NaOH.</li>
		<li>Align a long glass sheet across the container and fold (along the length) two long sheets of chromatography paper perpendicular across the glass sheet, extending into the container of NaOH.</li>
		<li>Wet the top of the paper with NaOH and carefully remove any air bubbles under its surface.</li>
		<li>Soak three sheets of chromatography paper with NaOH and place them over the folded paper, again removing any bubbles.</li>
		<li>Flip the second-dimension gel upside-down and transfer it over the papers.</li>
		<li>Wet a positively charged nylon membrane (0.45 &#956;m pore size) with DI water and place it over the gel.</li>
		<li>Finally, add three more sheets of paper, wet with DI water, over the membrane.</li>
		<li>Cover any exposed NaOH in the bottom container with plastic wrap to prevent evaporation. Place a stack of either napkins or paper towels over the blot, ensuring it is 0.3-0.5 m tall. Place a weight on the top, compressing the entire blot to facilitate tight capillary action. Allow at least 2 days for DNA to transfer to the membrane. 
		NOTE: The length and width of the chromatography paper and membrane depend on the size of the agarose gel used for the second dimension. For the most efficient transfer, use paper and membrane with the same dimensions as those of the gel.</li>
	</ol>
	</li>
	<li>Following transfer, crosslink the DNA to the membrane using a UV crosslinker at 120 &#956;J/cm2 for 1 min. 
	NOTE: The experiment may be paused here by placing the membrane in an airtight, dry, and clean sheet protector at room temperature.</li>
	<li>Wash the membrane 2x for 5 min with 2x saline sodium citrate (SSC buffer) (0.3 M NaCl, 0.03 M sodium citrate).</li>
	<li>Prehybridize the membrane with 0.18 mL/cm2 of Church &#38; Gilbert&#39;s hybridization buffer [1 mM EDTA, 1% bovine serum albumin (BSA), 0.5 M sodium phosphate, 7% SDS] at 65 &#176;C, rotating in a hybridization incubator for at least 2 h. 
	NOTE: The membrane may prehybridize for several days.</li>
	<li>Prepare the radiolabeled probe using &#945;-32P dATP or dCTP and a DNA labeling kit in accordance with the manufacturer&#39;s protocol. 
	CAUTION: Radiolabeled dNTPs are hazardous, and appropriate PPE should be worn when handling them. All radioactive work should be performed behind shielding, and trained individuals should be monitored for radiation uptake using dosimeters.
	<ol>
		<li>Design a 400-900 bp linear DNA fragment complementary to the restriction digested sequence (Figure 2) and amplify the fragment using polymerase chain reaction (PCR). 
		NOTE: We recommend having a stock PCR fragment of 50-100 ng/&#956;L</li>
		<li>Combine 100 ng of the complementary PCR fragment with DNA Pol I, Klenow fragment (3&#39; 5&#39; exo-) buffer, and random decanucleotide oligos.</li>
		<li>Denature the fragment at 100 &#176;C for 10 min.</li>
		<li>Add 5 units of DNA Pol I, Klenow fragment (3&#39; 5&#39; exo-), 50 &#956;Ci of &#945;-32P dNTP, and 30-50 &#956;mol of dNTP mix deficient in radiolabeled dNTP type to the sample.</li>
		<li>Incubate at 37 &#176;C for 10 min to allow for polymerization and radiolabeled dNTP incorporation.</li>
		<li>Add 30-50 &#956;mol of previously absent dNTP type to the sample and incubate at 37 &#176;C for 10 min.</li>
		<li>Purify the radiolabeled fragment using a spin column at 3,000 &#215; g for 2 min.</li>
	</ol>
	</li>
	<li>Add radiolabeled probe to 50 mL of hybridization buffer and incubate with the membrane overnight at 65 &#176;C rotating in a hybridization incubator.</li>
	<li>The following day, remove the probe and wash the membrane 2x with wash buffer 1 (0.1x SSC, 0.1% SDS) at 42 &#176;C and 2x with wash buffer 2 (2x SSC, 0.1% SDS) at 65 &#176;C. 
	&#8203;NOTE: All washes should be performed with fast rotations for 15 min in the incubator.</li>
	<li>Dry the membrane for 10 min and place it in a thin, transparent sheet protector. Store the sealed membrane in a controlled, radiation-resistant cassette with a phosphor-sensitive screen. Allow the membrane to be exposed to the screen for 1-10 days.</li>
	<li>Visualize the results using a biomolecular imager set to phosphor imaging. Wash and re-expose if necessary.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67229/67229fig04.jpg" /> 
Figure 4: Assembly of Southern blot. Comprehensive schematic of a typical apparatus used for Southern blot transfer of intermediates from the second dimension onto a nylon membrane. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

