In this study we demonstrate to be able to identify point mutations in human genome by Surface Plasmon Resonance Imaging (SPR-I) without PCR amplification. The method is based on the hybridization of sonicated genomic DNA to PNA probes and enhancement of the signal using gold nanoparticles linked to a second ODN probe. The efficiency and specificity of the detection of the beta°39 thalassemia point mutation was demonstrated by using two PNA probes complementary to the normal sequence (PNA-N) and the sequence carrying the °39 thal mutation (PNA-M), respectively. A close sequence located in 5’ is recognized by a standard 11-mer oligonucleotide (5’-AGC AGC CTA AG-3’) linked to a gold nanoparticle. When a 50 fM solution of normal 35-mer 5’-CTT AGG CTG CTG GTG GTC TAC CCT TGG ACC CAG AG-3’ sequence is added and the procedure completed, the results obtained demonstrated increased SPR-I responses only when the 35-mer target is adsorbed to the surface site exposing the PNA-N probe. The method was validated by detecting DNAs isolated from homozygous beta°39/beta°39 and heterozygous beta°39/N thalassemic patients and from N/N unaffected subjects. The experiments were carried out by using 300 microL of the DNA solutions, corresponding to about 450 genomic DNA molecules. The results obtained demonstrated efficient and specific detection of the beta°39 mutations using unamplified genomic DNA.This allows us to conclude that unamplified genomic DNA from low numbers of cells (few hundreds) might be analyzed by using a very simple protocol, thus avoiding enzymatic treatment or complex thermal cycling. This method is very efficient and to the best of our knowledge is the first reporting the possible use of SPR-I in detecting human genetic mutations without PCR-mediated amplification, but simply using shared genomic human DNA. This is a very important issue in biomedicine and biotechnology.
Surface Plasmon Resonance Imaging (SPR-I), peptide nucleic acid (PNA) probes and nanoparticle-enhancement for PCR-free ultrasensitive detection of beta-thalassemia mutations in human genomic DNA
BREVEGLIERI, Giulia;BORGATTI, Monica;GAMBARI, Roberto
2010
Abstract
In this study we demonstrate to be able to identify point mutations in human genome by Surface Plasmon Resonance Imaging (SPR-I) without PCR amplification. The method is based on the hybridization of sonicated genomic DNA to PNA probes and enhancement of the signal using gold nanoparticles linked to a second ODN probe. The efficiency and specificity of the detection of the beta°39 thalassemia point mutation was demonstrated by using two PNA probes complementary to the normal sequence (PNA-N) and the sequence carrying the °39 thal mutation (PNA-M), respectively. A close sequence located in 5’ is recognized by a standard 11-mer oligonucleotide (5’-AGC AGC CTA AG-3’) linked to a gold nanoparticle. When a 50 fM solution of normal 35-mer 5’-CTT AGG CTG CTG GTG GTC TAC CCT TGG ACC CAG AG-3’ sequence is added and the procedure completed, the results obtained demonstrated increased SPR-I responses only when the 35-mer target is adsorbed to the surface site exposing the PNA-N probe. The method was validated by detecting DNAs isolated from homozygous beta°39/beta°39 and heterozygous beta°39/N thalassemic patients and from N/N unaffected subjects. The experiments were carried out by using 300 microL of the DNA solutions, corresponding to about 450 genomic DNA molecules. The results obtained demonstrated efficient and specific detection of the beta°39 mutations using unamplified genomic DNA.This allows us to conclude that unamplified genomic DNA from low numbers of cells (few hundreds) might be analyzed by using a very simple protocol, thus avoiding enzymatic treatment or complex thermal cycling. This method is very efficient and to the best of our knowledge is the first reporting the possible use of SPR-I in detecting human genetic mutations without PCR-mediated amplification, but simply using shared genomic human DNA. This is a very important issue in biomedicine and biotechnology.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
