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

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…

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