scholarly journals Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type

The Analyst ◽  
2016 ◽  
Vol 141 (4) ◽  
pp. 1421-1433 ◽  
Author(s):  
Ceyhun E. Kirimli ◽  
Wei-Heng Shih ◽  
Wan Y. Shih
Keyword(s):  
Nucleic Acid ◽  
Point Mutation ◽  
Mutation Detection ◽  
Piezoelectric Plate ◽  
Locked Nucleic Acid ◽  
Wild Type ◽  
Single Nucleotide ◽  
Lna Probe

We have examined thein situdetection of a single-nucleotideKRASmutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35(PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to theKRASmutation.

scholarly journals Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

2009 ◽  
Vol 15 (12) ◽  
pp. 4009-4016 ◽  
Author(s):  
Nobutake Yamamichi ◽  
Ryoichi Shimomura ◽  
Ken-ichi Inada ◽  
Kouhei Sakurai ◽  
Takeshi Haraguchi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Hybridization Analysis ◽  
Locked Nucleic Acid ◽  
Cancer Development

scholarly journals Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

2006 ◽  
Vol 72 (8) ◽  
pp. 5311-5317 ◽  
Author(s):  
Kengo Kubota ◽  
Akiyoshi Ohashi ◽  
Hiroyuki Imachi ◽  
Hideki Harada
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescence Intensity ◽  
Dna Probes ◽  
Locked Nucleic Acid ◽  
Factors Affecting ◽  
Hybridization Efficiency ◽  
Probe Hybridization ◽  
Major Factors

ABSTRACT Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.

Distinguishing L from M photopigment coding sequences by hybridization to novel locked nucleic acid (LNA) oligonucleotide probes

2008 ◽  
Vol 25 (3) ◽  
pp. 283-287
Author(s):  
CHRISTINA PETTAN-BREWER ◽  
LI FU ◽  
SAMIR S. DEEB
Keyword(s):  
Nucleic Acid ◽  
Locked Nucleic Acid ◽  
Macaca Nemestrina ◽  
Oligonucleotide Probes ◽  
Coding Sequences ◽  
Cone Mosaic ◽  
Target Nucleic Acid ◽  
High Degree

Many attempts have been made over the years to distinguish human and primate L (long-wavelength sensitive) from M (middle-wavelength sensitive) cone photoreceptors using either immunohistochemistry or in situ hybridization. These attempts have been unsuccessful due to the very high degree of identity between the sequences of the L and M proteins and encoding mRNAs. The recent development of chemically modified oligonucleotide probes, referred to as locked nucleic acid (LNA) probes, has shown that they hybridize with much greater affinity and specificity to the target nucleic acid. This has greatly increased the potential for differentiating L from M cones by in situ hybridization. We have designed LNA oligonucleotide probes that are complementary to either the L or M coding sequences located in exon 5 of the Macaca nemestrina L and M pigment genes. We have shown that the LNA-M and LNA-L probes hybridize specifically to their respective target nucleic acid sequences in vitro. This result strongly suggests that these probes would be instrumental in rapidly distinguishing L from M cone in the entire retina, and in defining the cone mosaic during development and in adults.

scholarly journals High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules

2019 ◽  
Vol 116 (28) ◽  
pp. 13921-13926 ◽  
Author(s):  
Salvatore A. E. Marras ◽  
Yuri Bushkin ◽  
Sanjay Tyagi
Keyword(s):  
Single Molecule ◽  
Hybridization Chain Reaction ◽  
Wild Type ◽  
Allelic Discrimination ◽  
Single Nucleotide ◽  
Tissue Sections ◽  
Powerful Approach ◽  
Probe System ◽  
Single Nucleotide Variations

Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.

scholarly journals Locked Nucleic Acid Thymine Monomer Probe Identifies Four Single-Nucleotide Variants by Melting Temperature

2017 ◽  
Vol 112 (3) ◽  
pp. 331a-332a
Author(s):  
Judy M. Obliosca ◽  
Sara Y. Cheng ◽  
Yu-An Chen ◽  
Mariana F. Llanos ◽  
Yen-Liang Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Melting Temperature ◽  
Locked Nucleic Acid ◽  
Single Nucleotide Variants ◽  
Single Nucleotide

scholarly journals Specific in situ hepatitis B viral double mutation (HBVDM) detection in urine with 60 copies ml−1analytical sensitivity in a background of 250-fold wild type without DNA isolation and amplification

The Analyst ◽  
2015 ◽  
Vol 140 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
Ceyhun E. Kirimli ◽  
Wei-Heng Shih ◽  
Wan Y. Shih
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Isolation ◽  
Piezoelectric Plate ◽  
Wild Type ◽  
Double Mutation ◽  
B Virus

We have examinedin situdetection of hepatitis B virus 1762T/1764A double mutation (HBVDM) in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35(PMN-PT) piezoelectric plate sensor (PEPS) coated with a 16-nucleotide (nt) probe DNA (pDNA) complementary to the HBVDM.

scholarly journals Optimizing locked nucleic acid/2’-O-methyl-RNA fluorescence in situ hybridization (LNA/2’OMe-FISH) procedure for bacterial detection

PLoS ONE ◽  
2019 ◽  
Vol 14 (5) ◽  
pp. e0217689 ◽  
Author(s):  
Andreia S. Azevedo ◽  
Inês M. Sousa ◽  
Ricardo M. Fernandes ◽  
Nuno F. Azevedo ◽  
Carina Almeida
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescence In Situ Hybridization ◽  
Locked Nucleic Acid ◽  
Bacterial Detection

scholarly journals A Locked Nucleic Acid Probe Based on Selective Salt-Induced Effect Detects Single Nucleotide Polymorphisms

2015 ◽  
Vol 2015 ◽  
pp. 1-6
Author(s):  
Jing Zhang ◽  
Huizhe Wu ◽  
Qiuchen Chen ◽  
Pengfei Zhao ◽  
Haishan Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Nucleotide Polymorphisms ◽  
Room Temperature ◽  
Genetic Mutation ◽  
Locked Nucleic Acid ◽  
Nucleotide Polymorphisms ◽  
Buffer Solution ◽  
Single Nucleotide ◽  
Real Time Quantitative Pcr ◽  
Pcr Products

Detection of single based genetic mutation by using oligonucleotide probes is one of the common methods of detecting single nucleotide polymorphisms at known loci. In this paper, we demonstrated a hybridization system which included a buffer solution that produced selective salt-induced effect and a locked nucleic acid modified 12 nt oligonucleotide probe. The hybridization system is suitable for hybridization under room temperature. By using magnetic nanoparticles as carriers for PCR products, the SNPs (MDR1C3435T/A) from 45 volunteers were analyzed, and the results were consistent with the results from pyrophosphoric acid sequencing. The method presented in this paper differs from the traditional method of using molecular beacons to detect SNPs in that it is suitable for research institutions lacking real-time quantitative PCR detecting systems, to detect PCR products at room temperature.

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