• Title/Summary/Keyword: DNA oligonucleotides

Search Result 103, Processing Time 0.028 seconds

Sequence Verification of Synthetic Oligonucleotides by Exonuclease Digestion and Matrix Assisted Laser Desorption Ionization Mass Spectrometry

  • Kim, Jin-Sung;Jang, Jung-Suk;Choi, Jong-Soon;Chang, Yoon-Seok
    • BMB Reports
    • /
    • v.29 no.2
    • /
    • pp.122-126
    • /
    • 1996
  • A series of oligonucleotides were synthesized by automatic DNA synthesizer. The purity of crude products was checked and their molecular weights determined by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) with an accuracy of better than 0.05% deviation even without using an internal standard. This mass determining technology in combination with partial digestion of oligonucleotides by 5'- and 3'-exonuclease provides a straightforward and simple method to obtain sequence information of oligonucleotides. The extension of this technology to the sequencing of modified oligonucleotides and genomic DNA and RNA might become possible.

  • PDF

A Highly Effective and Long-Lasting Inhibition of miRNAs with PNA-Based Antisense Oligonucleotides

  • Oh, Su Young;Ju, YeongSoon;Park, Heekyung
    • Molecules and Cells
    • /
    • v.28 no.4
    • /
    • pp.341-345
    • /
    • 2009
  • MiRNAs are non-coding RNAs that play a role in the regulation of major processes. The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for the characterization and subsequent therapeutic targeting of miRNA function. Recent advances in ASO chemistry have been used to increase both the resistance to nucleases and the target affinity and specificity of these ASOs. Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity to DNA or RNA than natural nucleic acids and are resistant to nucleases, which is an essential characteristic for a miRNA inhibitor that will be exposed to serum and cellular nucleases. For increasing cell penetration, PNAs were conjugated with cell penetrating peptides (CPPs) at N-terminal. Among the tested CPPs, Tat-modified peptide-conjugated PNAs have most effective function for miRNA inhibition. PNA-based ASO was more effective miRNA inhibitor than other DNA-based ASOs and did not show cytotoxicity at concentration up to 1,000 nM. The effects of PNA-based ASOs were shown to persist for 9 days. Also, PNA-based ASOs showed considerable stability at storage temperature. These results suggest that PNA-based ASOs are more effective ASOs of miRNA than DNA-based ASOs and PNA-based ASO technology, compared with other technologies used to inhibit miRNA activity can be an effective tool for investigating miRNA functions.

Diversity of Repetitive Sequences in Toxigenic Cyanobacteria Detected by Repetitive Oligonucleotides-Primed PCR (반복염기 프라이머 PCR에 의해 탐색된 독성 남조류에 분포한 반복염기의 다양성)

  • Koo, Jung-Mo;Yoo, Soon-Ae;Park, Sang-Ho;Choi, Chang-Won
    • Korean Journal of Ecology and Environment
    • /
    • v.33 no.3 s.91
    • /
    • pp.206-212
    • /
    • 2000
  • Since some cyanobacterial isolates under selective culturing conditions are lacking of characteristic specialized cells or showing altered morphology, the morpho-taxonomic criteria are not accurate enough to discriminate between species. Instead of morphological parameters, a method based on the single or the combination of repetitive oligonucleotides in a single PCR, repetitive oligonucleotides-primed PCR (ROP-PCR), was applied to generate DNA profiles for members of the cyanobacterial genera Anabaena and Oscillatoria, both of which are responsible for causing poisonous blooms in various freshwater systems. ROP-PCR performed on 10 isolates of the cyanobacteria with ERIC and REP sequences from gram-negative bacteria, STRR1A and LTRR sequences derived from cyanobacterial genome, and eukaryotic repetitive sequences, led to the identification of distinct genotypes, and provided specific and repeatable DNA fingerprints for cyanobacterial isolates. Grouping analysis of cyanobacterial isolates showed a signifiant difference depending on the primer used in PCR.

