Name: genotyping of plant and animal samples without prior dna purification
Uploaded: 2012-09-24T12:00:00
Duration: 11 min
Description: Watch this Scientific Journal Video about Genotyping of Plant and Animal Samples without Prior DNA Purification at JoVE.com

For the Arabidopsis samples and the concerned PCR primers we thank Professor Jaakko Kangasj&auml;rvi and his group, The Plant Stress Group, Plant Biology, Department of Biosciences, University of Helsinki. The experiments with the traditional method were conducted by Mrs. Airi Lamminm&auml;ki.
Transgenic mouse samples were provided by Dr. Jaana Vesterinen, Institute of Biomedicine/Biochemistry, University of Helsinki.
Harris Uni-Core and Harris Cutting Mat are trademarks of Shunderson Communications Inc. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

1. Genotyping of Arabidopsis Plant Individuals with Direct Protocol
<ol>
 <li>To begin the dCAPS genotyping assay directly on the Arabidopsis plant leaf, first formulate 20 or 50 &mu;l PCR reactions using the Phire Plant Direct PCR Kit as described in Table 1. </li>
 <li>Next, cut a 0.50 mm punch from an Arabidopsis plant leaf using the Harris Uni-Core and Harris Cutting Mat. Holding the puncher firmly, press the cutting edge into the tissue and rotate the puncher back and forth. </li>
 <li>Press the plunger to eject the punch disc into a PCR reaction mixture. Ensure that the sample drops into the PCR solution and does not stick to the tube walls. </li>
 <li>Clean the cutting edge of the puncher between each sample to prevent cross-contamination by dipping it into 2% NaClO solution. Press the plunger up and down a few times and wipe the cutting edge with a clean paper towel. The cutting mat should also be rinsed between samples. </li>
 <li>Next, employ a Thermo Scientific Piko 24-well Thermal Cycler and Thermo Scientific Piko PCR Plate to perform the PCR reactions using the cycling conditions described in Table 2. The PCR reactions can also be performed in conventional PCR thermal cyclers. </li>
 <li>After PCR, spin down the plant material. Transfer 5 &mu;l of the supernatant to a new microcentrifuge tube. Add 4 &mu;l of water and 1 &mu;l of restriction enzyme, SspI. Mix gently and spin down briefly to collect contents in the bottom of the tube. Incubate the reaction mixture for one hour at 37 &deg;C. Inactivate the restriction enzyme by incubating at 65 &deg;C for 20 min. </li>
 <li>When amplifying DNA directly from plant tissues, the PCR products include plant and PCR-derived components that may interfere with the restriction digestion enzyme. Therefore, it may be necessary to either dilute (e.g. 1:2 or 1:3 in water) or purify the PCR product before the subsequent digestion, for example by using a suitable commercial kit such as Thermo Scientific GeneJET PCR Purification Kit. </li>
 <li>Following restriction enzyme digestion, analyze the resulting fragments on an agarose gel. Add 2.5 &mu;l of 5x loading buffer to the reaction and perform agarose gel electrophoresis with 10 &mu;l of the resulting mixture. </li>
</ol>
2. Amplifying Specific DNA Fragments from 15-year-old Oak Leaves using the Dilution Protocol
<ol>
 <li>To begin, cut a 2 mm punch of oak leaf.</li>
 <li>Place the sample into 20 &mu;l of Dilution Buffer supplied with the Phire Plant Direct PCR Kit. Clean the cutting edge of the puncher and cutting mat as before. </li>
 <li>Crush the leaf sample with a 100 &mu;l pipette tip by pressing it briefly against the tube wall. Depending on the plant material, the dilution protocol sometimes works better without crushing the sample. </li>
 <li>Spin the samples briefly in a microcentrifuge until the reactions are in the bottom of tubes. </li>
 <li>The supernatant can be used directly in the PCR, or it can be diluted 1:10 or 1:100 in sterile water depending on the sample type. Use 0.5 &mu;l to 1 &mu;l of the supernatant or dilution as a template for a 20 &mu;l PCR reaction. Prepare the reaction as described in Table 1.</li>
