Team:Calgary/Project/BsDetector/SamplePreparation

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<h1>Sample Preparation Protocols</h1>
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<h2>Target Sequence Amplification</h2>
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<h2>TwistDx RPA Kit Protocol*</h2>
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*Taken from the <a href=”http://www.twistdx.co.uk/images/uploads/docs/TA01cmanual_Combined_Manual_RevK.pdf”> TwistDx Protocol Manual</a>
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<ul>For each sample, prepare the rehydration solution as follows:
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<li> Primer A (10μM) 2.4 μl</li>
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<li>  Primer B (10μM) 2.4 μl</li>
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<li>  Rehydration Buffer 29.5 μl</li>
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<li> Template and dH2O 13.2 μl</li>
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<li> (Total Volume 47.5 μl) </li>
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Vortex and spin briefly.
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<ol type=”1”>
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<li> The components of the rehydration solution can be combined in a master-mix for the number of samples required. In some circumstances, for example when performing a primer screen, a number of different rehydration solutions have to be made (here according to the number of primer pairs being tested). In that case components common to all reactions (e.g. template, rehydration buffer, water) should be prepared as a master-mix, distributed in a corresponding volume into fresh tubes, and be combined with the required volume of the different primer pairs. The different rehydration solutions are then used as normal according to the protocol. </li>
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NOTE: Primers and probes should be added simultaneously to pellets to avoid any bias in recombination filament formation.www.twistdx.co.uk TwistAmp® Basic kit
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<li> For each sample, transfer 47.5 μl of the rehydration solution to  the reaction pellet. Mix by pipetting up and down until the entire pellet has been resuspended. </li>
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<li> For each sample, add 2.5 μl 280 mM magnesium acetate and mix  well. One way to do this simultaneously for many samples is to place the magnesium acetate into the lid of the reaction tubes (strip of 8) cap the tubes carefully and spin the magnesium acetate into the rehydrated material to initiate the reactions. Invert vigorously 8-10 times to mix and spin down once again. </li>
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<li> Insert the tubes into a suitable incubator block (optimum 37-39°C) and incubate for 4 minutes. </li>
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<li> After 4 minutes, take the samples out of the incubator, invert vigorously 8-10 times to mix, spin down and return the samples to the incubator block. (VARIATION IN THE EXACT TIME OF SAMPLE AGITATION CAN SOMETIMES IMPROVE PRODUCT FORMATION). </li>
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<li> Continue the incubation/detection for a total incubation time of 20-40 minutes. If a timecourse of TwistAmp® Basic reaction is being taken the incubation time has to be adjusted as required. At the end of the incubation proceed to “Monitoring TwistAmp® Basic amplification reactions”. </li>
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</ol>
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<h3>Optimized RPA Protocol</h3>
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The RPA Kit from TwistDx used in our experiments was designed to rapidly amplify short (~200bp) sequences. For our system we required longer amplicons and attempts were made to adjust the parameters to promote the amplification of longer sequences. Overall the adjustments were intended to reduce the reaction rate and prolong the lifetime of the reaction before exhaustion of resources. The following changes were made:
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<ul>
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<li> Addition of 1mM ATP</li>
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<li> Addition of 0.1mM dNTP’s</li>
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<li> 2.15 μl of 280mM Mg-acetate instead of 2.5 μl</li>
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<li> Vigorous shaking after 6 minutes after initiation rather than 4 minutes</li>
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</ul>
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<h2>KAPA Blood PCR Kit Protocol**</h2>
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**Taken from the <a href=”http://www.peqlab.com/wcms/de/pdf/07-KK7003-01_m.pdf”>KAPA Blood PCR Kit Manual</a>