  • PDF

Synthesis and Characterization of Oligonucleotides Containing Site-Specific Bulky $N^2$-Aralkylated Guanines and $N^6$-Aralkylated Adenines

  • Moon, Ki-Young;Kim, Yeong-Shik
    • Archives of Pharmacal Research
    • /
    • v.23 no.2
    • /
    • pp.139-146
    • /
    • 2000
  • 7- Bromomethylbenz[a]anthracene is a known mutagen and carcinogen. The two major DNA adducts produced by this carcinogen, i.e., $N^2$-(benz[a]anthracen-7-yl methyl)-2'-deoxyguanosine (2, b[a]$a^2$G) and $N^6$-(benz[a]anthracen-7-ylmethyl)-2'-deoxyadenosine (4, b[a]$a^6$/A), as wel 1 as the simpler benzylated analogs,$N^2$-benzyl-2'deoxyguanosine (1, $bn^2$G) and $N^6$-benzyl-2'-deoxyadenosine (3, $bn^6$/A), were prepared by direct aralkylation of 2'-deoxyguanosine and 2'-deoxyadenosine. To determine the site-specific mutagenicity of these bulky exocyclic amino-substituted adducts, the suitably protected nucleosides were incorporated into 16-base oligodeoxyribonucleotides in place of a normal guanine or adenine residues which respectively are part of the ATG initiation codon for the lac Z' \alpha-complementation gene by using an in situ activation approach and automated phosphite triester synthetic methods. The base composition and the incorporation of the bulky adducts into synthetic oligonucleotides were characterized after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis.

  • PDF

A Study on Match and Mismatch DNA Hybridization properties Using DNA Hybridization Detection Sensor (DNA Hybridization 검출 센서를 이용한 매치 및 미스매치 DNA hybridization 특성 연구)

  • Kim, Do-Kyun;Kwon, Young-Soo
    • Proceedings of the KIEE Conference
    • /
    • 2003.10a
    • /
    • pp.89-91
    • /
    • 2003
  • The determination of DNA hybridization reaction can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and other application area. So, the improvement of DNA detection system is very important for the determination of this hybridization reaction. In this study, we report the characterization of the probe and target oligonucleotide hybridization reaction using the evanescent field microscopy. First, we have fabricated DNA chip microarray. The particles which were immobilized oligonucleotides were arranged by the random fluidic self-assembly on the pattern chips, using hydrophobic interaction. Second, we have detected DNA hybridization reaction using evanescent field microscopy. The 5'-biotinylated probe oligonucleotides were immobilized on the surface of DNA chip microarray and the hybridization reaction with the Rhodamine conjugated target oligonucleotide was excited fluorescence generated on the evanescent field microscopy. In the foundation of this result, we could be employed as the basis of a probe olidonucleotide, capable of detecting the target oligonucleotide and monitoring it in a large analyte concentration range and various mismatching condition.

  • PDF

Real-Time Detection of DNA Hybridization Assay by Using Evanescent Field Microscopy

  • Kim, Do-Kyun;Choi, Yong-Sung;Murakami, Yuji;Tamiya, Eiichi;Kwon, Young-Soo
    • KIEE International Transactions on Electrophysics and Applications
    • /
    • v.11C no.3
    • /
    • pp.85-90
    • /
    • 2001
  • The determination of DNA hybridization reaction can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and other application area. So, the improvement of DNA detection system is very important for the determination of this hybridization reaction. In this study, we report the characterization of the probe and target oligonucleotide hybridization reaction using the evanescent field microscopy. First, we have fabricated DNA chip microarray. The particles which were immobilized oligonucleotides were arranged by the random fluidic self-assembly on the pattern chips, using hydrophobic interaction. Second, we have detected DNA hybridization reaction using evanescent field microscopy. The 5'-biotinylated probe oligonucleotides were immobilized on the surface of DNA chip microarray and the hybridization reaction with the Rhodamine conjugated target oligonucleotide was excited fluorescence generated on the evanescent field microscopy. In the foundation of this result, we could be employed as the basis of a probe olidonucleotide, capable of detecting the target oligonucleotide and monitoring it in a large analyte concentration range and various mismatching condition.

