Direct Detection of Point Mutations in Nonamplified Human Genomic DNA

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

Read-trough efficiency at UGA, UAG and UAA premature stop codons mediated by Gentamicin.

<p>Yeast transformants with a plasmid harboring each of the nonsense or the correspondent sense codon, were prepared and cultivated in quadruplicates as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154260#pone.0154260.g004" target="_blank">Fig 4</a>), in the absence or presence of aminoglycoside gentamicin, added at 200 μg/ml (lanes 2, 5 and 8) or 400 μg/ml (lanes 3, 6 and 9. A) Shown is a representative image of yEGFP acquired by a Typhoon 9600 FLA after 19h incubation at 30°C related to a gentamicin mediated read-through assay at the UGA stop codon (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154260#pone.0154260.s002" target="_blank">S2 Fig</a>). B) Read-through percentage is calculated as described in the Materials and Methods. Data are expressed as mean values and indicated with standard deviation.</p

Read-through reporter systems.

<p>(A) Plasmids of the YEpGR series harboring the yEGFP and yEmRFP coding sequences separated by either an in frame sense or nonsense codon; (B) Constructs bearing the same yEmRFP and yEGFP ORFs cloned in the inverted order; (C) Fluorescence microscope images of yeast cells transformed with plasmids expressing a yEGFP-sense-yEmRFP construct (CGA) or yEmRFP-nonsense-yEGFP (UGA) configuration; (D) plasmids expressing a yEmRFP-sense-yEGFP (CGA) or yEmRFP-nonsense-yEGFP (UGA) read-through cassette.</p

Expression products of the YepRG-UGA and YepRG-CGA reporters in the wild type and Δ<i>upf1</i>, Δ<i>upf2</i> and Δ<i>upf3</i> strains.

<p>(A) Quantification (RT-qPCR) of full length yEmRFP-yEGFP mRNAs in WT and Δ<i>upf1</i>, Δ<i>upf2</i> and Δ<i>upf3</i> mutants transformed with YepRG-UGA or YepRG-CGA. The relative values (RFP-GFP mRNA/β-actin) are indicated with black boxes (UGA clones) and white boxes (CGA clones). (B) Western blotting with cellular extracts obtained from WT and upfs mutants transformants. Western blotting was performed using an antibody against RFP protein, detecting the full-length yEmRFP-yEGFP (blue arrowed) as well as truncated yEmRFP-stop (red arrowed) proteins. Yeast anti-Actin antibody was used for densitometry normalization (data not shown). UGA, clones containing the read-through UGA cassette; CGA, clones containing the sense control CGA cassette; V, clones containing the empty vector; M, markers.</p

List of primers.

<p>List of primers.</p

Read-through assay as a function of abolished NMD.

<p>WT and Δ<i>upf1</i>, Δ<i>upf2</i> and Δ<i>upf3</i> strains were transformed with the YepRG-UGA or YepRG-CGA reporters. A) Yeast transformants were inoculated incubated at 30°C for 24 h in microplate and analysed by the dual fluorescence scanner (Typhoon FLA9600) as described in the previous experiments. B) Read-through percentage was calculated as described in the Materials and Methods. Data are related to identical clones replicates and expressed as mean values and indicated with standard deviation. Wild type and upfs deleted strains are described in Materials and Methods.</p

List of primers employed to amplify and sequence the β-globin gene.

<p>List of primers employed to amplify and sequence the β-globin gene.</p

Read-through mediated by G418 determined by using a dual-laser fluorescence scanner.

<p>(A) Yeast strain CW04 was transformed with plasmids (YEpGR series) harboring the indicated read-through cassette UGA or the corresponding sense control CGA. Independent clones were inoculated in quadruplicates in 96 wells microplates to perform two read-through assays (RT1 and RT2). Geneticin (G418) was added at the concentrations indicated. Microplates OD was measured at 595 nm and fluorescence was acquired by using the dual-laser scanner Typhoon 8600 after 24h incubation at 30°C. (B) Read-through levels as a function of the presence of increasing concentrations of G418 were quantitate as described in the Materials and Methods. Quantitative data were obtained from two independent experiments and are expressed as mean values and indicated with standard deviation.</p

Allelic discrimination plots obtained from 94 total samples of genomic DNA.

<p>The genomic DNAs extracted from healthy donors, healthy carriers and β-thalassemia patients, were analyzed with β⁰39 (A), β⁺IVSI-110 (B), β⁺IVSI-6 (C), or β⁰IVSI-1 (D) genotyping assays. For each plot, the normalized end-point fluorescence (Rn) values generated by the VIC^®-labeled probe for the normal allele and the FAM^™-labeled probe for the mutated allele are reported along the x-axis and the y-axis, respectively.</p

List of oligonucleotides used as primers or probes for genotyping experiments.

<p>List of oligonucleotides used as primers or probes for genotyping experiments.</p