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<p> Whole human EDTA blood may be added to a final volume of 1 - 20% in the PCR reaction (i.e. 0.5 - 10.0 µl in a 50 µl reaction). Reaction volumes ranging from 10 to 50 µl may be used. Thorough mixing of the blood and other reaction components prior to thermal cycling is important. </p>
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<p> A typical reaction is set up by mixing the components in the order listed in the table below. Once pipetting has been completed, shake or spin tubes briefly to collect all components in the bottom of the tube. Vortex to mix but do not spin again before reactions are placed in the thermocycler. </p>
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<img src=”https://static.igem.org/mediawiki/2014/5/5b/2014UCalgary_Blood_PCR_Table.jpg” align="center">
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<p>A major advantage of our diagnostic system is an internal sample preparation stage. All that is required by the end-user is inputting the whole blood sample into the device. </p>
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<p> A typical 3-step cycling profile for Whole Blood PCR with KAPA Blood PCR Mix A or B is given in the table below. </p>
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<p>Two technical challenges were identified regarding sample preparation, and amplification of our target DNA sequence. First, we must be capable of amplifying a target DNA sequence in a resource poor environment in addition to a potentially hostile climate. Conventional PCR methods are not feasible due to the inhibitory cost of thermocyclers, the necessary refrigeration of PCR components such as enzymes and buffers, and the training required to carry out and analyze PCR experiments. Secondly, we must be capable of amplifying the target DNA sequence directly from whole blood, a complex environment containing inhibitors of Taq polymerase.</p>
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<p><span class="Orange">Isothermal PCR (isoPCR)</span> serves as an alternative to conventional PCR. <span class="Orange">isoPCR</span> refers to the many molecular strategies that allow for amplification of a DNA sequence at a constant, relatively low temperature. Although there are several approaches to <span class="Orange">isoPCR</span>, all strategies employ a molecular mechanism to circumvent the need for thermal denaturation of double-stranded DNA. In our system we utilized <span class="Orange">Recombinase Polymerase Amplification (RPA)</span>. <span class="Orange">RPA</span> utilizes a strand-displacing polymerase to synthesize a complimentary strand of DNA using a double-stranded substrate; the recombinase and a single-stranded DNA-binding protein allow for oligonucleotide primers to stably bind to the template DNA. Because <span class="Orange">RPA</span> is not dependent on a thermocylcer, it is capable of cycling through the amplification process continuously until the exhaustion of resources such as ATP and primers. <span class="Orange">Continuous amplification in RPA</span> results in an amplified signal that is comparative to conventional PCR in a fraction of the time. For our purposes, in developing the sample preparation stage of our system, we used the commercially-available RPA kit purchased from <span class="Orange">TwistDx</span>.</p>
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<img src=”https://static.igem.org/mediawiki/2014/a/ab/2014UCalgary_Blood_PCR_Table_cycling.png” align="center">
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<p>To eliminate the need for refrigeration, and further improve the shelf-life of our system, the components of <span class="Orange">isoPCR</span> are freeze-dried, allowing activation by rehydration immediately prior to use. This was demonstrated to be effective as the <span class="Orange">TwistDx isoPCR kit</span> provided us with freeze-dried pellets and rehydration buffer (Fig. 1). </p>
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<h2> Hybrid Blood & isoPCR Protocol </h2>
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<img src="https://static.igem.org/mediawiki/2014/7/7b/2014UCalgary_Freeze-dried_pellets.png" width="700" class="Center">
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<p> The sample preparation stage in our final system primarily consists of the RPA kit components; the optimized RPA protocol was used as the initial mixture. 25% v/v substitution of the total volume of the RPA reaction mixture was substituted with the Blood PCR Kit Mix A – a mixture containing all necessary components of the blood-compatible PCR except for template and primers.</p>
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<p class="Center"><b>Figure 1:</b> Freeze-dried pellets provided by the <span class="Orange">RPA Basic Kit from TwistDx</span. Pellets contain all components necessary for <span class="Orange">isoPCR</span>, reaction is activated upon addition of hydration buffer and magnesium-acetate also provided in the kit.</p>
