Liquid biopsies provide a non-invasive approach to tracing tumor-derived biomarkers in blood, with broad applications in medicine. During the past decade, circulating free DNA (cfDNA) has been turned into an informative resource for cancer management. Mutation-enrichment methods enhance the detection of tumor-derived, low-level mutations in blood. These methods increase the frequency of low-level mutations insofar as they become detectable via routine diagnostic techniques. Enriching mutations prior to PCR (pre-PCR) offers distinctive advantages. Applying the mutation-enrichment process directly to genomic DNA or cfDNA circumvents PCR errors and provides enriched mutation-containing products that different technologies can detect without any required changes in their protocols. In this review, we discuss the recent developments in pre-PCR enrichment methods from the perspective of their applications in liquid biopsies.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 , emphasizing their applications in liquid biopsies. Over the past decade, liquid biopsies have become an informative complement to tumor tissue biopsies, and provide a minimally invasive approach for repeated interrogation of tumor status. Liquid biopsy encompasses circulating tumor cells (CTCs) and circulating free DNA (cfDNA), including DNA originating from the tumor (ctDNA); circulating free RNA (cfRNA); platelets; and exosomes (EXOs) ,2. AmongAdditionally, while the clinical significance of low-level mutations of tumor origin in cfDNA is evident, their measurement remains technically challenging. For example, the routine use of next-generation sequencing technology has a lower limit of detection (LOD) for a MAF of ~0.5–1%, below which false-positive signals often emerge due to polymerase mis-incorporations (‘PCR errors’) introduced during sample preparation, and during sequencing-by-synthesis. This unavoidable ‘mutational noise’ is independent of interrogation depth, i.e., increasing the number of reads does not improve the NGS detection limit ,41,42,43This review focuses on mutation-enrichment approaches that can be applied prior to performing a PCR step . Such prNuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO) is a scalable enrichment technique where the genotypic selection step is applied directly on genomic DNA or cfDNA without pre-amplification . After DTo avoid protocols that require the opening of PCR vials and the addition of probes and DSN following the initial DNA denaturation, a modified closed-tube approach called no-denaturation nuclease-assisted minor-allele enrichment with probe-overlap (ND-NaME-PrO) was also developed . This apNaME-PrO with the Amplification Refractory Mutation System (ARMS)—called a NAPA assay—boosts the analytical specificity of ARMS [PIK3CA mutations (H1047R and E545K) in cfDNA and circulating tumor cells (CTCs) from metastatic BC patients. PIK3CA is one of the frequently mutated genes in different cancers with valuable predictive potential [PIK3CA at a frequency of 0.1–0.5% with high specificity. The NAPA assay was similarly applied to detect hotspot ESR1 mutations in both CTC and cfDNA samples from BC patients with improved sensitivity and specificity [The application of NaME-PrO directly on genomic DNA/cfDNA prior to PCR enables straightforward combination with established mutation-enrichment technologies operating during PCR amplification. The combination of  of ARMS . ARMS-PC of ARMS  showed totential ,62. NAPAcificity .PIK3CA gene  in cfDNA from BC samples. This study applied NaME-PrO following a target pre-amplification to accurately detect PIK3CA mutations in cfDNA via real-time qPCR or ddPCR. Using SYBR Green real-time qPCR makes this approach accessible and affordable for many laboratories. Moreover, a simple prediction approach has been introduced to determine the initial mutation frequency in blood samples using a logarithmic regression method. This approach increases the mutant allele frequency up to 30-fold, improves detection sensitivity, and provides close-to-quantitative results, which is important for the clinical interpretation of cfDNA samples.Keraite et al.  applied Microsatellite instability (MSI) refers to frequent indels within repetitive genomic sequences, resulting from deficiencies in cellular DNA mismatch repair . TracingAnother modification of NaME-PrO for enriching MSI prior to PCR amplification is nuclease-assisted microsatellite instability enrichment (NaMSIE) . NaMSIE Tracing aberrant methylation signatures as “epimutations” in liquid biopsies can boost the utility and clinical value of cfDNA in different diseases, including cancer ,70,71. AUV-mediated cross-linking minor-allele enrichment (UVME) is a newly developed technique for pre-PCR mutation enrichment that utilizes UV irradiation to block wtDNA from subsequent amplification . The UVMUVME can operate in two different settings, pre-PCR-UVME and UVME-PCR. The former  is direcThe latter approach, UVME-PCR, employs UV irradiation during each PCR