<b>Page under construction as of 2/22/2023</b> ---+ Custom Primer Design While there are tools available for automatically designing primers (such as the [[https://www.ncbi.nlm.nih.gov/tools/primer-blast/][NCBI Primer BLAST]]) often specialized PCR applications, such as amplifying fragments for cloning or using [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsTaq][different polymerases]], will necessitate specific primer placements and modifications. In these cases it is important to take extra care to ensure the primers will anneal to the template and amplify well. This page presents guidelines and tools for specifically designing primers from DNA sequences, using the Benchling platform as an example. In short, you will need to: </br><b>1)</b> determine where to place your primers within the template sequence to amplify your desired product </br><b>2)</b> adjust your primer sequences to ideal annealing temperatures </br><b>3)</b> modify the sequences with any necessary adapters and check for inhibitory secondary structures. Designing primers may require trade-offs and revisions. As such it's important to understand the needs of your specific experiment, such as specific locations in the genome, annealing temperatures, and adapter sequences, and keep those in mind while designing your primers. The examples presented here illustrate designing primers to amplify the downstream region of the <i>Acinetobacter baylyi</i> ADP1 <i>ACIAD2049</i> gene for use in [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsAcinetobacterGoldenTransformation][Golden Transformation]]. ---++ 1) Determining Primer Placement <b>I.</b> Start by identifying the gene or region you are amplifying.</br> <b>II.</b> Determine the upstream and downstream locations where you primers will have to anneal to amplify your target. Depending up on your application this may require specific locations (for example, immediately next to the stop codon of a gene if designing primers to knock it out or at a particular base pair in a coding region if your goal is to introduce a point mutation) or a general area. * When possible, avoid repeat or low-complexity sequences as they can cause non-specific binding or the formation of secondary structures. <b>III.</b> Highlight ~30 base pairs of sequence where you want to place your primer. On Benchling, right-click the highlighted region and select "Create primer" to mark it. You will need to select whether it is a forward or reverse primer. The final design will be refined in the following steps. <p style="text-align:center;"><b>Figure 1</b> Placing a primer downstream of the <i>ACIAD2049</i> gene.<br/> <img width="600" alt="ACIAD2049_DoF_v3.png" src="%ATTACHURLPATH%/ACIAD2049_DoF_v3.png" height="300"/></p> <b>Note</b> GC content can be a useful guide for choosing a good primer location, as equal base distributions (i.e. ~50% GC) are less likely to form detrimental secondary structures. However, this may not be a realistic guideline for all primers as different genomes and locations (such as near promoters) can vary considerably in GC content. ---++ 2) Calculating Annealing Temperatures Calculate the annealing temperature for your PCR reaction using the [[https://tmcalculator.neb.com/#!/main][NEB Tm Calculator]].</br> <b>I.</b> Select the polymerase for your PCR reaction from the dropdown menu.</br> <b>II.</b> Copy ~30 bp of the forward and reverse primer sequences into the Primer 1 and Primer 2 boxes.</br> <b>III.</b> Adjust the annealing temperature (top right box) by removing bases from the 3' end of each primer until:</br> * The annealing temperature is in your desired range. This is typically ~57-60°C. * The individual Tms of the two primers (shown in the boxes on the right) are less than 5° apart. * Each primer is 18-25 bp long. Shorter primers are preferred because they reduce the possibility of forming secondary structures that may inhibit your reaction. * Ending a primer with a C or G nucleotide can improve its ability to bind to the template. <p style="text-align:center;"><b>Figure 2 A)</b> Annealing temperature calculations for <i>ACIAD2049</i> downstream primers. <b>B)</b> ACIAD2049 downstream forward primer from Figure 1 shortened to account for annealing temperature.<br/> <img width="1000" alt="annealing_temperature_combined.png" src="%ATTACHURLPATH%/annealing_temperature_combined.png" height="350"/></p> ---++ 3) Modifying Primer Sequences and Checking Secondary Structures <b>I.</b> Add any additional modifications or adapters to the end of your sequences. Restriction sequences used for cloning need to be added to the 5' end of the primer sequence.</br> <b>II.</b> Copy and paste each primer sequence into IDT's [[https://www.idtdna.com/calc/analyzer][OligoAnalyzer]] program. Press "HAIRPIN" to analyze secondary structure formations. Structures with melting temperatures (Tm) below the annealing temperature for your reaction will melt in the thermocycler and not interfere with your reaction (<b>Figure 3</b>, left). Note any structures with Tms within a few degrees of your annealing temperature (<b>Figure 3</b>, right).</br> <b>III.</b> If problematic structures were detected, modify the primer sequence to remove them. Potential solutions include:</br> * Shortening the primer. * Relocating the primer binding site to a nearby region of the template DNA (if possible according to the needs of your experiment). This may be especially helpful if your original target site was particularly A/T or G/C rich. * Choosing a different restriction site or ligation overhang (if possible). <b>d.</b> If you have changed one or both primer sequences you will need to recalculate your annealing temperature as above and confirm that it is still within the desired range (make sure to remove any adapter sequences before calculating your annealing temperature. Use only the sequence that anneals to the template DNA). Add or remove additional bases if necessary to bring the annealing temperature back to the desired range. <p style="text-align:center;"><b>Figure 3</b> OligoAnalyzer results showing primers without (left) and with (right) problematic secondary structures. The structures on the left would all melt in a typical PCR reaction, the first one on the right would form in a reaction with a 57°C annealing temperature.<br/> <img width="1000" alt="OligoAnalyzer_combined.png" src="%ATTACHURLPATH%/OligoAnalyzer_combined.png" height="400"/></p> ---++ Further Reading * [[https://www.benchling.com/primer-design-for-pcr#chapter4][Benchling primer design guidelines]]
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Topic revision: r5 - 2023-02-24 - IsaacGifford