2D Gel Electrophoresis for Analyzing DNA Replication and Fork Stalling Events

<ol>
	<li>Liao, X., 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=Repetitive+DNA+sequence+detection+and+its+role+in+the+human+genome.">Repetitive DNA sequence detection and its role in the human genome.</a> Commun Biol. 6 (1), 1-21 (2023).</li><li>Fotsing, S. F., 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+impact+of+short+tandem+repeat+variation+on+gene+expression.">The impact of short tandem repeat variation on gene expression.</a> Nat Genet. 51 (11), 1652-1659 (2019).</li><li>Fan, H., Chu, J. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+short+tandem+repeat+mutation.">A brief review of short tandem repeat mutation.</a> GPB. 5 (1), 7-14 (2007).</li><li>Khristich, A. N., Mirkin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+wrong+DNA+track:+Molecular+mechanisms+of+repeat-mediated+genome+instability.">On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability.</a> J Biol Chem. 295 (13), 4134-4170 (2020).</li><li>Samadashwily, G. M., Raca, G., Mirkin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trinucleotide+repeats+affect+DNA+replication+in+vivo.">Trinucleotide repeats affect DNA replication in vivo.</a> Nat Genet. 17 (3), 298-304 (1997).</li><li>Khristich, A. N., Armenia, J. F., Matera, R. M., Kolchinski, A. A., Mirkin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+contractions+of+Friedreich's+ataxia+GAA+repeats+in+yeast+occur+during+DNA+replication+due+to+their+triplex-forming+ability.">Large-scale contractions of Friedreich's ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability.</a> Proc Natl Acad Sci USA. 117 (3), 1628-1637 (2020).</li><li>Shishkin, A. 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=Large-scale+expansions+of+Friedreich's+ataxia+GAA+repeats+in+yeast.">Large-scale expansions of Friedreich's ataxia GAA repeats in yeast.</a> Mol Cell. 35 (1), 82-92 (2009).</li><li>Sundararajan, R., Gellon, L., Zunder, R. M., Freudenreich, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+break+repair+pathways+protect+against+CAG/CTG+repeat+expansions,+contractions+and+repeat-mediated+chromosomal+fragility+in+Saccharomyces+cerevisiae.">Double-strand break repair pathways protect against CAG/CTG repeat expansions, contractions and repeat-mediated chromosomal fragility in Saccharomyces cerevisiae.</a> Genetics. 184 (1), 65-77 (2010).</li><li>Kim, H. -. M., 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=Chromosome+fragility+at+GAA+tracts+in+yeast+depends+on+repeat+orientation+and+requires+mismatch+repair.">Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair.</a> EMBO J. 27 (21), 2896-2906 (2008).</li><li>Polleys, E. J., House, N. C. M., Freudenreich, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+recombination+and+replication+fork+restart+in+repeat+instability.">Role of recombination and replication fork restart in repeat instability.</a> DNA Repair. 56, 156-165 (2017).</li><li>Gold, M. 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=Restarted+replication+forks+are+error-prone+and+cause+CAG+repeat+expansions+and+contractions.">Restarted replication forks are error-prone and cause CAG repeat expansions and contractions.</a> PLoS Genet. 17 (10), e1009863 (2021).</li><li>Lambert, S., 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=Homologous+recombination+restarts+blocked+replication+forks+at+the+expense+of+genome+rearrangements+by+template+exchange.">Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange.</a> Mol Cell. 39 (3), 346-359 (2010).</li><li>Burssed, B., Zamariolli, M., Bellucco, F. T., Melaragno, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+structural+chromosomal+rearrangement+formation.">Mechanisms of structural chromosomal rearrangement formation.</a> Mol Cytogenet. 15 (1), 23 (2022).</li><li>Paulson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeat+expansion+diseases.">Repeat expansion diseases.</a> Handb Clin Neurol. 147, 105-123 (2018).</li><li>Malik, I., Kelley, C. P., Wang, E., Todd, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+underlying+nucleotide+repeat+expansion+disorders.">Molecular mechanisms underlying nucleotide repeat expansion disorders.</a> Nat Rev Mol Cell Biol. 22 (9), 589-607 (2021).</li><li>Emanuel, B. S., Zackai, E. H., Medne, L., Adam, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emanuel+Syndrome.">Emanuel Syndrome.</a> GeneReviews&#174;. , (1993).</li><li>Brewer, B. J., Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+localization+of+replication+origins+on+ARS+plasmids+in+S.+cerevisiae.">The localization of replication origins on ARS plasmids in S. cerevisiae.</a> Cell. 51 (3), 463-471 (1987).</li><li>Bell, L., Byers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+branched+from+linear+DNA+by+two-dimensional+gel+electrophoresis.">Separation of branched from linear DNA by two-dimensional gel electrophoresis.</a> Anal Biochem. 130 (2), 527-535 (1983).</li><li>Voineagu, I., Narayanan, V., Lobachev, K. S., Mirkin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+stalling+at+unstable+inverted+repeats:+Interplay+between+DNA+hairpins+and+fork+stabilizing+proteins.">Replication stalling at unstable inverted repeats: Interplay between DNA hairpins and fork stabilizing proteins.</a> Proc Natl Acad Sci USA. 105 (29), 9936-9941 (2008).</li><li>Nguyen, J. H. G., 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=Differential+requirement+of+Srs2+helicase+and+Rad51+displacement+activities+in+replication+of+hairpin-forming+CAG/CTG+repeats.">Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.</a> Nucleic Acids Res. 45 (8), 4519-4531 (2017).</li><li>Krasilnikova, M. M., Mirkin, S. M., Kohwi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+triplet+repeat+replication+by+two-dimensional+gel+electrophoresis.">Analysis of triplet repeat replication by two-dimensional gel electrophoresis.</a> Trinucleotide Repeat Protocols. , (2004).</li><li>Follonier, C., Oehler, J., Herrador, R., Lopes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friedreich's+ataxia-associated+GAA+repeats+induce+replication-fork+reversal+and+unusual+molecular+junctions.">Friedreich's ataxia-associated GAA repeats induce replication-fork reversal and unusual molecular junctions.</a> Nat Struct Mol Biol. 20 (4), 486-494 (2013).</li><li>Chandok, G. S., Patel, M. P., Mirkin, S. M., Krasilnikova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Friedreich's+ataxia+GAA+repeats+on+DNA+replication+in+mammalian+cells.">Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells.</a> Nucleic Acids Res. 40 (9), 3964-3974 (2012).</li><li>Rastokina, 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=Large-scale+expansions+of+Friedreich's+ataxia+GAA&#8226;TTC+repeats+in+an+experimental+human+system:+role+of+DNA+replication+and+prevention+by+LNA-DNA+oligonucleotides+and+PNA+oligomers.">Large-scale expansions of Friedreich's ataxia GAA&#8226;TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers.