Detection of Single-Base Substitutions in Amplified Fragments via Ligation of a Tandem of Short Oligonucleotides in Solution and on a Solid Carrier

Ligation of a tandem of short oligonucleotides was proposed for detecting single-base substitutions in amplified DNA fragments. An octamer–tetramer–octamer tandem was ligated on a 20-mer template with T4 DNA ligase. As shown with radiolabeled oligonucleotides, the efficiency and selectivity of ligation did not change with an octamer linked to a water-soluble carrier based on polyethylene glycol (MPEG), while ligation was somewhat lower with the octamer immobilized on methacrylate beads (DMEG). In both cases, polymer attachment improved the discrimination of 20-mer templates with single-base substitutions in the binding site for the tetramer or for the immobilized octamer. Tandems with a radiolabeled or biotinylated component were also efficiently ligated on amplified DNA fragments. The data obtained with DNA fragments of HIV-1 strains bru and rf demonstrate the possibility of reliable detection of single-base substitutions via ligation of a tandem and colorimetric detection of the immobilized ligation product with the streptavidin–alkaline phosphatase technique.     

A new approach to revealing point mutations in DNA analyzed by colorimetric detection

A new approach to detection of point mutations in an amplified DNA was developed. The approach is based on highly selective ligation (T4 DNA ligase) of a tandem of short oligonucleotides one of which contains the biotin group. The ligation product is formed only when the hybridization complex DNA/tandem is formed and the tandem is perfect. The hybridization complex DNA/(biotinylated ligation product) was separated from the biotinylated component of the tandem by UV-immobilization of the reaction mixture on a nylon membrane. The immobilized hybridization complex was detected colorimetrically by a streptavidin-alkaline phosphatase cojugate with a chromogenic substrate.     

Mini-antisense Oligonucleotides

Abstract

A new strategy of selective DNA target modification was proposed. The using of reactive derivatives of short oligonucleotides in the presence of flanking effector pair allows one to modify DNA target only when the perfect complementary complex of DNA target and oligonucleotide tandem is formed.     

A New Approach to Detect a Particular DNA Sequence by UV-Immobilization of Its Hybridization Complex with a Highly Specific Probe Resulting from Ligation of a Tandem of Short Oligonucleotides in Solution

A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN₈ + pN₄ + pN₈ ^"(Bio)) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin–alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was fully complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.     

Synthesis of DNA Dumbbells: Chemical vs. Enzymatic Ligation of Self-Complementary Oligonucleotides

Abstract

The chemical (cyanogen bromide) and enzymatic (T4 DNA ligase) ligation of five different self-complementary oligonucleotide sequences which form 14-or 16-base pair dumbbells are described and compared here. A review of both chemical and enzymatic methods is presented; an improved enzymatic method is described as well. While both methods of ligation are effective, chemical ligation may be preferred since it is faster and less costly.     

A New Approach to Revealing Point Mutations in DNA Analyzed by Colorimetric Detection

A new approach to detection of point mutations in an amplified DNA was developed. The approach is based on highly selective ligation (T4 DNA ligase) of a tandem of short oligonucleotides one of which contains the biotin group. The ligation product is formed only when the hybridization complex DNA/tandem is formed and the tandem is perfect. The hybridization complex DNA/(biotinylated ligation product) was separated from the biotinylated component of the tandem by UV‐immobilization of the reaction mixture on a nylon membrane. The immobilized hybridization complex was detected colorimetrically by a streptavidin‐alkaline phosphatase cojugate with a chromogenic substrate.     

Inline index helped in cleaning up data contamination generated during library preparation and the subsequent steps

Background

High-throughput sequencing involves library preparation and amplification steps, which may induce contamination across samples or between samples and the environment.

Methods

We tested the effect of applying an inline-index strategy, in which DNA indices of 6 bp were added to both ends of the inserts at the ligation step of library prep for resolving the data contamination problem.

Results

Our results showed that the contamination ranged from 0.29 to 1.25% in one experiment and from 0.83 to 27.01% in the other. We also found that contamination could be environmental or from reagents besides cross-contamination between samples.

Conclusions

Inline-index method is a useful experimental design to clean up the data and address the contamination problem which has been plaguing high-throughput sequencing data in many applications.

     

The NMR Structure of Estrone (Es)-tethered Tandem DNA Duplex: [d(5′pCAGCp3′)-Es] + [Es-d(5′ pTCCA3′)]: d(5′ pTGGAGCTG3′)

Abstract

The solution structure of an estrone (Es)-tethered tandem DNA duplex consisting of two Estethered tetranucleotides and a target octameric DNA sequence is reported. The structure of this Es-tethered tandem duplex has been compared with a corresponding natural tandem duplex without estrones. The T_m of the 3′-Es-tethered tetranucleotide part of the tandem duplex increases by 5°C, whereas the T_m of the 5′-Es-tethered tetranucleotide part increases by 7°C, compared with the corresponding natural counterpart. The NMR structures of both the Es-tethered tandem duplex and the natural counterpart have been based on 24 experimental NMR constraints per residue. Despite the fact that there is considerable distortion at the junction of two Es-tethered tetranucleotides in the major groove of the Es-tethered DNA duplex compared to the natural counterpart, both duplexes do take up B-type DNA structures. It is likely that the spatial proximity of two Es residues, and the resulting hydrophobic interaction between them might be responsible for the increase of the thermal stability of the Es-tethered tandem duplex in comparison with the natural counterpart.     

Synthesis of Circular Oligodeoxynucleotide Conjugates VIA Transient Abasic Sites

Abstract

New chemical ligation or cyclisation reactions, using high reactivity of abasic sites with amines, are reported for the synthesis of oligonucleotide clamps and singlestranded circular oligonucleotides. Thermal denaturation experiments show that these molecules display very high binding affinities for complementary DNA oligomer by forming triple-helical complexes.     

