Anchored Multiplex PCR: Key Insights and Targeted Sequencing

Advancements in molecular diagnostics have significantly improved the detection of genetic variations with high specificity and sensitivity. Anchored multiplex PCR (AMP) has gained attention for its role in targeted sequencing, particularly in detecting gene fusions and structural variants that traditional methods may miss.

This method enhances the accuracy of next-generation sequencing by selectively amplifying specific genomic regions without requiring prior knowledge of both primer binding sites. Understanding its principles, components, and advantages over conventional approaches is essential for researchers and clinicians utilizing AMP for precision medicine and genetic analysis.

Core Principles Of Anchored Multiplex PCR

Anchored multiplex PCR (AMP) overcomes the limitations of traditional PCR methods by enabling the amplification of unknown or highly variable genomic regions. Unlike conventional multiplex PCR, which requires both forward and reverse primers for known sequences, AMP uses a single fixed primer alongside random or semi-random primers. This approach allows for the detection of structural variations such as gene fusions, insertions, and deletions that standard amplification techniques often miss.

AMP relies on an anchored primer within a well-characterized genomic region, while the second primer is generated through methods such as random priming, ligation-mediated amplification, or template switching. This design ensures amplification can proceed even if one end of a genetic rearrangement is unknown. AMP is particularly effective in identifying fusion genes, which play a significant role in tumorigenesis. Studies have shown its reliability in detecting ALK, ROS1, and NTRK fusions in non-small cell lung cancer (NSCLC), even when breakpoints occur unpredictably.

A key advantage of AMP is its ability to maintain high specificity while capturing a broad range of genetic alterations. Molecular barcoding and unique molecular identifiers (UMIs) help distinguish true genetic variants from sequencing artifacts, reducing false positives and improving variant calling accuracy. Compatible with next-generation sequencing (NGS) platforms, AMP allows for high-throughput analysis of complex genomic rearrangements with minimal input DNA.

Key Molecular Components

The effectiveness of AMP depends on the careful selection of molecular components. At its core are primers: an anchored primer targeting a conserved sequence in a known genomic region and a second primer that amplifies unknown or variable sequences. The anchored primer ensures reliable binding, while the second primer, generated through template switching, random priming, or ligation-based methods, captures diverse genomic rearrangements.

AMP also relies on high-fidelity DNA polymerases optimized for complex genomic templates. Enzymes such as Phusion or Q5 DNA polymerase, with low error rates and strong proofreading activity, help maintain sequence integrity. These polymerases are particularly important when analyzing clinical samples, where input DNA may be limited or degraded. Optimized reaction buffers and magnesium ion concentrations further support amplification efficiency.

Unique molecular identifiers (UMIs) and molecular barcoding enhance specificity by reducing errors during library preparation and sequencing. UMIs, short random nucleotide sequences incorporated into primers or adapters, differentiate true genetic variants from PCR duplicates and sequencing errors. This improves variant calling accuracy, particularly in oncology and inherited disease screening, where distinguishing true variants from background noise is critical.

Steps In Laboratory Setup

Establishing an effective AMP workflow requires meticulous optimization from sample preparation to amplification and sequencing library construction. High-quality nucleic acid extraction is essential, as degraded or fragmented DNA can compromise amplification. Formalin-fixed paraffin-embedded (FFPE) tissue samples, commonly used in clinical oncology, present challenges due to cross-linking and degradation. Specialized extraction kits incorporating enzymatic digestion and purification steps help maximize DNA integrity. Fluorometric quantification methods, such as Qubit assays, ensure accurate DNA concentration measurement for reaction setup.

Primer design is crucial. The anchored primer must be positioned within a well-characterized genomic region to provide a stable starting point, while the secondary primer strategy—whether random priming, template switching, or ligation-mediated amplification—must be validated to capture a broad range of structural variants. Computational tools such as Primer3 or NCBI Primer-BLAST aid in designing primers with optimal melting temperatures and minimal secondary structures, reducing off-target amplification. Pilot experiments using control DNA samples with known rearrangements help refine primer efficiency before applying AMP to clinical or research samples.

Reaction conditions must balance specificity and sensitivity. Polymerase selection is crucial, as proofreading enzymes reduce sequencing errors while maintaining robust amplification across complex genomic regions. Thermal cycling parameters, including annealing temperature and extension time, are adjusted based on primer characteristics and target fragment size. A two-step or nested PCR approach may enhance specificity, particularly when working with low-input or degraded DNA. Incorporating unique molecular identifiers (UMIs) at the library preparation stage mitigates PCR bias and sequencing errors by enabling bioinformatic correction of duplicate reads.

Differences From Conventional Multiplex Methods

Traditional multiplex PCR amplifies multiple target sequences using specific primer pairs designed for known genomic regions. While effective for detecting predefined mutations, it requires both forward and reverse primers, limiting its ability to identify structural variants with unknown breakpoints. AMP addresses this limitation by using a single fixed primer with a secondary primer that enables amplification of variable or unknown sequences.

This flexibility is particularly useful in clinical oncology, where gene rearrangements influence diagnosis and treatment. For example, in non-small cell lung cancer (NSCLC), conventional multiplex PCR struggles to identify ALK or ROS1 rearrangements if the precise fusion partners are unknown. AMP anchors amplification to a well-characterized gene region while allowing the second primer to capture diverse fusion partners. This improves sensitivity in detecting rare or novel variants, making AMP a preferred method for precision oncology applications.

Uses In Targeted Next-Generation Sequencing

AMP is a powerful tool in targeted next-generation sequencing (NGS), particularly for detecting structural variants and gene rearrangements that conventional approaches may miss. By enabling amplification of unknown fusion partners and complex genetic alterations, AMP enhances the resolution of sequencing assays used in oncology, inherited genetic disorders, and infectious disease research.

A primary use of AMP in targeted NGS is detecting oncogenic gene fusions, common in cancers such as lung, thyroid, and hematologic malignancies. Traditional NGS panels often struggle to identify fusion breakpoints when the partner gene is unknown or highly variable. AMP overcomes this challenge by anchoring amplification to a known exon of a frequently rearranged gene, allowing for unbiased capture of its fusion counterpart. In NSCLC, AMP-based assays have demonstrated superior sensitivity in identifying ALK, ROS1, RET, and NTRK fusions, which are critical biomarkers for selecting targeted therapies such as crizotinib and entrectinib. AMP-based sequencing can detect fusions in as little as 10 ng of RNA, making it suitable for clinical samples with limited or degraded material.

Beyond oncology, AMP is increasingly applied in diagnosing inherited genetic disorders involving large deletions, duplications, or complex rearrangements. Many traditional PCR-based methods lack the flexibility to capture these structural variations, leading to missed diagnoses. AMP allows for the amplification of breakpoints without prior knowledge of both primer binding sites, making it particularly useful in conditions such as Duchenne muscular dystrophy and congenital syndromes. Additionally, in infectious disease research, AMP has been adapted for pathogen detection and antimicrobial resistance profiling, where genomic variability among bacterial and viral strains complicates standard PCR-based identification methods.