 <li>Once the reactions are prepared, employ a Thermo Scientific Piko 24-well Thermal Cycler and Thermo Scientific Piko PCR Plate to perform the PCR reactions using the cycling conditions described in Table 2. The PCR reactions can also be performed in conventional PCR thermal cyclers. </li>
 <li>After PCR, add 5 &mu;l of 5x loading buffer to the reaction and perform agarose gel electrophoresis with 15 &mu;l of the resulting mixture. </li>
</ol>
3. Genotyping of Transgenic Mice using the Dilution Protocol
<ol>
 <li>Begin the dilution protocol on transgenic mice by placing a 2 mm punch of the mouse ear in 20 &mu;l of Dilution Buffer containing 0.5 &mu;l of DNARelease Additive supplied with the Phire Animal Tissue Direct PCR Kit. </li>
 <li>Incubate the samples at room temperature for 2 min, followed by 2 min incubation at 98 &deg;C. </li>
 <li>Spin the samples briefly in a microcentrifuge until the reactions are in the bottom of tubes. </li>
 <li>Use 1 &mu;l of supernatant as the template for a 20 &mu;l PCR reaction, prepared as described in Table 3. Any supernatant that is not to be used right away can be transferred to a new tube and stored at -20 &deg;C. </li>
 <li>Next, employ a Thermo Scientific Piko 24-well Thermal Cycler and Thermo Scientific Piko PCR Plate to perform the PCR reactions using the cycling conditions described in Table 4. Again, the PCR reactions can also be performed in conventional PCR cyclers. </li>
 <li>Following PCR, add 5 &mu;l of 5x loading buffer to the reaction and perform agarose gel electrophoresis with 15 &mu;l of the resulting mixture. </li>
</ol>
4. Representative Results
In the dCAPS technique the restriction site within the SNP of interest is either introduced or destroyed using a PCR primer with one or more mismatches to the target DNA. Genotyping of Arabidopsis plant individuals was performed with the dCAPS technique directly from leaf punches. The amplified products were digested and the resulting fragments revealing the genotype of each individual were analyzed by gel electrophoresis (Figure 2). 
In this example, the 160 bp PCR products containing the SNP site of interest (G or A allele) in the Arabidopsis genome were amplified from plant leaf punches with the Phire Plant Direct PCR Kit. The forward primer contained one mismatch at the 3' end, creating an SspI-specific restriction site in the target DNA, which included the SNP of interest (A allele) but not in the other allele of the SNP (Figure 2B). 
Dilution protocols are necessary for challenging plant material, such as the oak leaf, due to its high concentration of phenolic compounds. Using the dilution protocol described here, the 297 bp fragment of chloroplastic DNA was amplified using the 3-step protocol. Oak leaf specimens with different dilutions are shown in Figure 3, in addition to positive and negative controls. 
The Phire Animal Tissue Direct PCR Kit was employed with a challenging protocol where only one primer set was used for amplification of two fragments with a large size difference, resulting in abundant yields of both correctly sized products of 1,500 bp for transgenic mice and 200 bp for wild type mice (Figure 4). The weaker upper band with heterozygote mice is due to the competition of both templates (alleles) for the same primer pair; however, the genotyping results are unambiguous. 
The stability of the samples prepared using the dilution protocol was also tested. The results show that the dilution samples can be stored at -20 &deg;C for at least one year. Furthermore, repeated freeze/thaw cycles did not significantly affect the reaction (Figure 5). Also shown is the robust amplification from mouse ear tissue samples using Phire DNA Polymerase and the Direct PCR method compared to hot start Taq polymerase and purified DNA (Figure 6). 