Structurally Altered Substrates for DNA Topoisomerase. I. Effects of Inclusion of a Single 3′-Deoxynucleotide Within the Scissile Strand†

Abstract

A partial DNA duplex containing a high efficiency topoisomerase I cleavage site was substituted singly at each of three sites with 3′-deoxyadenosine. Depending on the site of substitution, the facility of the topoisomerase I-mediated cleavage or ligation reactions was altered. Inclusion of the modified nucleoside at the 5′-end of the acceptor oligonucleotide diminished the rate of religation following substrate cleavage by the enzyme.

     

Rational Methods of DNA Synthesis,Exemplified for the Preparation of Parts of the Human Proopiomelanocortin Gene

Abstract

Complete genes can now be prepared in single-batch procedures a) as one long oligonucleotide strand, b) as a number of simultaneously synthesized fragments. For further increase of overall efficiency DNA fragments terminated by a 3′-ribonucleoside can be joined by solid-phase single strand ligation using RNA ligase. This paper describes the application of these techniques to the preparation of parts of the human proopiomelanocortin gene.     

Autoligation of Oligonucleotides via Nucleophilic Substitution Reaction

Abstract

A Fast and efficient template - driven autoligation reaction between oligonucleotides derivatized with bromoacetyl and thiol groups at their opposing termini is described. The product of reaction is capable of forming a stable duplex with a complementary DNA strand.     

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.     

PNA∗Synthesis and Diagnostic Application

Abstract

An optimized automated PNA synthesis protocol is reported. Under optimal conditions the product yield of a test 17-mer PNA is approximately 90 %. The average coupling yield is 99.4 %. The synthesis strategy is Boc/Z. The protocol is developed in a 5 pmole scale but is easily scaled up to 10–50 μmole scale syntheses on the automated synthesizer (ABI 433A). DNA capture experiments by PNA was used to develop a method for PNA-mediated purification of genomic Chlamydia DNA from urine. This purification removed efficiently substances that impeded DNA amplification.

     

Measurement of oxidatively induced DNA damage and its repair,by mass spectrometric techniques

Abstract

Oxidatively induced damage caused by free radicals and other DNA-damaging agents generate a plethora of products in the DNA of living organisms. There is mounting evidence for the involvement of this type of damage in the etiology of numerous diseases including carcinogenesis. For a thorough understanding of the mechanisms, cellular repair, and biological consequences of DNA damage, accurate measurement of resulting products must be achieved. There are various analytical techniques, with their own advantages and drawbacks, which can be used for this purpose. Mass spectrometric techniques with isotope dilution, which include gas chromatography (GC) and liquid chromatography (LC), provide structural elucidation of products and ascertain accurate quantification, which are absolutely necessary for reliable measurement. Both gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), in single or tandem versions, have been used for the measurement of numerous DNA products such as sugar and base lesions, 8,5’-cyclopurine-2’-deoxynucleosides, base-base tandem lesions, and DNA-protein crosslinks, in vitro and in vivo. This article reviews these techniques and their applications in the measurement of oxidatively induced DNA damage and its repair.     

Mechanisms of free radical-induced damage to DNA

Abstract

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5′-cyclopurine-2′-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

Assessment of DNA damages in lymphocytes of agricultural workers exposed to pesticides by comet assay in a cross-sectional study

Purpose: To assess the predictive power of the comet assay in the context of occupational exposure to pesticides.

Materials and methods: The recruited subjects completed a structured questionnaire and gave a blood sample. Exposure to pesticides was measured by means of an algorithm based on Dosemeci’s work (Agricultural Health Study). Approximately 50 images were analyzed for each sample via fluorescence microscopy. The extent of DNA damage was estimated by tail moment (TM) and is the product of tail DNA (%) and tail Length.

Results: Crude significant risks (odds ratios, ORs) for values higher than the 75th percentile of TM were observed among the exposed subjects (score?>?1). The frequency of some confounding factors (sex, age and smoking) was significantly higher among the exposed workers. A significant dose–effect relationship was observed between TM and exposure score. Significant high-risk estimates (ORs), adjusted by the studied confounding factors, among exposure to pesticides and TM, % tail DNA and tail length were confirmed using unconditional logistic regression models.

Conclusions: The adjusted associations (ORs) between the comet parameters and exposure to pesticides were significant. The sensitivity of the comet test was low (41%), the specificity (89%) and the predictive positive value (0.77) were found acceptable.     

Nucleosides and Nucleotides. 165. Chemical Ligation of Oligodeoxynucleotides Having a Mercapto Group at the 5-Position Of 2′-Deoxyuridine via a Disulfide Bond #

Abstract

We describe the nonenzymatic ligation of oligodeoxynucleotides (ODNs) containing a mercapto group at the 5-position of 2′-deoxyuridine via a disulfide bond. Two ODNs containing different sequences were efficiently ligated in the presence of a template by this method.

     

A Serendipitous Synthesis of 8-Dimsyl-2′-deoxyguanosine

Abstract

A serendipitous synthesis of 8-dimsyl-dG (2) has been achieved along with the known 8-benzyloxy-dG (3) in a nucleophilic substitution reaction of 8-bromo-dG (1) with in situ generated dimsyl and benzyloxy sodium. Compound 3 was directly converted into the mutagenic oxidative DNA damage product, 8-oxo-dGTP (4).     

A New Cyclic Phosphoramidate D4T Prodrug Approach cycloAmb-D4T-Phosphoramidates

Abstract

A new potential phosphoramidate prodrug approach for d4T 1 is described. In hydrolyses studies the cycloAmb-d4T-phosphoramidates 2 and 3 proved to deliver d4TMP following a tandem reaction.