Wild-Type Blocking PCR to Detect Low-Frequency Somatic Mutations
Published: May 31, 2023
Abstract
Source: Albitar, A. Z. et al., Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations. J. Vis. Exp. (2017).
This video describes wild-type blocking polymerase chain reaction, or WTB-PCR, which detects somatic mutations. The PCR-based technique uses a locked nucleic acid, or LNA, an oligonucleotide that binds to its complementary wild-type allele and blocks its elongation. The blocking selectively allows the amplification of low-level mutant alleles over their wild-type variants in the sample.
Protocol
1. DNA Extraction from FFPE Tissue, Peripheral Blood, and Bone Marrow Aspirate For bone marrow FFPE tissue with DNA FFPE extraction kit 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. Place slides in a slide basket and prepare four wash reservoirs (two for Xylene and two for 100% Alcohol). Add a minimum volum…
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The authors have nothing to disclose.
Materials
| 1.5 or 2 mL Safe-Lock microcentrifuge tubes | Eppendorf | 05-402-25 | |
| 100% alcohol | VWR | 89370-084 | Histology grade; 91.5% Ethanol, 5% Isopropyl alcohol, 4.5% Methyl alcohol |
| Aluminium sealing foils | GeneMate | T-2451-1 | For PCR and cold storage |
| Centrifuge 5804 Series | Eppendorf | A-2-DWP rotor (for PCR plate) | |
| Cold plate for 96-well plates | Eppendorf | Z606634 | |
| DNAse, RNAse-free, ultra-pure water | |||
| dNTPs (100 mM) | Invitrogen | 10297-117 | |
| Ethanol Absolute | Sigma | E7023 | 200 proof, for molecular biology |
| Exiqon website Oligo Tools | http://www.exiqon.com/oligo-tools | ||
| FastStart Taq DNA polymerase (5 U/µL) | Roche | 12032937001 | With10x concentrated PCR reaction buffer, with 20 mM MgCl2 |
| Gel electrophoresis apparatus | 2% agarose gel | ||
| LNA oligonucleotide | Exiqon | 500100 | 5'-TCAGA+AG+C+G+A+C+T+G+A +T+CC/invdT/ (+N = LNA bases) |
| Mastercycler Pro S Thermocycler | Eppendorf | E950030020 | |
| Microcentrifuge Model 5430 | Eppendorf | FA-45-30-11 rotor (for 1.5/2 mL microcentrifuge tubes) | |
| NanoDrop 2000 Spectrophotometer | Thermo Fisher Scientific | ||
| PCR forward primer | IDT | 5'-tgt aaa acg acg gcc agt TGC CAG GGG TAC TTA GAT GG | |
| PCR reverse primer | IDT | 5'-cag gaa aca gct atg acc GGT TGG TGT AGT CGC AGA CA | |
| PCR plates | GeneMate | T-3107-1 | |
| Pipettors | 20, 200, 1,000 µL | ||
| QIAamp DNA Mini Kit | Qiagen | 51304 | For BM aspirate and peripheral blood |
| Vortex genie | Scientific Industries | SI-0236 | |
| Slide basket |