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<p> Template may be either whole cells, as performed in typical colony PCR, or a purified DNA extract (e.g. via plasmid miniprep or genomic DNA extraction).</p>
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<p>Whole-blood compatibility in our amplification protocol was achieved using a Taq polymerase tolerant of inhibitors typically found in whole blood. Our sponsor KAPA provided us with KAPA Blood PCR Kits containing an engineered, second-generation Taq polymerase. By supplementing the RPA mixture with the blood-compatible Taq we achieved a greater signal output of isoPCR in the presence of blood. This demonstrated the efficacy of our system to amplify a target DNA sequence in the presence of blood, without the use of a thermocycler (Fig. 2). </p>
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<img src="https://static.igem.org/mediawiki/2014/9/96/2014UCalgary_Sept12_isoBloodHybrid_positiveCntrl_EDIT.png" width="500" class="Center">
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<p class="Center"><b>Figure 2:</b> Results of isoPCR using a 132bp positive control sequence provided with the RPA Basic Kit from TwistDx. Amplification was performed with blood concentrations of 0%, 1%, 5% or 10% v/v sheeps blood treated with the anti-coagulant citrate. Amplification was performed with either RPA exclusively, or with a 25% v/v substitution using the KAPA Blood PCR Kit mix containing the blood- compatible Taq polymerase. At 10% v/v blood, RPA failed to produce the expected amplicon; the addition of blood-compatible Taq produced the expected 132bp amplicon in the presence of 10% v/v blood.</p>
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<p>Isolation of genomic DNA was not necessary for successful amplification of the target DNA sequence. Typically, conventional PCR requires a prolonged initial denaturation step (e.g. 10 minutes at 95C) to lyse the bacterial cells and release DNA. However, our experiments demonstrate that isoPCR is effective on whole <i>E. coli</i> cells without prior lysis (Fig. 3). </p>
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<img src="https://static.igem.org/mediawiki/2014/e/ed/2014UCalgary_%28colony_vs_miniprep%29Aug28_isoPCR_DilutionTest_EDIT.png" width="500" class="Center">
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<p class="Center"><b>Figure 3:</b>  Results of isoPCR under standard isothermal conditions using BioBrick primers. Two different sequences were targeted for amplification; C1-λ is a promoter sequence with an expected amplicon size of 406bp; RFP is a coding sequence with an expected amplicon size of 1100bp. Templates used in this experiment were either whole colonies labelled ‘C’, or purified plasmid extracts labelled ‘P’. </p>
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<h2>Target Sequence Specificity</h2>
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<p>To selectively detect the presence of <i>N. meningititis</i>, we identified a unique yet conserved sequence present in all known <i>N. meningititis</i> genomes. The selected sequence was approximately 400bp, the maximum length of DNA our isoPCR was capable of amplifying. The specificity of our chosen sequence was confirmed by conventional PCR; a ~2.8kb amplicon was produced from the genomic DNA of six different <i>N. meningititis</i> strains, no amplicons were produced from the genomic DNA of five other pathogens including a commensal strain of <i>Neisseria</i> (Fig. 4). These results were replicated using isoPCR, amplifying a shorter target sequence of ~450bp. Although other smaller amplicons are produced by isoPCR these are the result of ‘primer noise’ inherent to the isoPCR method of <span class="Orange">RPA</span> and may be reduced in the future by optimizing conditions such as primer concentration and screening primer variants for reduced primer noise (Fig. 5).</p>
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<img src="https://static.igem.org/mediawiki/2014/1/1e/2014UCalgary_Sept10_MeningPrimer_SpecificityPCR_EDIT.png" width="700" class="Center">
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<p class="Center"><b>Figure 4:</b> Results of PCR on genomic DNA samples from eleven pathogenic parasites. All six <i>N. meningititis</i> strains produced the expected amplicon at ~2.8kb as indicated by white asterisks. The five other pathogens did not produce any products of the appropriate size.</p>
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<img src="https://static.igem.org/mediawiki/2014/c/c0/2014UCalgary_Oct1_isoPCR_targetSeq400bp%28pure%29_EDIT.png" width="700" class="Center">
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<p class="Center"><b>Figure 5:</b> Results of isoPCR using <span class="Orange">RPA</span> on the eleven genomic DNA samples as described in Fig. 3. Bands below 500bp are indicative of primer noise inherent to the <span class="Orange">RPA</span> system. </p>