</a> Nucleic Acids Res. 51 (16), 8532-8549 (2023).</li><li>Giannattasio, M., 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=Visualization+of+recombination-mediated+damage-bypass+by+template+switching.">Visualization of recombination-mediated damage-bypass by template switching.</a> Nat Struct Mol Biol. 21 (10), 884-892 (2014).</li><li>Hisey, J. 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=Pathogenic+CANVAS+(AAGGG)n+repeats+stall+DNA+replication+due+to+the+formation+of+alternative+DNA+structures.">Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures.</a> Nucleic Acids Res. 52 (8), 4361-4374 (2024).</li><li>Kalejta, R. F., Lin, H. B., Dijkwel, P. A., Hamlin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+replication+intermediates+in+the+amplified+CHO+dihydrofolate+reductase+domain+by+two+novel+gel+electrophoretic+techniques.">Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques.</a> Mol Cell Biol. 16 (9), 4923-4931 (1996).</li><li>Little, R. D., Platt, T. H., Schildkraut, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+and+termination+of+DNA+replication+in+human+rRNA+genes.">Initiation and termination of DNA replication in human rRNA genes.</a> Mol Cell Biol. 13 (10), 6600-6613 (1993).</li><li>Fanning, E., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SV40+DNA+replication:+From+the+A+gene+to+a+nanomachine.">SV40 DNA replication: From the A gene to a nanomachine.</a> Virology. 384 (2), 352-359 (2009).</li><li>Sogo, J. M., Stahl, H., Koller, T., Knippers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+replicating+simian+virus+40+minichromosomes:+The+replication+fork,+core+histone+segregation+and+terminal+structures.">Structure of replicating simian virus 40 minichromosomes: The replication fork, core histone segregation and terminal structures.</a> J Mol Biol. 189 (1), 189-204 (1986).</li><li>Weisshart, K., Taneja, P., Fanning, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+replication+protein+A+binding+site+in+simian+virus+40+(SV40)+T+antigen+and+its+role+in+the+initial+steps+of+SV40+DNA+replication.">The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication.</a> J Virol. 72 (12), 9771-9781 (1998).</li><li>Sowd, G. A., Fanning, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wolf+in+sheep's+clothing:+SV40+co-opts+host+genome+maintenance+proteins+to+replicate+viral+DNA.">A Wolf in sheep's clothing: SV40 co-opts host genome maintenance proteins to replicate viral DNA.</a> PLoS Pathog. 8 (11), e1002994 (2012).</li><li>. BLAST: Basic Local Alignment Search Tool Available from: <a target="_blank" href="https://blast.ncbi.nlm.nih.gov/Blast.cgi">https://blast.ncbi.nlm.nih.gov/Blast.cgi</a> (2024)</li><li>Hirt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extraction+of+polyoma+DNA+from+infected+mouse+cell+cultures.">Selective extraction of polyoma DNA from infected mouse cell cultures.</a> J Mol Biol. 26 (2), 365-369 (1967).</li><li>Lopes, M., Foiani, M., Sogo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+mechanisms+control+chromosome+integrity+after+replication+fork+uncoupling+and+restart+at+irreparable+UV+lesions.">Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.</a> Mol Cell. 21 (1), 15-27 (2006).</li></ol>

