Lambda exonuclease pre-treatment for improved DNA-chip performance depends on the relative probe-target position

Brinker, Anja; Schulze, Holger; Bachmann, Till T.; Möller, Robert

doi:10.1016/j.bios.2010.07.011

Cited by 21 publications

(19 citation statements)

References 24 publications

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“…We further evaluated the performance of enzymatically generated silver nanoparticle (EGNP) deposition by immobilizing biotinylated single‐stranded DNA in the hydrogels. As seen in Figure , the silver deposition as an alternative readout increases with higher concentrations of biotinylated DNA in the gel.…”

Section: Resultsmentioning

confidence: 99%

Easy Daylight Fabricated Hydrogel Array for Colorimetric DNA Analysis

Beyer

Pollok

Berg

et al. 2014

Macromolecular Bioscience

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The fabrication of 3D hydrogel microarrays for DNA analytics that allow simple visual signal readout for on-site applications is described. A convenient one-step polymerization of the hydrogel including in situ capture oligonucleotide immobilization is accomplished by using N,N'-dimethylacrylamide/polyethylene glycol (PEG1900 )-bisacrylamide monomers. The implementation of an acylphosphine-oxide photoinitiator even allows polymerization at daylight, whereas other approaches require exposure with light in the UV-range. This minimizes the risk of UV-caused DNA damages within the capture DNA-strand that could adversely affect the subsequent hybridization step. The porous network of these gel segments allows DNA as well as protein penetration. Thus, the successful in-gel DNA hybridization is monitored by the deposition of silver nanoparticles. These metal particles allow naked eye signal readout.

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Section: Resultsmentioning

confidence: 99%

Easy Daylight Fabricated Hydrogel Array for Colorimetric DNA Analysis

Beyer

Pollok

Berg

et al. 2014

Macromolecular Bioscience

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“…To enable the electrical detection, the reverse primer was labeled at the 5′ end with biotin and for the pretreatment with lambda exonuclease, the forward primer was labeled with phosphate on the 5′ end. Before hybridization, amplicons were digested with lambda exonuclease as described elsewhere 34. After the enzymatic digestion, the hybridization buffer containing 6×SSPE/0.04% SDS was added at a ratio of 1:2.…”

Section: Methodsmentioning

confidence: 99%

Chip‐based detection system for the on‐site analysis of animal diseases

Seise¹,

Brinker²,

Kretschmer³

et al. 2011

Engineering in Life Sciences

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A portable and robust system which is suitable for the automated analysis of DNA or RNA of selected pathogens such as foot‐and‐mouth disease virus (FMDV) is developed. The system incorporates a stationary PCR chip and is coupled with a DNA chip and an electrical detection for the sequence‐specific identification of the PCR products. The PCR chip represents a miniaturized form of the classical thermocyclers and enables a fast and sensitive amplification as well as labeling of specific DNA sequences with minimal space and energy requirements. The detection and identification of the PCR products is performed on a DNA chip with an electrical detection scheme. The combination of the two technologies allows a very fast and highly specific sequence‐based detection and differentiation of pathogens. Further, it combines the accuracy of sequence analysis with the speed of chip technologies. For the total analysis including DNA amplification and DNA detection, less than 2 h are required.

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“…Lastly, lambda exonuclease preferentially hydrolyzes the 5′ terminus phosphoryl strand of duplex DNA, while it displays greatly reduced activity for 5′ terminus hydroxyl strand of native and denatured DNA [19], [20]. Also, the enzyme does not cleave 5′- modified termini (digoxigenin, biotin and cyanine dyes) and has been used to prepare single-stranded DNA for hybridization assays [21]–[23], sequencing [24], [25] and systematic selection of aptamers (SELEX, Systematic Evolution of Ligands by EXponential enrichment) [26].…”

Section: Introductionmentioning

confidence: 99%

Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

2014

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Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

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Lambda exonuclease pre-treatment for improved DNA-chip performance depends on the relative probe-target position

Cited by 21 publications

References 24 publications

Easy Daylight Fabricated Hydrogel Array for Colorimetric DNA Analysis

Easy Daylight Fabricated Hydrogel Array for Colorimetric DNA Analysis

Chip‐based detection system for the on‐site analysis of animal diseases

Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

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