Direct Detection of Point Mutations in Nonamplified Human Genomic DNA

Abstract

Ultrasensitive detection protocols not requiring polymerase chain reaction (PCR)-mediated target DNA amplification are expected to significantly improve our possibilities in several research and diagnostic applications for which minute cell quantities are available. For this reason we have tested a nanoparticle-enhanced surface plasmon resonance imaging (SPRI) sensing strategy to detect point mutations in nonamplified genomic DNA. We have used genomic DNAs, not subject to costly, time-consuming, and prone to contamination PCR-based amplification procedures, obtained from both healthy individuals and homozygous or heterozygous patients affected by β-thalassemia, in order to demonstrate the specificity and the sensitivity of the described sensing strategy. The assay we describe is ultrasensitive and convenient. Attomolar concentrations of target genomic DNA are detected, DNAs from healthy individuals and homozygous or heterozygous patients affected by β-thalassemia are discriminated, and only simple manipulations of the genetic samples are required before the analysis. The proposed ultrasensitive detection of DNA point mutations involved in genomic disorders possibly represents an important advantage in several biomedical applications.

Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

“…Biotinylated and unbiotinylated 11-mer oligo deoxyribonucleotide (BiotinDNA, 5’-AGCAGCCTAAG-3’-Biotin, and DNA, 5’-AGCAGCCTAAG-3’, respectively. T m = 34.0 °C), successfully employed in previous studies for the detection of non-amplified human genomic DNA [13], were purchased from Thermo Fisher Scientific, Inc. Streptavidin from Streptomyces avidinii , provided in lyophilized form in 10 mM PBS, pH 7.4, was purchased from Invitrogen (Italy). Mixed cellulose ester membrane filters were purchased from Whatman (UK).…”

“…The ultrasensitive detection of nucleic acids has been recently achieved by using streptavidin (SA)-conjugated AuNPs and SPR imaging (SPRI) [11–12]. In this case, the enhanced sensitivity enables the detection of point mutations in non-amplified human genomic DNA with attomolar sensitivity [13], thus offering an excellent cost-effective alternative to time consuming and prone to sample contamination nucleic acid amplification protocols [14]. In this context, the interaction of SA-conjugated AuNPs with large DNA fragments immobilized on the surface of SPRI sensors has been hypothesized to induce an AuNP aggregation process which could contribute to further enhance the sensitivity of nanoparticle-enhanced SPRI DNA detection assays [12].…”

Recent patents and technology transfer for molecular diagnosis of β-thalassemia and other hemoglobinopathies

“…Finally the direct detection of b-thalassemia mutations in non-PCR-amplified human genomic DNA has been recently demonstrated by surface plasmon resonance imaging (SPR-I). Attomolar concentrations of target genomic DNA were detected from healthy individuals and homozygous or heterozygous patients affected by b-thalassemia, representing an important advantage in several biomedical applications, including prenatal diagnosis [99].…”

Artificial DNA and surface plasmon resonance

“…A similar detection strategy was successfully used for the direct detection of a point mutation related to the β-thalassemia disease in non-amplified genomic DNA obtained from blood samples 89 . In this case normal, homozygous and heterozygous genomic DNA samples were directly fluxed into each of six microchannels of the SPRi fluidic system in order to allow the direct interaction of each of the three samples with two different PNA probes complementary to the normal (PNA-N) and mutated (PNA-M) DNA sequences, respectively (Fig.…”