<table border="1">
 <tbody>
 <tr>
 <td fo:width="2in">Components</td>
 <td fo:width=".75in">20 &mu;l reaction</td>
 <td fo:width=".75in">50 &mu;l reaction</td>
 <td fo:width=".75in">Final Conc.</td>
 </tr>
 <tr>
 <td>H2O</td>
 <td>add to 20 &mu;l</td>
 <td>add to 50 &mu;l</td>
 <td> </td>
 </tr>
 <tr>
 <td>2x Phire Plant PCR Buffer</td>
 <td>10 &mu;l</td>
 <td>25 &mu;l</td>
 <td> 1x</td>
 </tr>
 <tr>
 <td>primer A</td>
 <td>X &mu;l</td>
 <td>X &mu;l</td>
 <td>0.5 &mu;M</td>
 </tr>
 <tr>
 <td>primer B</td>
 <td>X &mu;l</td>
 <td>X &mu;l</td>
 <td>0.5 &mu;M</td>
 </tr>
 <tr>
 <td>Phire Hot Start II DNA Polymerase</td>
 <td>0.4 &mu;l</td>
 <td> 1 &mu;l </td>
 <td> </td>
 </tr>
 <tr>
 <td>Sample/direct protocol</td>
 <td> </td>
 <td> 0.50 mm punch </td>
 <td> </td>
 </tr>
 <tr>
 <td>Sample/dilution protocol</td>
 <td>0.5-1 &mu;l</td>
 <td></td>
 <td> </td>
 </tr>
</tbody>
</table>
Table 1. Reaction conditions for Plant PCR used in this protocol.
<table border="1">
<tbody>
 <tr>
 <td fo:width="1in">Cycle Step</td>
 <td fo:width=".5in">Temp.</td>
 <td fo:width=".5in">Time</td>
 <td fo:width=".4in">Cycles</td>
 </tr>
 <tr>
 <td>Initial denaturation</td>
 <td>98 &deg;C</td>
 <td>5 min</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Denaturing 
 Annealing* 
 Extension</td>
 <td>98 &deg;C 
 X &deg;C 
 72 &deg;C</td>
 <td>5 sec 
 5 sec 
 20 sec</td>
 <td> 40</td>
 </tr>
 <tr>
 <td>Final Extension</td>
 <td>72 &deg;C 
 4 &deg;C</td>
 <td>1 min 
 hold</td>
 <td>1</td>
 </tr>
 </tbody>
</table>
Table 2. Cycling conditions for Plant PCR used in this protocol.
* The Tm calculator at <a href="http://www.thermoscientific.com/pcrwebtools" target="_blank">www.thermoscientific.com/pcrwebtools</a> is recommended when determining the Tm for primers to be used with Phire Hot Start II DNA Polymerase. The recommended annealing temperature for primers &le; 20 nt is equal to the lower Tm given by the webtool. For primers &gt; 20 nt, use an annealing temperature 3 &deg;C higher than the lower Tm given by the webtool. 
<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Components</td>
 <td fo:width=".75in">20 &mu;l reaction</td>
 <td fo:width=".75in">Final Conc.</td>
 </tr>
 <tr>
 <td>H2O</td>
 <td>add to 20 &mu;l</td>
 <td> </td>
 </tr>
 <tr>
 <td>2x Phire Animal Tissue PCR Buffer</td>
 <td>10 &mu;l</td>
 <td>1x</td>
 </tr>
 <tr>
 <td>primer A</td>
 <td>X &mu;l</td>
 <td>0.5 &mu;M</td>
 </tr>
 <tr>
 <td>primer B</td>
 <td>X &mu;l</td>
 <td>0.5 &mu;M</td>
 </tr>
 <tr>
 <td>Phire Hot Start II DNA Polymerase</td>
 <td>0.4 &mu;l</td>
 <td> </td>
 </tr>
 <tr>
 <td>Sample/dilution protocol</td>
 <td>1 &mu;l</td>
 <td> </td>
 </tr>
 </tbody>
</table>
Table 3. Reaction conditions for Animal Tissue PCR used in this protocol.
