Identification and quantification of GC-rich oligodeoxynucleotides in tissue extracts by capillary gel electrophoresis

Noll, Bernhard; Debelak, Harald; Uhlmann, Eugen

doi:10.1016/j.jchromb.2006.09.046

Cited by 9 publications

(10 citation statements)

References 42 publications

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“…Heat and addition of denaturants to the separation buffer are often used to denature DNA molecules in the capillary [34]. In our separation system, the effect of increasing the temperature of the capillary during the separation was found helpful in the detection of ligation products (Fig.…”

Section: Characterization Of the Ligation Products With Cge-lifmentioning

confidence: 89%

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

García‐Cañas¹,

Mondello²,

Cifuentes³

2011

J of Separation Science

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New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.

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Section: Characterization Of the Ligation Products With Cge-lifmentioning

confidence: 89%

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

García‐Cañas¹,

Mondello²,

Cifuentes³

2011

J of Separation Science

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“…Recovery of phosphorothioate oligonucleotides were reported to be approximately 40% with the majority of the losses attributed to irreversible binding to the C 18 SPE column. To date, this procedure has been successfully applied to several studies and clinical trials (Geary et al ., ; Yu et al ., ; Shang et al ., ; Soucy et al ., ; Noll et al ., ; Graham et al ., ; Szekely et al ., ). Tissue extraction procedures in these studies were similar, except that a proteinase K digestion and a liquid–liquid extraction using phenol–chloroform (1:1 v/v) were added prior to solid‐phase extraction to remove the proteins from tissues more thoroughly (Geary et al ., ; Agrawal et al ., ).…”

Section: Sample Extractionmentioning

confidence: 99%

“…For this reason, hydrodynamic injections have been generally preferred for quantitative analysis (Guttman and Schwartz, ). However, more recent studies also showed that hydrodynamic injection is associated with several disadvantages compared with electrokinetic injection, such as smaller sample amount injected, lower reproducibility and difficulty of detection when applied to biological samples (Noll et al ., ). Most of the quantitative work of therapeutic oligonucleotides done so far utilizes ‘fixed’ gels as the sieving matrix owing to their higher resolution, leaving electrokinetic injection the only feasible sample introduction mode for most CGE applications.…”

Section: Sample Introductionmentioning

confidence: 97%

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Determination of therapeutic oligonucleotides using capillary gel electrophoresis

Chen

Bartlett

2011

Biomedical Chromatography

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Oligonucleotides have developed into highly versatile and selective therapeutics over the past 20 years. More than five discrete mechanisms of action have been reported and more than 10 different chemical modifications have been used to extend their in vivo half-life and reduce their toxicity. Capillary gel electrophoresis (CGE) has been used extensively for the quantitative analysis of oligonucleotide therapeutics in both preclinical and clinical studies since the 1990s. The success of CGE is based on its extraordinary resolving power, which allows for the simultaneous determination of the parent drug and its metabolites. More recently, capillary gel electrophoresis has seen renewed interest with the emergence of replaceable gels with single-base resolving power and new capillary electrophoresis-mass spectrometry interfaces. This review discusses the bioanalysis of therapeutic oligonucleotides showing the evolution of the field over the past two decades leading to the current new approaches. Included in this review are topics such as different gel types, sample introduction modes, sample extraction procedures, separation conditions and detection methods used in CGE, along with discussions of the successes and limitations associated with each.

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“…The metabolism of C-class CpG ODNs differs from that of B-class ODNs in that the usually observed degradation by 30-exonucleases is slowed down because of duplex formation of the 30-palindromic sequence [16,17]. The cancer and antiviral therapy can be conducted both as a monotherapy and combination therapies.…”

Section: Therapeutic Applications Of Cpg Oligonucleotidesmentioning

confidence: 99%

Immunostimulatory Oligonucleotides

Bodera

2011

IAD

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RNA-protein interactions have been characterized often. Above all, the proteins which are associated with the studied RNA should be precisely identified. The pharmaceutical compositions containing nucleic acids and/or other compounds can be administered by any suitable route for administering medications. The immunostimulatory oligonucleotides play the role of antisense drugs which are being researched to treat cancers (including lung cancer, colorectal carcinoma, pancreatic carcinoma, malignant glioma and malignant melanoma), diabetes, Amyotrophic lateral sclerosis (ALS), and diseases such as asthma and arthritis with an inflammatory component. The immunostimulatory oligonucleotides may contain one or more natural or unnatural amino acid residues which are connected to the polymer by peptide (amide) linkages. The vaccine against cancer which has been produced during this work can be prophylactic or therapeutic. Since most studies so far have been performed with first-generation antisense oligonucleotides (ODNs), it is interesting to observe how second-generation immune stimulatory drug candidates with enhanced potency and efficacy can further improve the utility of this class of therapeutic agents. The aim of this article is to review most significant patents on immunostimulatory oligonucleotides.

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Identification and quantification of GC-rich oligodeoxynucleotides in tissue extracts by capillary gel electrophoresis

Cited by 9 publications

References 42 publications

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

Determination of therapeutic oligonucleotides using capillary gel electrophoresis

Immunostimulatory Oligonucleotides

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