Latest revision as of 03:59, 18 October 2014

Target Sequence Amplification

A major advantage of our diagnostic system is an internal sample preparation stage. All that is required by the end-user is inputting the whole blood sample into the device.

Two technical challenges were identified regarding sample preparation, and amplification of our target DNA sequence. First, we must be capable of amplifying a target DNA sequence in a resource poor environment in addition to a potentially hostile climate. Conventional PCR methods are not feasible due to the inhibitory cost of thermocyclers, the necessary refrigeration of PCR components such as enzymes and buffers, and the training required to carry out and analyze PCR experiments. Secondly, we must be capable of amplifying the target DNA sequence directly from whole blood, a complex environment containing inhibitors of Taq polymerase.

Isothermal PCR (isoPCR) serves as an alternative to conventional PCR. isoPCR refers to the many molecular strategies that allow for amplification of a DNA sequence at a constant, relatively low temperature. Although there are several approaches to isoPCR, all strategies employ a molecular mechanism to circumvent the need for thermal denaturation of double-stranded DNA. In our system we utilized Recombinase Polymerase Amplification (RPA). RPA utilizes a strand-displacing polymerase to synthesize a complimentary strand of DNA using a double-stranded substrate; the recombinase and a single-stranded DNA-binding protein allow for oligonucleotide primers to stably bind to the template DNA. Because RPA is not dependent on a thermocylcer, it is capable of cycling through the amplification process continuously until the exhaustion of resources such as ATP and primers. Continuous amplification in RPA results in an amplified signal that is comparative to conventional PCR in a fraction of the time. For our purposes, in developing the sample preparation stage of our system, we used the commercially-available RPA kit purchased from TwistDx.

To eliminate the need for refrigeration, and further improve the shelf-life of our system, the components of isoPCR are freeze-dried, allowing activation by rehydration immediately prior to use. This was demonstrated to be effective as the TwistDx isoPCR kit provided us with freeze-dried pellets and rehydration buffer (Fig. 1).

Figure 1: Freeze-dried pellets provided by the RPA Basic Kit from TwistDxisoPCR, reaction is activated upon addition of hydration buffer and magnesium-acetate also provided in the kit.

Whole-blood compatibility in our amplification protocol was achieved using a Taq polymerase tolerant of inhibitors typically found in whole blood. Our sponsor KAPA provided us with KAPA Blood PCR Kits containing an engineered, second-generation Taq polymerase. By supplementing the RPA mixture with the blood-compatible Taq we achieved a greater signal output of isoPCR in the presence of blood. This demonstrated the efficacy of our system to amplify a target DNA sequence in the presence of blood, without the use of a thermocycler (Fig. 2).

Figure 2: Results of isoPCR using a 132bp positive control sequence provided with the RPA Basic Kit from TwistDx. Amplification was performed with blood concentrations of 0%, 1%, 5% or 10% v/v sheeps blood treated with the anti-coagulant citrate. Amplification was performed with either RPA exclusively, or with a 25% v/v substitution using the KAPA Blood PCR Kit mix containing the blood- compatible Taq polymerase. At 10% v/v blood, RPA failed to produce the expected amplicon; the addition of blood-compatible Taq produced the expected 132bp amplicon in the presence of 10% v/v blood.

Isolation of genomic DNA was not necessary for successful amplification of the target DNA sequence. Typically, conventional PCR requires a prolonged initial denaturation step (e.g. 10 minutes at 95C) to lyse the bacterial cells and release DNA. However, our experiments demonstrate that isoPCR is effective on whole E. coli cells without prior lysis (Fig. 3).

Figure 3: Results of isoPCR under standard isothermal conditions using BioBrick primers. Two different sequences were targeted for amplification; C1-λ is a promoter sequence with an expected amplicon size of 406bp; RFP is a coding sequence with an expected amplicon size of 1100bp. Templates used in this experiment were either whole colonies labelled ‘C’, or purified plasmid extracts labelled ‘P’.

Target Sequence Specificity

To selectively detect the presence of N. meningititis, we identified a unique yet conserved sequence present in all known N. meningititis genomes. The selected sequence was approximately 400bp, the maximum length of DNA our isoPCR was capable of amplifying. The specificity of our chosen sequence was confirmed by conventional PCR; a ~2.8kb amplicon was produced from the genomic DNA of six different N. meningititis strains, no amplicons were produced from the genomic DNA of five other pathogens including a commensal strain of Neisseria (Fig. 4). These results were replicated using isoPCR, amplifying a shorter target sequence of ~450bp. Although other smaller amplicons are produced by isoPCR these are the result of ‘primer noise’ inherent to the isoPCR method of RPA and may be reduced in the future by optimizing conditions such as primer concentration and screening primer variants for reduced primer noise (Fig. 5).

Figure 4: Results of PCR on genomic DNA samples from eleven pathogenic parasites. All six N. meningititis strains produced the expected amplicon at ~2.8kb as indicated by white asterisks. The five other pathogens did not produce any products of the appropriate size.

Figure 5: Results of isoPCR using RPA on the eleven genomic DNA samples as described in Fig. 3. Bands below 500bp are indicative of primer noise inherent to the RPA system.