<table border="1">
<tbody>
 <tr>
 <td fo:width="1in">Cycle Step</td>
 <td fo:width=".5in">Temp.</td>
 <td fo:width=".5in">Time</td>
 <td fo:width=".4in">Cycles</td>
 </tr>
 <tr>
 <td>Initial denaturation</td>
 <td>98 &deg;C</td>
 <td>5 min</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Denaturing 
 Annealing* 
 Extension </td>
 <td>98 &deg;C 
 X &deg;C 
 72 &deg;C </td>
 <td>5 sec 
 5 sec 
 20 sec &le; 1 kb 
 20 sec/kb &gt; 1 kb</td>
 <td> 40</td>
 </tr>
 <tr>
 <td>Final Extension</td>
 <td>72 &deg;C 
 4 &deg;C</td>
 <td>1 min 
 Hold</td>
 <td>1</td>
 </tr>
 </tbody>
</table>
Table 4. Cycling conditions for Animal Tissue PCR used in this protocol.
* The Tm calculator at <a href="http://www.thermoscientific.com/pcrwebtools" target="_blank">www.thermoscientific.com/pcrwebtools</a> is recommended when determining the Tm for primers to be used with Phire Hot Start II DNA Polymerase. The recommended annealing temperature for primers &le; 20 nt is equal to the lower Tm given by the webtool. For primers &gt; 20 nt, use an annealing temperature 3 &deg;C higher than the lower Tm given by the webtool. 
<img src="/files/ftp_upload/3844/3844fig1.jpg" alt="Figure 1" /> 
Figure 1. Direct PCR workflow. Direct PCR can be performed using two alternative protocols. In the direct protocol a tiny amount of sample is added directly to the PCR reaction. The dilution protocol employs a brief pre-incubation step before PCR to release DNA from the sample material.
<img src="/files/ftp_upload/3844/3844fig2.jpg" alt="Figure 2" /> 
 Figure 2. Genotyping of Arabidopsis plant individuals with dCAPS technique directly from leaf punches. A) Figure 2A shows the principle of the dCAPS assay performed. The restriction site within the SNP of interest is either introduced or removed by PCR using a primer with one or more mismatches to the target DNA. The PCR products are digested and the resulting fragments reveal the genotype of each individual when analyzed by electrophoresis. B) 0.50 mm punches from plant leaves were placed directly in 50 &mu;l PCR reactions. The 160 bp PCR products containing the SNP site (G or A allele) were amplified with primers introducing a unique SspI site to the A-allele. The unpurified PCR products were digested with SspI restriction enzyme. Resulting fragments were analyzed on a 3% agarose gel. Lane M is the size marker; lanes A and G correspond to the SNP alleles of each sample. The obtained results were confirmed by conventional analysis of DNA extraction followed by PCR and restriction digestion (data not shown). The negative control reactions without template DNA were included in test set up and run on a separate agarose gel in parallel with the actual PCR reactions before performing the digestions (data not shown).
<img src="/files/ftp_upload/3844/3844fig3.jpg" alt="Figure 3" fo:content-width="5.5in" fo:src="/files/ftp_upload/3844/3844fig3highres.jpg" /> 
 Figure 3. Dilution protocol using oak leaf samples. The dilution protocol was used to amplify the 297 bp fragment of chloroplastic DNA in oak leaf samples (3-step protocol, annealing at 62 &deg;C) at different dilutions (1:100, 1:10, 1:1 and 2:1) and positive and negative controls [C+: positive control from purified DNA (Arabidopsis), C-: negative control with no template DNA]. The sample nomenclature represents the sample collection site (towns in Finland) and the sample numbering. 
<img src="/files/ftp_upload/3844/3844fig4.jpg" alt="Figure 4" /> 
 Figure 4. Genotyping of transgenic mice with one primer pair using the dilution protocol. Ear tissues of nine individual mice (lanes 1-9) were placed into 20 &mu;l of Dilution Buffer including DNARelease Additive. After incubations at room temperature and 98 &deg;C, 1 &mu;l of supernatant was used as a template in the 20 &mu;l PCR reaction. Fragment sizes: 1,500 bp (transgenic) and 200 bp (wildtype).
<img src="/files/ftp_upload/3844/3844fig5.jpg" alt="Figure 5" /> 
 Figure 5. Samples prepared according to the dilution protocol are stable for long term storage. Samples of mouse ear tissues were incubated in 20 &mu;l of Dilution Buffer including DNARelease Additive and subjected to repeated freezing/thawing (lane 1), stored at -20 &deg;C for one year (lane 2), or used immediately for PCR (lane 3). The amplified fragment sizes were 900 bp, 1,500 bp and 3,200 bp.
