Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection.

Ana Lázaro,Luis A Tortajada-Genaro,Ángel Maquieira

doi:10.1021/acssensors.1c02220

Ana Lázaro, Luis A Tortajada-Genaro + Show 1 more

Open Access

https://doi.org/10.1021/acssensors.1c02220

Copy DOI

Abstract

Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands.

Highlights

Massive DNA testing requires novel technologies to support a sustainable health system
There have been two critical limitations: (i) given the structural complexity of genomic DNA, only short DNA sequences from clinical samples are amplified by hybridization chain reaction (HCR), such as microRNAs,[23] short gene sequences,[24] and specific circulating tumor DNA25 and (ii) none of the reported studies identify the change in a single nucleotide in gDNA due to the hybridization complexity associated with secondary structures of nucleic acids.[26]
■ RESULTS AND DISCUSSION Principle of the Genotyping of single-nucleotide variations (SNV) Based on ASHCR

Summary

Introduction

Massive DNA testing requires novel technologies to support a sustainable health system. C ertain changes in a specific position in the genome sequence, called single-nucleotide variations (SNV), are closely associated with genetic diseases and cancer.[1] disease-related SNVs serve as biomarkers for efficient clinical diagnosis and prognosis based on the genomic profile of primary tumors.[2] the genotyping of SNVs is difficult due to close molecular similarity and their presence at trace levels.[3] A specific copy number increase is required to detect and avoid inference from high-abundant variants.[4] The most widely used amplification methods are thermocycling techniques, including a polymerase reaction chain (PCR)[5] and a ligase chain reaction.[6] Despite their good performance, the requirement of sophisticated equipment considerably limits applications for point-of-care testing (POC).[7] Important signs of progress have been achieved in the last years, developing biosensing methods[8−10] and microfluidics-integrated sensors[11] with excellent single-base specificity. There have been two critical limitations: (i) given the structural complexity of genomic DNA (gDNA), only short DNA sequences from clinical samples are amplified by HCR, such as microRNAs,[23] short gene sequences,[24] and specific circulating tumor DNA25 and (ii) none of the reported studies identify the change in a single nucleotide in gDNA due to the hybridization complexity associated with secondary structures of nucleic acids.[26]

Methods

Results

Conclusion