cycle. Following 10 cycles pre-amplification without UV, a second PCR stage is initiated, that blocks wtDNA at the primer annealing temperature of each PCR cycle, resulting in robust mutation enrichment. UVME-PCR technically shows higher enrichment efficiency in comparison to pre-PCR-UVME. However, pre-PCR UVME is easier to perform, using a standard UV lamp and a PCR machine or a simple heating block. UVME-PCR, on the other hand, employs UV at each PCR step. The use of an external UV lamp manually during PCR annealing steps, as carried for demonstrating the UVME principle, is laborious. Accordingly, incorporating a UV source within PCR instruments is imperative if UVME-PCR is to be utilized broadly. The combination of pre-PCR-UVME with UVME-PCR shows remarkable mutation enrichment, which converts the original mutation frequencies of 0.1% and 1% to 85% and 98%, respectively .−3–10−5% could become detectable by applying DEASH prior to amplification. Although the originally described DEASH method was not applied on cfDNA samples, a recent development uses a modified DEASH principle to track thousands of low-level mutations in cfDNA. Minor-allele-enriched sequencing through recognition oligonucleotides (MAESTRO), employs short probes to capture up to 10,000 low-level mutations simultaneously, and then, incorporates duplex sequencing to detect mutation fingerprints in cfDNA for tracking minimal residual disease with high sensitivity and specificity  cleave DNA strands through a specific recognition site . These e–3 frequency, while improvements in digestion efficiency have led to higher sensitivity up to 10−6. RSM is simple, cost-effective, and sensitive; however, the method is subject to false positives when enzymatic digestion is incomplete or when the sample contains impurities that inhibit the enzyme. RSM should be optimized carefully for a specific sequence and enzyme combination. In 1992, Sandy et al. [8 to 109 WT strands. However, this sensitivity comes at a cost. There is a preparative step of restriction digestion to enrich the target sequence from genomic DNA. In the next step, the main digestion process with specific RE is performed, and undigested targets, including mutations, are amplified. The PCR products are cloned and detected by reporter probes [In 1990 Parry et al. proposed the RSM technique for detecting mutations in restriction-enzyme recognition sites . Althougy et al.  developer probes . The abo8 WT alleles). It also provides simultaneous and quantitative analysis of mutations in multiple genes. This method has been used to investigate random point-mutations in normal and malignant human tissues [Random Mutation Capture assay is a derivation of the RSM technique that incorporates restriction-enzyme-mediated enrichment and bead-based capture ,86. Rand tissues . The resCRISPR-based mutation-enrichment approaches have emerged over the past decade ,90,91. TThermus thermophilus (NAVIGATER) technique is based on nucleic acid-guided endonuclease activity of the TtAgo enzyme to efficiently cleave DNA targets complementary to its single-strand guide DNA (gDNA) [KRAS G12D/V/R mutations. The results revealed that twice-NAVIGATER, followed by xenonucleic acid clamp PCR (XNA-PC), could detect rare mutations at a mutation allelic frequency <0.2%, with a specificity comparable to tissue NGS genotyping. The authors also applied NAVIGATER directly to genomic DNA and cfDNA without pre-amplification to track KRAS G12D mutant alleles. The results indicate that this method could potentially enrich cell-free mutant alleles without pre-amplification with acceptable efficiency, since NAVIGATER can operate with short size DNA strands, such as those in cfDNA ~160 bp, or formalin-degraded samples [The nucleic acid enrichment via DNA-Guided Argonaute from A (gDNA) . TtAgo iA (gDNA)  applied  samples . Neverth samples .Pyrrole-imidazole (PI) polyamide bead-based capture may also potentially enrich rare specific mutant alleles in cfDNA samples in a pre-PCR setting. By employing the affinity of PI polyamides to attach to a particular sequence of the minor groove of double helix B-DNA, the hotspot KRAS mutations in cfDNA samples are successfully captured and detected by ddPCR . For thiLiquid biopsy harbors valuable information for the clinical management of disease and has become a hot topic in cancer research and diagnostics. cfDNA is an informative molecular biomarker that can be tracked to detect tumor-related mutations, or other DNA alterations. Notable advancements in mutation-enrichment methods provide the ability to increase the frequency of low-level mutations in liquid biopsies so that they become detectable by commonly used detection techniques. Among all enrichment techniques, pre-PCR enrichment methods offer distinct advantages such as the avoidance of PCR errors. Polymerase mis-incorporations take place whenever DNA is amplified, and the rate of mis-incorporation depends on the proof-reading ability of the polymerase used, as well as the sequence context ,98. PCR