<img src="/files/ftp_upload/3844/3844fig6.jpg" alt="Figure 6" /> 
 Figure 6. Phire Animal Tissue Direct PCR Kit outperforms a commercial DNA purification system combined with a hot start Taq DNA polymerase. Four amplicons (0.5-1.5 kb) were amplified from mouse ear tissues using the dilution protocol of Phire Animal Tissue Direct PCR Kit. For comparison, DNA was first purified using a commercial DNA extraction kit and the same fragments were amplified using a hot start Taq DNA polymerase according to manufacturers' recommendations. Only with the Phire Animal Tissue Direct PCR Kit were all four amplicons successfully amplified.
<img src="/files/ftp_upload/3844/3844fig7.jpg" alt="Figure 7" /> 
 Figure 7. Universal comparison of the workflow with estimation of times/consumed reagents. Thermo Scientific Direct PCR approach is compared to the DNA isolation approach, the Extraction buffer approach, and the PCR buffer approach. The estimated time for each approach prior to the PCR step is listed in red text. The Thermo Scientific Direct PCR approach takes only 0-4 min for sample preparation prior to PCR. Below each method, the consumed reagents are listed. The Thermo Scientific Direct PCR approach uses the least amount of reagents in comparison to the other methods. <a href="https://www.jove.com/files/ftp_upload/3844/3844fig7large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Doyle, J. J., Doyle, 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=A+rapid+DNA+isolation+procedure+for+small+quantities+of+fresh+leaf+tissue.">A rapid DNA isolation procedure for small quantities of fresh leaf tissue.</a> Phytochemical Bulletin. 19, 11-15 (1987).</li><li>Kim, C. S., Lee, C. H., Shin, J. S., Chung, Y. S., Hyung, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+isolation+of+high+quality+genomic+DNA+from+fruit+trees+and+conifers+using+PVP.">A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP.</a> Nucleic Acids Research. 25, 1085-1086 (1997).</li><li>Neff, M. M., Neff, J. D., Chory, J., Pepper, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dCAPS,+a+simple+technique+for+the+genetic+analysis+of+single+nucleotide+polymorphisms:+experimental+applications+in+Arabidopsis+thaliana+genetics.">dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics.</a> The Plant Journal. 14, 387-392 (1998).</li><li>Michaels, S. D., Amasino, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+method+for+detecting+single-nucleotide+changes+as+polymorphic+markers+by+PCR.">A robust method for detecting single-nucleotide changes as polymorphic markers by PCR.</a> The Plant Journal. 14, 381-385 (1998).</li><li>John, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+isolation+of+RNA+and+DNA+from+plants+containing+polyphenolics.">An efficient method for isolation of RNA and DNA from plants containing polyphenolics.</a> Nucleic Acids Research. 20, 2381-23 (1992).</li><li>Wang, Z., Storm, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+mouse+tails.">Extraction of DNA from mouse tails.</a> BioTechniques. 41, 410-412 (2006).</li><li>Malumbres, M., Mangues, R., Ferrer, N., Lu, S., Pellicer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+High+Molecular+Weight+DNA+for+Reliable+Genotyping+of+Mice.">Isolation of High Molecular Weight DNA for Reliable Genotyping of Mice.</a> BioTechniques. 22, 1114-1119 (1997).</li><li>Yang, Y., Kim, J. Y., Soh, M. S., Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+gene+amplification+from+Arabidopsis+Leaves+Using+AnyDirect+system.">A simple and rapid gene amplification from Arabidopsis Leaves Using AnyDirect system.</a> Journal of Biochemistry and Molecular Biology. 40, 444-447 (2007).</li><li>Sambrook, J., Fritsch, E., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory. , (1989).</li><li>Meldgaard, M., Bollen, P. J. A., Finsen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+method+for+sampling+and+extraction+of+mouse+DNA+for+PCR.">Non-invasive method for sampling and extraction of mouse DNA for PCR.</a> Laboratory Animals. 3, 413-441 (2004).</li></ol>

Production and Free Access to this article is sponsored by Thermo Fisher Scientific, Inc.

