Pre-PCR Mutation-Enrichment Methods for Liquid Biopsy Applications

Abstract

Liquid biopsy is having a remarkable impact on healthcare- and disease-management in the context of personalized medicine. Circulating free DNA (cfDNA) is one of the most instructive liquid-biopsy-based biomarkers and harbors valuable information for diagnostic, predictive, and prognostic purposes. When it comes to cancer, circulating DNA from the tumor (ctDNA) has a wide range of applications, from early cancer detection to the early detection of relapse or drug resistance, and the tracking of the dynamic genomic make-up of tumor cells. However, the detection of ctDNA remains technically challenging, due, in part, to the low frequency of ctDNA among excessive circulating cfDNA originating from normal tissues. During the past three decades, mutation-enrichment methods have emerged to boost sensitivity and enable facile detection of low-level mutations. Although most developed techniques apply mutation enrichment during or following initial PCR, there are a few techniques that allow mutation selection prior to PCR, which provides advantages. Pre-PCR enrichment techniques can be directly applied to genomic DNA and diminish the influence of PCR errors that can take place during amplification. Moreover, they have the capability for high multiplexity and can be followed by established mutation detection and enrichment technologies without changes to their established procedures. The first approaches for pre-PCR enrichment were developed by employing restriction endonucleases directly on genomic DNA in the early 1990s. However, newly developed pre-PCR enrichment methods provide higher sensitivity and versatility. This review describes the available pre-PCR enrichment methods and focuses on the most recently developed techniques (NaME-PrO, UVME, and DEASH/MAESTRO), emphasizing their applications in liquid biopsies.

Keywords: cell-free DNA; circulating tumor DNA; liquid biopsy; low-level mutation detection; mutation enrichment; pre-PCR enrichment.

Figure 1

Applications of nuclease-assisted minor-allele enrichment (NaME) method in cell-free DNA (cfDNA) or genomic DNA: (A) Workflow for NaME using probe overlap (NaME-PrO) for enrichment of mutations. The overlapping probes are designed to match sense and antisense strands of wild-type (WT) targets. The nucleotides within the probe overlap region determine the target sequence. Following denaturation step and probe hybridization, duplex-specific nuclease (DSN) preferentially digests fully matched double-stranded sequences, thereby retaining intact the mutated strands; (B) workflow for enrichment of indel-containing sequences to enhance detection of microsatellite instability (MSI-NaME-PrO). The overlapping probes for targeting microsatellite NR27, as an example, are illustrated. The polyA homopolymer has a low Tm, such that the probe–probe and the probe–target hybrids show a ~5 °C difference. Following probe hybridization, DSN digests WT alleles, while indel-containing sequences remain substantially undigested because of bulges within probe–target hybrids; (C) workflow of methylation-sensitive NaME (MS-NaME): U probes are designed to bind uracil-containing sequences (which are generated via bisulfite-mediated cytosine deamination). M probes are designed to bind 5mC-containing DNA. Following denaturation and whole-genome amplification (WGA), either U probes or M probes are applied to enrich methylated and unmethylated targets, respectively.

Figure 2

Workflow of pre-PCR-UV-mediated cross-linking minor-allele enrichment (pre-PCR-UVME): (A) A UV lamp is employed to induce selective cross-linking of wtDNA. Target-specific CNVK-modified probes bind sense strand of wtDNA, and common CNVK-modified probes attach to antisense strands of both wtDNA and mutated DNA; (B) after UV irradiation, fully matched probe–target hybrids undergo photo-crosslinking of CNVK with C or T on the target sequence, thereby inhibiting the proliferation of wtDNA strands and mutant antisense strand during PCR. Mismatch-containing probes undergo significantly lower crosslinking.

Figure 3

(A) Principles of DNA enrichment via allele-specific hybridization (DEASH), and minor-allele-enriched sequencing through recognition oligonucleotides (MAESTRO). The biotinylated allele-specific oligonucleotide (bio-ASO) is designed to bind mutant strands, while competitor ASO attaches to wild-type strands. Following denaturation and hybridization, the bio-ASO/target hybrids are captured by streptavidin-coated beads. The unbound DNA can undergo further cycles of DEASH. The enriched single-stranded targets are eluted and purified; (B) workflow for minor-allele-enriched sequencing through recognition oligonucleotides (MAESTRO) technique. The underlying principle is similar to DEASH, although MAESTRO operates without competitive blockers and provides genome-wide massively parallel enrichment. Following NGS library construction and barcoding top and bottom DNA strands, the biotinylated probes complementary for up to 10,000 genome-wide mutations are employed to bind mutant strands. Afterwards, the mutation-containing strands are preferentially captured by streptavidin-coated beads, are amplified, and undergo duplex sequencing.