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…

開示

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

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記事を引用
Wild-Type Blocking PCR to Detect Low-Frequency Somatic Mutations. J. Vis. Exp. (Pending Publication), e21356, doi: (2023).

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