The Direct PCR approach demonstrated here allows for PCR amplification directly from small amounts of unpurified samples, reducing the time needed and simplifying the workflow for plant and animal genotyping (Figure 7). Also demonstrated here is the use of the dilution protocol in combination with the Direct PCR approach (Figure 3). The dilution protocol is recommended with difficult samples (e.g. aged plant leaves, plant species that contain interfering compounds) or with long (or GC-rich) amplicons. This protocol is particularly useful when starting a new Direct PCR experiment, allowing for optimization of the reaction. The protocol can serve as a tool to address occasional difficulties such as low product yields due to differences in tissue material and/or primers used. Furthermore, when running multiple reactions from the same sample, use of the dilution protocol ensures the entire sample is not consumed in one reaction. 
In order to purify DNA from plants for PCR, the convention was to perform cell lysis followed by DNA isolation using various components that could potentially interfere with PCR analysis8. For particularly challenging plants with high phenolic content, addition of polyvinylpyrrolidone was traditionally used to remove the compounds that bind to DNA following cell lysis2,5. As evident here, these complicated, time-intensive steps can be avoided through use of the Phire Plant Direct PCR Kit to detect the target DNA. Cell lysis and DNA isolation steps can be omitted using the sensitive dCAPS techniques directly from Arabidopsis plant leaves (Figure 2). As demonstrated here, the dilution protocol effectively manages the presence of problematic components that can interfere with PCR (Figure 3). 
Traditionally, a prerequisite to animal genotyping was isolation of genomic DNA from animal tissue through a toxic, time-consuming process characterized by a lengthy incubation with proteinase K to digest skin and connective tissue, followed by organic solvent extraction, alcohol precipitation, and centrifugation steps6,9,10. As demonstrated here, simplification of this process is achieved through use of the Phire Animal Tissue Direct PCR Kit, allowing transgenic mice to be genotyped without prior DNA purification (Figure 4). The example demonstrated in this article is particularly challenging as only one primer set was used for amplification of two fragments with a large size difference. Despite the protocol's innate difficulty, use of the Direct PCR approach in conjunction with the simple dilution protocol resulted in high yields of the two PCR products (Figure 4).
PCR-based target DNA detection has many applications in research, including genotype analysis to identify the roles of genes in development, physiology and disease. Due to the inhibitor tolerance of the specialized DNA polymerases, the protocols can be completed in minimal time without prior DNA purification.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Phire Plant Direct PCR Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>F-130</td>
			<td>200 reactions (50 &mu;l each)</td>
		</tr>
		<tr>
			<td>Phire Animal Tissue Direct PCR Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>F-140</td>
			<td>200 reations (50 &mu;l each)</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 0.35 mm (pink)</td>
			<td>Thermo Fisher Scientific</td>
			<td>F-180S/L</td>
			<td>Qty: 5/25</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 0.50 mm (blue)</td>
			<td>Thermo Fisher Scientific</td>
			<td>F-185S/L</td>
			<td>Qty: 5/25</td>
		</tr>
		<tr>
			<td>Harris Cutting Mat 6.4 &times; 7.6 cm</td>
			<td>Thermo Fisher Scientific</td>
			<td>F-190</td>
			<td>Qty: 5</td>
		</tr>
		<tr>
			<td>SspI</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER0771</td>
			<td>100 &mu;l (100 reactions)</td>
		</tr>
		<tr>
			<td>Piko 24-Well Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>TCP0024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>24-Well Piko PCR Plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>SLP0241</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GeneJET PCR 
			Purification Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K0701 
			K0702</td>
			<td>50 preps 
			250 preps</td>
		</tr>
	</tbody>
</table>

genotyping of plant and animal samples without prior dna purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

Watch this Scientific Journal Video about Genotyping of Plant and Animal Samples without Prior DNA Purification at JoVE.com

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

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Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.