Simple Multiplex Genotyping by Surface-Enhanced Resonance Raman Scattering

Abstract

The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.

Clinical SERS: are we there yet?

“…This not only substantially improves detection sensitivity, but also retains SERS's advantages of being highlymultiplexed as well as photo-stable [28]. As further demonstrated by Gambhir and coworkers, simultaneous detection of up to as many as ten differently SERS-labeled bio-analytes is possible with this particular immunoassay method [114]. These works, however, do not eliminate the common issues of material wastages (since at least two antibodies are required to detect each targeted antigen), multiple washing steps and long analysis time encountered in a conventional immunoassay [27].…”

Surface-enhanced Raman scattering as a detection technique for molecular diagnostics

“…These approaches all focused on using functionalized surfaces, which were then exposed to the DNA sample. In an alternative approach, the transconductance membrane gene for cystic fibrosis was detected using a multiplexed 'amplification refractory mutational system' assay and silver nanoparticles [16]. This allowed the detection of the two homozygotes and heterozygote using two differently labeled probes in the same reaction vessel.…”

Identification and characterization of pharmaceuticals using Raman and surface‐enhanced Raman scattering

“…A major challenge for the spectroscopy is the sequencing of the human genome, where SERS is already involved. 54 Basically, colloids, island films or roughened electrodes of noble metals exhibit the necessary morphological features that are essential for the enhancement of the Raman cross-section of the adsorbed molecule. The type and morphology of the metallic surface are responsible for the specific adsorption properties, 55 the enhancement of the Raman signal being largely dependent on the excitation frequency 56 and the chemical nature of the adsorbed species.…”