The authors have no conflicts of interest to disclose.

2DGE provides a semi-quantitative and comprehensive image of the relative populations of intermediates that arise during the replication of a particular sequence. Given that the fragile molecular structures of replication forks must be maintained throughout this procedure, great care should be implemented to prevent physical shearing and chemical denaturation. Therefore, it is highly recommended that any alkaline treatment be avoided during plasmid isolation. To avoid this, we and others have implemented a modified form ...

Short tandem repeats (STR) are small, typically 2-9 base pairs (bp), repetitive sequences of DNA that constitute around 3% of the human genome1. STR play an important role in gene regulation2; however, their repetitive composition leaves them prone to non-canonical DNA secondary structure formation and subsequent genetic instability3,4. From left-handed helices to hairpins/cruciforms, to three and four-stranded helices, these alternative DNA structures cause intrinsic challenges for the replisome. A natural prerequisite for secondary structure formation is DNA unwinding, which is a prerequisite for DNA replication. This presents a unique conundrum for genome functioning as many of these structures can form during replication, hindering replisome progression and ultimately causing replication fork stalling5,6,7, or in severe cases, fork collapse and DNA breakage8,9. Both the restart of stalled forks and DNA repair pathways have been shown to lead to repeat instability, such as repeat expansions10,11 and complex genome rearrangements (CGR)12,13. These events can result in the development of roughly 60 human diseases known as repeat expansion disorders, including Fragile X syndrome, Huntington&#39;s disease, Friedreich&#39;s ataxia, and others14,15 as well as CGR diseases, such as Emmanuel syndrome16. Therefore, to better understand the mechanisms of human disease driven by repeat instability, it is imperative to study the details of replication fork progression through those repeats.
A technique for studying replication progression emerged in the mid-1980s when Brewer and Fangman sought to provide direct evidence that replication initiation in Saccharomyces cerevisiae occurs at autonomous replication sequence (commonly known as ARS) elements17. In doing so, they separated structures of yeast replication intermediates in agarose, adapting an earlier method from Bell and Byers known as 2-dimensional Neutral/Neutral gel-electrophoresis (2DGE)18. This technique utilized the fact that nonlinear DNA travels differently in agarose gel than its linear equivalent of the same mass. More specifically, in 2DGE, isolated DNA is separated in two perpendicular dimensions, first primarily by size and then primarily by shape, to create a comprehensive map of replication at a particular region of interest. In their original paper, Brewer and Fangman demonstrated this as an arc composed of &#34;simple Y&#34; structures or replication forks bridging unreplicated DNA to their replicated counterparts. They further describe other observed intermediates as &#34;bubbles&#34; and &#34;double Ys,&#34; representing replication origins and converging forks, respectively.
2DGE can be used to study the relative populations of DNA replication intermediates at a given time. Therefore, if one population of intermediates is more prevalent than another, this would be evident upon visualization. This makes 2DGE an especially useful tool for studying replication progression through challenging sequences, like structure-forming repeats. For example, if the region analyzed contains a sequence capable of inducing replication fork stalling, this would present as a bulge on the arc (Figure 1A), indicating an accumulation of replication forks at that locus. This can be seen with the replication of both hairpin-forming repeat sequences in yeast19,20,21 and triplex-forming repeats in human cells22,23,24. In addition to stalling, 2DGE can be used to observe DNA structures that do not conform to the standard simple Ys formed during replication, as in the case of recombinant intermediates25. These intermediates have a heavier and more branched X-shaped structure and, therefore, travel slower in both the first- and second dimensions than standard replication forks. Similar results can also be observed with regards to replication fork reversal20,24,26. In response to strong replication stress, eukaryotic cells have been shown to utilize replication fork reversal to rescue stalled forks. These reversed forks have similar molecular weight to stalled forks; yet their chicken-foot structure results in slower electrophoretic mobility in the second dimension relative to their Y-shaped complements, resulting in an extension up and out from the arc.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67229/67229fig01.jpg" /> 
Figure 1: 2D Gel electrophoresis analysis of DNA replication. (A)&#160;Schematic of a typical 2DGE depicting replication through a structure-forming repeat capable of inducing fork stalling. Intermediate size and structure will influence electrophoretic mobility. (B)&#160;Sample Y-arc with ascending and descending arms respectively labeled. Abbreviation: 2DGE = Two-dimensional neutral/neutral gel-electrophoresis. