Wild-Type Blocking PCR to Detect Low-Frequency Somatic Mutations

1. DNA Extraction from FFPE Tissue, Peripheral Blood, and Bone Marrow Aspirate

1. For bone marrow FFPE tissue with DNA FFPE extraction kit
   1. Begin with FFPE tissue from unstained slides (5 – 10 sections at 5 – 10 µm thickness).
      NOTE: If beginning with tissue shavings, use 3 – 6 sections at 5 – 10 µm thickness and skip to step 1.1.6.
   2. Place slides in a slide basket and prepare four wash reservoirs (two for Xylene and two for 100% Alcohol). Add a minimum volume of 600 mL of the solution to each basket.
   3. Deparaffinize the slides by doing a 5 min xylene wash in the first tray. Transfer the slides to the second xylene wash reservoir for an additional 5 min.
   4. After deparaffinization, wash the slides with 100% alcohol for 5 min. Transfer the slides to the second alcohol wash reservoir for an additional 5 min.
   5. Allow the slides to dry completely under a hood before scraping them with a razor blade into a microcentrifuge tube.
      NOTE: If an H&E slide with the tumor region indicated is available, align the slides and scrape only the tumor region.
   6. Proceed with instructions from the manufacturer handout included in the kit.
2. For BM aspirate and Peripheral Blood
   1. Extract according to the manufacturer's instructions handout with these specifications.
      NOTE: There are numerous commercially available kits and methods for DNA extraction from FFPE tissues and cells. Practically, all can be used. Use a method that is established in the laboratory.
      1. Use 200 µL peripheral blood (PB) or 100 µL BM + 100 µL PBS.
      2. Use 4 µL RNase A stock solution.
      3. Elute with 100 µL elution buffer.
3. DNA Quantification
   1. Measure DNA concentrations using a spectrophotometer ensuring a 260 nm/280 nm ratio of approximately 1.8 (for pure DNA). If the ratio is appreciably lower, it may indicate the presence of protein, phenol, or other contaminants that may interfere with downstream applications.
   2. Adjust DNA concentrations to approximately 50 – 100 ng/µL with appropriate elution buffer.

2. Wild-Type Blocking PCR

1. Primer Design
   1. Design and obtain primers for genes of interest according to previously described general guidelines of PCR primer design. Include M13-forward and reverse universal sequencing primers in PCR primers.
      NOTE: The MYD88 assay was developed to amplify exon 5 of MYD88 (G259 – N291) and 110 nucleotides located in the 5' intronic region to cover the splice site and part of intron 4. The forward and reverse primers were designed with a 5'-M13 sequence (M13- forward: tgt aaa acg acg gcc agt; M13-reverse: cag gaa aca gct atg acc) to allow for annealing of complementary sequencing primers (see Materials Table).
2. Locked Nucleic Acid Oligonucleotide Design
   1. Design the blocking oligonucleotide to be approximately 10 – 15 bases in length and complementary to the WT template where mutant enrichment is desired.
      NOTE: A shorter oligo will improve mismatch discrimination. To achieve high target specificity, it is important not to use too many blocking nucleotides since this can result in a very "sticky" oligonucleotide. The content and the length and blocking nucleotide should be optimized according to the specific nucleotide that needs to be blocked. It is important to achieve high binding specificity without compromising the secondary structure and risking self-complementarity.
   2. Design the blocking oligo to have a T_m 10 – 15 °C above the extension temperature during thermocycling. Adjust the T_m by adding or removing blocking bases or by substituting blocking bases for DNA while avoiding long stretches (3 – 4 bases) of blocking G or C bases.
      1. Navigate to the Oligo tools website (see Table of Materials) and select the "blocking Oligo T_m Prediction" tool.
      2. Paste the sequence of the WT template that is desired to be blocked into the "Oligo Sequence" box. Add "+" in front of bases to indicate blocker bases.
      3. Select the "Calculate" button to determine the approximate T_m of the DNA-Blocker hybrid.
   3. Design the blocking oligo to avoid secondary structure formation or self-dimers.
      1. Navigate to the Oligo tools website (see Table of Materials) and select the "blocking Oligo Optimizer" tool.
      2. Paste the sequence of the WT template that is desired to be blocked into the box. Add "+" in front of bases to indicate blocking bases.
      3. Select the two boxes for "Secondary Structure" and "Self Only". Press the Analyze button to review the oligo for potentially troublesome secondary structures or self-dimers.
         NOTE: The scores represent very rough estimates of the T_m's of the secondary structures and self-dimers. Lower scores are therefore optimal and can be achieved by limiting blocker-blocker pairing. The blocking oligonucleotide for MYD88 [MYD88 blocker (TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/)] was designed to cover amino acids Q262-I266 and features a 3'-inverted dT to inhibit both extensions by DNA polymerase and degradation by 3'-exonuclease. This specific blocking oligo is a 17 mer with 10 blocking bases which are denoted as "+N". The remaining 7 bases are ordinary DNA nucleotides.
3. WTB-PCR Setup and Thermocycling
   1. Prepare a WTB-PCR master mix (MMX) using 2.5 µL PCR reaction buffer 10x w/ 20 mM MgCl₂, 250 µM dNTPs, 0.2 µM forward and reverse primer, 1.2 µM MYD88 blocker, and DNAse, RNAse-free, ultra-pure H₂O to create a final solution volume of 21.75 µL per reaction.
      NOTE: Working concentrations are for the final reaction volume of 25 µL (after the addition of the DNA template and polymerase). Conventional PCR MMX is prepared by simply omitting the addition of a blocker. All protocol steps remain identical for both WTB and conventional PCR. This can be used in validation and determining enrichment achieved by the addition of a blocker.
      1. When calculating the amount of MMX to prepare, make sure to account for 3 additional reactions (positive and negative controls and a non-template control to check for contamination) and at least 1 additional reaction for pipetting error.
   2. Vortex MMX thoroughly. The MMX can be stored at -80 °C for up to a year.
   3. Add 0.25 µL Taq DNA polymerase per reaction to the MMX and invert to mix. Once the polymerase has been added to the MMX, keep it on ice.
   4. Put a new 96-well PCR plate on a cold plate and pipette 22 µL of MMX with the polymerase to each reaction well.
   5. Add 3 µL genomic DNA (50 – 100 ng/µL to each of the wells containing the MMX with the polymerase.
   6. Seal the plate and centrifuge briefly to ensure the solution reaches the bottom of each well.
   7. Load the PCR plate on a thermocycler.
      1. Set the thermocycling conditions for the WTB-PCR reaction as follows: initial denaturation at 95 °C for 6 min; 40 cycles of denaturation at 95 °C for 30 s, primer annealing at 56 °C for 30 s, and extension at 72 °C for 1 min 20 s; final extension at 72 °C for 10 min.
         NOTE: Best practice involves completing the remainder of the protocol in a physically separate area to avoid amplicon contamination in future setups.