Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Summary

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Abstract

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Introduction

Sanger sequencing has traditionally been the gold standard in testing for both known and unknown somatic mutations. One of the limitations of Sanger sequencing is its limit of detection (~ 10 - 20% mutant allele in a background of WT)¹. This level of sensitivity is inappropriate for detecting low level somatic mutations that may be present in samples from premalignant tissues or patients with few circulating tumor cells, or when bone marrow (BM) is patchy. This also makes assessing residual disease after therapy or detecting emerging resistance mutations during therapy difficult by conventional sequencing alone². By repl....

Protocol

Ethics Statement: All testing of human samples was performed after obtaining Institutional Review Board (IRB) approval.

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 solu....

Results

A conceptual overview of WTB-PCR during extension is presented in Figure 1. Because a single nucleotide mismatch in the blocker-DNA hybrid greatly decreases its melting temperature (ΔT_m=20 - 30 °C), amplification of the WT allele is blocked while mutant template DNA is free to complete extension¹⁷. In this manner, mutant DNA is amplified exponentially while WT DNA is amplified linearly.

Discussion

The WTB-PCR assay described here uses a generic set of primers with a blocking oligo designed to block amplification of WT DNA during extension (Figure 1). The WTB-PCR product is then sequenced for mutational analysis. The utility of WTB-PCR/Sanger lies in its simplicity, high-sensitivity, and high-throughput. Using the guidelines described here, most existing Sanger based assays can be simply modified via the addition of a blocking oligonucleotide to greatly increase sensitivity. In the example assay pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
3730XL sequencer	ABI		or equivalent
Agencourt AMPure XP	Beckman Coulter	A63881	For magnetic bead PCR purification
Aluminum sealing foils	GeneMate	T-2451-1	For PCR and cold storage
BigDye Terminator v3.1 Cycle sequencing kit	Life Technologies	4337455	For bi-directional sequencing. With 5x Sequencing Buffer
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
DynaMag-96 Side-Skirted Magnet	Thermo Fisher Scientific	12027	For use in PCR Purification.
Ethanol Absolute	Sigma	E7023	200 proof, for molecular biology
Exiqon website Oligo Tools			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
GeneRead DNA FFPE extraction Kit	Qiagen	180134	Contains uracil DNA glycosylase necessary for reducing sequencing artifacts
Hi-Di Formamide	ABI	4311320	For sequencing.
LNA oligonucleotide	Exiqon	500100	5'-TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/ (+N = LNA bases)
M13-F Sequencing Primer	ABI		5'-tgt aaa acg acg gcc agt
M13-R Sequencing Primer	ABI		5'-cag gaa aca gct atg acc
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
Plate septa, 96 well	ABI	4315933
QIAamp DNA Mini Kit	Qiagen	51304	For BM aspirate and peripheral blood
SeqScape Sortware v3.0	ABI	4474978	For sequencing analysis
Slide basket
Sodium Acetate (3 M, pH 5.2)	Sigma	S7899
Sterile filtered pipette tips			20, 200, 1,000 µL
Thermomixer C	Eppendorf	5382000023
Vortex genie	Scientific Industries	SI-0236
Wash reservoir			~ 1,000 mL
Xylene	VWR	89370-088	Histology grade