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Naturally, one of the most important aspects of 2DGE concerns the quality and quantity of replication intermediates. However, the resolution of 2DGE analysis of replication through endogenous loci in mammalian cells is insufficient for a single copy target sequence within the 6 &#215; 109 bp diploid human genome, though it has been done for multi-copy genes, such as heavily amplified DHFR locus27 or ribosomal RNA28. SV40-based replication is an efficient and well-characterized means of studying replication in eukaryotic cells29. It provides a reliable model of eukaryotic replication that utilizes most of the host replisome machinery to replicate the viral genome, which is parceled into nucleosomes upon infection30,31. Two notable exceptions from the mammalian replisome are that the T-antigen (Tag), instead of the host CMG complex, serves as replicative DNA helicase, and DNA polymerase delta synthesizes both leading and lagging DNA strands32. We have taken advantage of this system by placing pathogenic stretches of structure-forming repeats downstream from an SV40 origin of replication within a plasmid that was originally created in the Massimo Lopes lab22. Importantly, this plasmid also contains the gene encoding for Tag itself, thus resulting in its constitutive and extremely potent replication upon transfection into a variety of cultured human cells. This feature gives rise to a large quantity of products, ideal for 2DGE analysis of the intermediates formed during and in response to the replication of pathogenic repeats in human cells. Here, we describe a detailed method of visualizing the replication of structure-forming repeats within the SV40-based human episome using 2-dimensional gel electrophoresis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x TBE Buffer</td>
			<td>Bio Rad</td>
			<td>1610733</td>
			<td></td>
		</tr>
		<tr>
			<td>20x SSC Buffer</td>
			<td>Fisher Scientific</td>
			<td>BP1325-1</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>a-32P dATP, 3000 Ci/mmol&#160;</td>
			<td>Revvity</td>
			<td>BLU512H250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Hybond-N+</td>
			<td>Fisher Scientific</td>
			<td>RPN303B</td>
			<td></td>
		</tr>
		<tr>
			<td>BAS Storage Phosphor Screens</td>
			<td>Fisher Scientific</td>
			<td>28956482</td>
			<td></td>
		</tr>
		<tr>
			<td>Church and Gibert&#39;s hybriddization buffer</td>
			<td>Fisher Scientific</td>
			<td>50-103-5408</td>
			<td></td>
		</tr>
		<tr>
			<td>DecaLabel DNA labeling kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K0622</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high gluctose, GltaMAX Supplement, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs</td>
			<td>R0176S</td>
			<td>Additional restriction enzymes will need to be purchased as well</td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8</td>
			<td>Fisher Scientific</td>
			<td>BP2482500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 70%</td>
			<td>Fisher Scientific</td>
			<td>BP82031GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution, 12 M</td>
			<td>Millipore Sigma</td>
			<td>13-1683</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26184</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPRIME DNA and siRNA Transfection Reagent with Buffer</td>
			<td>VWR</td>
			<td>101000027</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoZap Plus-CL</td>
			<td>VWR</td>
			<td>75870-448</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Millipore Sigma</td>
			<td>746398-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Oak Ridge High-Speed Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>3139-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline, pH 7.4</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline, pH 7.5</td>
			<td>ThermoFisher Scientific</td>
			<td>10010024</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>ThermoFisher Scientific</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>ThermoFisher Scientific</td>
			<td>EO0492</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure Cellulose Chromatography Paper</td>
			<td>Fisher Scientific</td>
			<td>05-714-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure Cellulose Chromatography Paper</td>
			<td>Fisher Scientific</td>
			<td>05-714-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Fisher Scientific</td>
			<td>09-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fisher Scientific</td>
			<td>12-460-451</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Millipore Sigma</td>
			<td>436143-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S25548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorval LYNX 4000 Superspeed Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75006580</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-cell Horizontal Electrophoresis System</td>
			<td>Bio Rad</td>
			<td>1704401</td>
			<td></td>
		</tr>
		<tr>
			<td>TH13-6 x 50 Swinging Bucket Rotor</td>
			<td>ThermoFisher Scientific</td>
			<td>75003010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl 1 M, pH 7.5</td>
			<td>Fisher Scientific</td>
			<td>BP1757-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophoretic analysis of replication through structure-prone dna repeats within the sv40-based human episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

If successful, upon visualization, a sharp arc of replication forks can be observed extending up and out from the massive 1n spot (Figure 5A). The size of a fragment, or percentage that is replicated, determines the fragment&#39;s mobility in the first dimension. As the intermediates develop a more jointed structure, they will begin to travel more slowly in the second dimension. Therefore, if an intermediate has traveled slowly in both dimensions, it can be asserted that it is a highly repli...

Author Spotlight: Characterizing DNA Replication of Pathogenic Repeats to Uncover Mechanisms of Replication Fork Stalling and Expansion

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

electrophoretic-analysis-replication-through-structure-prone-dna

Research

JoVE Journal

Genetics

580 Views.  Tufts University. Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

Video: Author Spotlight: Characterizing DNA Replication of Pathogenic Repeats to Uncover Mechanisms of Replication Fork Stalling and Expansion

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5&#8217;UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.