Encyclopedia of Experiments: Biological Techniques
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The forward and reverse primers were designed with a 5'-M13 sequence to allow for annealing of complementary sequencing primers. Design the blocking oligonucleotide to be approximately 10 to 15 bases in length and complementary to the wild-type template where mutant enrichment is desired.
A shorter oligo will improve mismatch discrimination. To achieve high target specificity, it's important not to use too much of the blocking nucleotides, as this will result in a very "sticky" oligonucleotide.
To design the blocking oligonucleotide, begin by navigating to the Oligo Tools website. Select the "Oligo TM Prediction" tool. A new window will open up. Paste the sequence of the wild-type template to be blocked into the "Oligo Sequence" box. Add a plus sign in front of the blocker bases to mark them. Click on the "Calculate" button to determine the approximate TM of the DNA blocker hybrid. The calculated melting temperatures will appear in the boxes below.
Design the blocking oligo to have a melting temperature 10 to 15 degrees Celsius above the extension temperature during thermocycling. Here, the extension temperature is 72 degrees Celsius. To adjust the melting temperature, add, remove, or substitute blocking bases. Avoid long stretches of three to four blocking C or G bases.
Next, to avoid secondary structure formation or self-dimerization, go back to the Oligo Tools website home screen and select the "Oligo Optimizer" tool. A new window will open. Paste the sequence of the wild-type template to be blocked into the box. Add a plus sign to indicate blocking bases. Select the two boxes for "Secondary Structure" and "Self Only" and press the "Analyze" button to see the scores for hybridization and secondary structure.
These scores represent very rough estimates of the melting temperatures of the self-dimers and secondary structures, respectively. Lower scores are optimal and can be achieved by limiting blocker-blocker pairing. Remove or reposition blocking nucleotides in order to achieve lower scores. The optimized blocking oligonucleotide for MyD88, shown here, strikes a balance between the DNA blocker hybrid melting temperature and low enough hybridization and secondary structure scores.
It was designed to cover amino acids Q262 to I266 and features a 3'-inverted dT to inhibit both extension by DNA polymerase and degradation by 3'-exonuclease. Once the primers have been designed, set up the wild-type blocking PCR and perform thermocycling, as described in the accompanying document.
