On-chip detection of a single nucleotide polymorphism without polymerase amplification - PubMed (original) (raw)
On-chip detection of a single nucleotide polymorphism without polymerase amplification
Jinhee Han et al. Nano Res. 2014.
Abstract
A nanoparticle-assembled photonic crystal (PC) array was used to detect single nucleotide polymorphism (SNP). The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair (bp) difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease (PKD) with a sensitivity of 0.9 fg/mL (50 aM)-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA (gDNA) extracted from the urine and blood of two PKD- wild type and three PKD positive cats. The standard curves for PKD positive (PKD+) and negative (PKD-) DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection (LOD) was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction (PCR). The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analysis is desirable.
Keywords: DNA; array; photonic crystal; polycystic kidney disease; real time polymerase chain reaction (PCR); single nucleotide polymorphisms.
Figures
Figure 1
Detection of synthesized DNA oligos with systematically varied number of bases complementary to the probe oligo in the nanowells. The sequence of the probe, target DNA oligos with different number of complementary bases (3, 6, 9, and 12), and negative control were respectively 5′-ATCCTTAAAAGGTGACA-3′, 5′-CGCGCGC
GAT
-3′, 5′-CGCG
AAGGAT
-3′, 5′-C
TTTAAGGAT
-3′, 5′-
CCTTTTAAGGAT
-3′, and 5′-GCGCGCGCGC-3′. Error bars are based on the standard deviations of four replicates. The dashed line represents the background signal.
Figure 2
The sequences of PKD positive (PKD+), negative (PKD−), probe DNA and signal probe DNA. PKD+ (57 bases) and PKD− (57 bases) DNA have a single polymorphism of cytosine (C) → adenine (A) transversion in exon 29. The synthesized target DNAs are conjugated with Alexa 532 for fluorescent detection. Probe DNA (20 bases) was designed and synthesized to discriminate a single mismatched base-pair (A and C) between PKD+ and PKD−. Signal probe DNA conjugated with Alexa 532 (20 bases) is used for second hybridization to emit the fluorescent signal.
Figure 3
Schematic of PKD SNPs detection on the nanoparticle-assembled PC array. The 40 nm-nanoparticles are coated with streptavidin and then mixed with biotinylated probe DNA. Nanoparticles—probe DNA are trapped into the nanowells of the array by using a electrophoretic particle entrapment system. Synthesized PKD+/PKD− target DNAs are conjugated with Alexa 532 for rapid detection after hybridization with probe DNA. For detection of SNP of feline urine/blood gDNA, second hybridization of signal probe DNA-Alexa 532 with the target DNA provided a fluorescence signal.
Figure 4
SNPs detection of the synthesized feline PKD+ and PKD− DNA on the PC array. Six different concentrations were detected: 0.9, 9, 90, 9 × 102, 9 × 103 and 9 × 104 fg/mL (5 × 10−5, 5 × 10−4, 5 × 10−3, 5 × 10−2, 5 × 10−1 and 5 pM). LODs were determined from the mean plus three standard deviations of the background noise. Error bars are based on the standard deviations of four replicates.
Figure 5
SNPs detection of the PKD+/PKD− gDNA extracted from feline urine. The samples were obtained from five Persian cats with known PKD positivity and negativity: Ken (+, •), Marcus (−, ■), Barbie (+, ▲), MJ (+, ×) and Polo (−, ◆). The dashed line indicates the mean plus three standard deviations of the background noise. The concentrations used for the standard curve: 5 × 10−4, 5 × 10−3, 5 × 10−1, and 50 ng/mL. The signal from target urine-gDNA of MJ, Barbie and Polo were compared to the standard curve created from the signals of Ken and Marcus. Error bar: standard deviation determined from three replicates.
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