  • PDF

A Simple and Economical Short-oligonucleotide-based Approach to shRNA Generation

  • Kim, Jin-Su;Kim, Hyuk-Min;Lee, Yoon-Soo;Yang, Kyung-Bae;Byun, Sang-Won;Han, Kyu-Hyung
    • BMB Reports
    • /
    • v.39 no.3
    • /
    • pp.329-334
    • /
    • 2006
  • RNAi (RNA interference) has become a popular means of knocking down a specific gene in vivo. The most common approach involves the use of chemically synthesized short interfering RNAs (siRNAs), which are relatively easy and fast to use, but which are costly and have only transient effects. These limitations can be overcome by using short hairpin RNA (shRNA) expression vectors. However, current methods of generating shRNA expression vectors require either the synthesis of long (50-70 nt) costly oligonucleotides or multi-step processes. To overcome this drawback, we have developed a one-step short-oligonucleotides-based method with preparation costs of only 15% of those of the conventional methods used to obtain essentially the same DNA fragment encoding shRNA. Sequences containing 19 bases homologous to target genes were synthesized as 17- and 31-nt DNA oligonucleotides and used to construct shRNA expression vectors. Using these plasmids, we were able to effectively silence target genes. Because our method relies on the onestep ligation of short oligonucleotides, it is simple, less error-prone, and economical.

Fabrication of Mesoporous Hollow TiO2 Microcapsules for Application as a DNA Separator

  • Jeon, Sang Gweon;Yang, Jin Young;Park, Keun Woo;Kim, Geon-Joong
    • Bulletin of the Korean Chemical Society
    • /
    • v.35 no.12
    • /
    • pp.3583-3589
    • /
    • 2014
  • This study evaluated a simple and useful route to the synthesis of mesoporous $TiO_2$ microcapsules with a hollow macro-core structure. A hydrophilic precursor sol containing the surfactants in the hydrophobic solvents was deposited on PMMA polymer surfaces modified by non-thermal plasma to produce mesoporous shells after calcination. The surface of the PMMA polymer spheres was coated with $NH_4F$ and CTAB to control the interfacial properties and promote the subsequent deposition of inorganic sols. These hollow type mesoporous $TiO_2$ microcapsules could be applied as an efficient substrate for the immobilization of DNA oligonucleotides.

Detection and Analysis of DNA Hybridization Characteristics by using Thermodynamic Method (열역학법을 이용한 DNA hybridization 특성 검출 및 해석)

  • Kim, Do-Gyun;Gwon, Yeong-Su
    • The Transactions of the Korean Institute of Electrical Engineers C
    • /
    • v.51 no.6
    • /
    • pp.265-270
    • /
    • 2002
  • The determination of DNA hybridization reaction can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and application area. So, the improvement of DNA hybridization detection method is very important for the determination of this hybridization reaction. Several molecular biological techniques require accurate predictions of matched versus mismatched hybridization thermodynamics, such as PCR, sequencing by hybridization, gene diagnostics and antisense oligonucleotide probes. In addition, recent developments of oligonucleotide chip arrays as means for biochemical assays and DNA sequencing requires accurate knowledge of hybridization thermodynamics and population ratios at matched and mismatched target sites. In this study, we report the characteristics of the probe and matched, mismatched target oligonucleotide hybridization reaction using thermodynamic method. Thermodynamic of 5 oligonucleotides with central and terminal mismatch sequences were obtained by measured UV-absorbance as a function of temperature. The data show that the nearest-neighbor base-pair model is adequate for predicting thermodynamics of oligonucleotides with average deviations for $\Delta$H$^{0}$ , $\Delta$S$^{0}$ , $\Delta$G$_{37}$ $^{0}$ and T$_{m}$, respectively.>$^{0}$ and T$_{m}$, respectively.