Temperature- and flow-enhanced detection specificity of mutated DNA against the wild type with reporter microspheres.

  title={Temperature- and flow-enhanced detection specificity of mutated DNA against the wild type with reporter microspheres.},
  author={Ceyhun E. Kirimli and Wei-Heng Shih and Wan Y. Shih},
  journal={The Analyst},
  volume={138 20},
Detection of mutated (MT) deoxyribonucleic acid (DNA) amongst the wild type (WT) requires the probe DNA (pDNA) that is complementary to the MT to discriminate the WT by one or two nucleotide mismatches. Traditionally this is achieved by raising the temperature to above the melting temperature (Tm) of the WT (TWT) but below that of the MT (TMT). However, a raised temperature is also accompanied by a weakened binding of the MT to the pDNA which can reduce the detection sensitivity. In this study… 
7 Citations

Figures and Tables from this paper

Piezoelectric Plate Sensor (PEPS) for Analysis of Specific KRAS Point Mutations at Low Copy Number in Urine Without DNA Isolation or Amplification.
Under these conditions PEPS was shown to specifically detect KRAS MT in situ within 30 min with an analytical sensitivity of 60 copies/mL in a clinically relevant background of WT with concentrations 1000-fold greater than that of MT without DNA isolation, amplification, or labeling.
Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type.
Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies per mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling.
Specific in situ hepatitis B viral double mutation (HBVDM) detection in urine with 60 copies ml(-1) analytical sensitivity in a background of 250-fold wild type without DNA isolation and amplification.
In situ detection of hepatitis B virus 1762T/1764A double mutation (HBVDM) in urine using a (PMN-PT) piezoelectric plate sensor coated with a 16-nucleotide (nt) probe DNA (pDNA) complementary to the HBVDM is examined.
DNA hybridization detection with 100 zM sensitivity using piezoelectric plate sensors with an improved noise-reduction algorithm.
Real-time, in situ hybridization detection of target DNA (tDNA) in a buffer solution and in urine using 8 μm-thick lead magnesium niobate-lead titanate (PMN-PT) piezoelectric plate sensors (PEPSs) and a new multiple-parabola (>50) resonance peak position fitting algorithm is examined.


35S-labelled probes improve detection of mismatched base pairs by chemical cleavage.
A refinement of this method in the detection of a 13-thalassaemia mutation utilizing 35S-labelled probes rather than 32P-lab labelled probes is reported.
DNA melting analysis for detection of single nucleotide polymorphisms.
DNA melting analysis (DM) was used successfully for variant detection, and two previously unknown SNPs were discovered by this approach, making it highly suitable for the large-scale detection of sequence variants.
Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP.
RNA-SSCP was generally superior to SSCP, especially for the 307 bp segment, and the abundance of transcript produced as a result of rSSCP allows the rapid, nonradioactive detection of mutations by staining the gel with ethidium bromide.
Surface Structure and Coverage of an Oligonucleotide Probe Tethered onto a Gold Substrate and Its Hybridization Efficiency for a Polynucleotide Target
Atomic force microscopy and flow-injection quartz crystal microbalance were used in tandem to study the immobilization of the probe, to estimate the extent and efficiency of the hybridization of M13 phage DNA, and to examine the effect of using a different alkanethiol to reorient the preformed film for a higher hybridization efficiency.
Electrochemical quantitation of DNA immobilized on gold.
The hybridization efficiency of immobilized single-stranded DNA to complementary strands as a function of the immobilized DNA surface density is measured and it is found that it exhibits a maximum with increasing surface density.
Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.
Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes.
Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis.
High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques.
Design of LNA probes that improve mismatch discrimination
Forescence experiments using 2-aminopurine suggest that LNA modifications enhance base stacking of perfectly matched base pairs and decrease stabilizing stacking interactions of mismatched base pairs, and new guidelines are suggested for design of LNA probes, which significantly improve mismatch discrimination in comparison with unmodified DNA probes.
Label-free flow-enhanced specific detection of Bacillus anthracis using a piezoelectric microcantilever sensor.
The use of the same flow that is used for detection to further distinguish the specific binding (BA to anti-BA spore antibody) from nonspecific binding (BT, BC, and BS toAnti- BA spore antibodies) is unique and has great potential in the detection of general biological species.