Naked-Eye Detection of Rare Point Mutations in DNA

Summary

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

Abstract

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAF^V600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.

The test detects 0.5% of BRAF^V600E in an excess of BRAF^WT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

Introduction

The purpose of the method is to detect underrepresented point mutations in a DNA sample with a minimally instrumented methodology and a naked-eye readout. The final aim is to have a proof-of-principle assay, suitable for future applications in rapid tests for the detection of somatic mutations in circulating cell-free DNA (ccf-DNA) (e.g., from blood biopsy samples) for the early diagnostics and monitoring of cancer¹. Cancer-related somatic mutations represent an important cancer biomarker² and are present in a minor (yet very variable)³ fraction of ccf-DNA, making their identification challenging<....

Protocol

1. Synthesis of gold nanoparticle probes

1. Synthesize 40 nm citrate capped gold nanoparticles, using two-steps standard seeding growth method as detailed below.
   1. Synthesize 15 nm citrate capped gold nanoparticles (AuNPs seeds) using the classical Turkevich–Frens method^13,14.
      1. Wash all glassware with aqua regia (HCl:HNO₃ in a 3:1 v/v ratio).
      2. Heat 250 mL of 0.25 mM HAuCl₄ to boil while unifor.......

Discussion

The core aspect of the method is the ability to discriminate a target DNA in the context of an excess of interfering non-target DNA, where target and non-target DNA only differ for one single nucleotide. Thus, the design of the probes and the hybridization conditions are critical to achieve a sensitive discrimination. The assay is designed to use universal colorimetric probes to be adapted to the detection of any point mutations of interest. However, it is possible that some minor optimization of the reaction conditions .......

Materials

Name	Company	Catalog Number	Comments
Bench Top Centrifuge- Allegra X 30	Beckman Coulter	A99473
DL-Dithiothreitol	Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany	D0632-25G
Dynabeads M-280 streptavidin paramagnetic microparticles	Invitrogen	11205D
Hydroxylamine sulfate	Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany	379913-25G
KDS 100 Legacy Syringe Pump	kdScientific	789100
NanoDrop OneC spectrophotometer	Thermo Fisher Scientific Inc.,Waltham, MA, USA)
Phosphate Buffered Saline	Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany	806552-500ML
Pierce™ TCEP-HCl, No-Weigh™ Format	Thermo Fisher Scientific Inc.,Waltham, MA, USA)	A35349
Polyethylene glycol 600	Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany	202401
PTFE 0,22 µm filters, Fluoropore	Millipore	FGLP04700
Quant-iT™ OliGreen™ ssDNA Assay Kit	Thermo Fisher Scientific Inc.,Waltham, MA, USA)	O11492
Sodium citrate dihydrate	Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany	W302600
Synthetic oligonucleotides	Integrated DNA Technologies, Inc. (IDT DNA)
Tetrachloroauric(III) acid	Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany	520918
Thiolated polyT DNA probes	Integrated DNA Technologies, Inc. (IDT DNA)
Transmission electron microscopy (TEM)	JEOL JEM 1011 microscope
Zetasizer Nano S - Dynamic Light Scattering System	Malvern Panalytical