Why restriction enzymes

Journal of Molecular Biology 51 , — Southern, E. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98 , — Restriction Enzymes. Genetic Mutation. Functions and Utility of Alu Jumping Genes. Transposons: The Jumping Genes. DNA Transcription. What is a Gene? Colinearity and Transcription Units. Copy Number Variation. Copy Number Variation and Genetic Disease.

Copy Number Variation and Human Disease. Tandem Repeats and Morphological Variation. Chemical Structure of RNA. Eukaryotic Genome Complexity. RNA Functions. Restriction Enzymes By: Leslie A.

Pray, Ph. Citation: Pray, L. Nature Education 1 1 Restriction enzymes are one of the most important tools in the recombinant DNA technology toolbox.

But how were these enzymes discovered? And what makes them so useful? Aa Aa Aa. When I come to the laboratory of my father, I usually see some plates lying on the tables. These plates contain colonies of bacteria. These colonies remind me of a city with many inhabitants. In each bacterium there is a king. He is very long, but skinny. The king has many servants. These are thick and short, almost like balls. My father calls the king DNA , and the servants enzymes.

My father has discovered a servant who serves as a pair of scissors. If a foreign king invades a bacterium, this servant can cut him in small fragments, but he does not do any harm to his own king.

Initial Steps in Restriction Enzyme Research. Figure 1. Figure Detail. Learning to Use Restriction Enzymes. Cutting with Restriction Enzymes. Figure 2. Recombining with Restriction Enzymes.

Figure 3. References and Recommended Reading Arber, W. Annual Review of Biochemistry 38 , — Brownlee, C. Journal of Bacteriology 64 , — Mertz, J. Journal of Molecular Biology 51 , — Southern, E.

Article History Close. Share Cancel. Revoke Cancel. Keywords Keywords for this Article. Save Cancel. Exposure of biotin-functionalized Ag nanotriangles to nM streptavidin SA caused a Comparison of the data with the theor. Several control expts. These include the following: 1 electrostatic binding of SA to a nonbiotinylated surface, 2 nonspecific interactions of prebiotinylated SA to a biotinylated surface, 3 nonspecific interactions of bovine serum albumin to a biotinylated surface, and 4 specific binding of anti-biotin to a biotinylated surface.

The LSPR nanobiosensor provides a pathway to ultrasensitive biodetection expts. Elsevier B. Recent reports have indicated that aberrant expression of microRNAs is highly correlated with occurrence of lung cancer.

Therefore, highly sensitive detection of lung cancer specific microRNAs provides an attractive approach in lung cancer early diagnostics. Herein, we designed 3D DNA origami structure that enables electrochem.

The top portion hybridized with the lung cancer correlated microRNA, while the bottom portion was self-assembled on gold disk electrode surface, which was modified with gold nanoparticles Au NPs and blocked with mercaptoethanol MCH.

The prepn. Under the optimal conditions, the developed genosensor had a detection limit of 10 pM and a good linearity with microRNA concn. Actuators, B , , DOI: A review. This review discusses main techniques and methods which use nanoscale materials for construction of electrochem. Described approaches include nanotube and nanoparticle-based electrodes relying on aligned nanotube arrays, direct electron transfer between biomol.

Specific issues related to electrochem. Various applications for genomic and proteomic anal. Nano Lett. We examine through anal. Specifically, we det. In all cases, sensor size and shape profoundly affect the total analyte flux. The calcns. We conclude that without directed transport of biomols. There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current assocd.

While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biol. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mech. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not obsd.

Instead, the protein analyte binds to a biochem. Three different mol. Acta , , DOI: Small , 1 , DOI: Analyst , , DOI: It is possible to generate sequences of oligomeric nucleic acids which will preferentially assoc.

These structures are predicted on the maximization of Watson-Crick base pairing and the lack of sequence symmetry customarily found in their analogs in living systems. Criteria which oligonucleotide sequences must fulfill to yield these junction structures are presented. The generable junctions are nexuses, from which double helices may emanate. Each junction may be treated as a macromol.

This covalent linkage can be done with enormous specificity, using sticky-ended ligation techniques. It appears to be possible to generate covalently joined 3-dimensional networks of nucleic acids which are periodic in connectivity and perhaps in space. Institute of Physics Publishing. Recent trends and challenges in the electrochem.

Basic criteria for electrochem. DNA biosensor technol. ACS Sens. Brown, Carl W. DNA nanostructures provide a reliable and predictable scaffold for precisely positioning fluorescent dyes to form energy transfer cascades. Furthermore, these structures and their attendant dye networks can be dynamically manipulated by biochem. However, the complexity of DNA structures that have undergone such types of manipulation for direct biosensing applications is quite limited.

Here, the authors study four different modification strategies to effect such dynamic manipulations using a DNA dendrimer scaffold as a testbed, and with applications to biosensing in mind. The dendrimer has a branching ratio that organizes the dyes into a FRET-based antenna in which excitonic energy generated on multiple initial Cy3 dyes displayed at the periphery is then transferred inward through Cy3.

Advantages of this design included good transfer efficiency, large spectral sepn. Of the approaches to structural rearrangement, the first two mechanisms employed either toehold-mediated strand displacement or strand replacement and their impact was mainly via direct transfer efficiency, while the other two were more global in their effect using either a belting mechanism or an 8-arm star nanostructure to compress the nanostructure and thereby modulate its spectral response through an enhancement in parallelism are considered.

The performance of these mechanisms, their ability to reset, and how they might be used in biosensing applications are discussed. ACS Appl. Interfaces , 9 , DOI: Science , , DOI: American Association for the Advancement of Science. We demonstrate the ability to engineer complex shapes that twist and curve at the nanoscale from DNA. Through programmable self-assembly, strands of DNA are directed to form a custom-shaped bundle of tightly crosslinked double helixes, arrayed in parallel to their helical axes.

Targeted insertions and deletions of base pairs cause the DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quant. We also combined multiple curved elements to build several different types of intricate nanostructures, such as a wireframe beach ball or square-toothed gears. Nature , , DOI: Douglas, Shawn M. Nature Publishing Group. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra.

Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase scaffold strand' that is folded into a flat array of antiparallel helixes by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helixes constrained to a honeycomb lattice.

We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concns.

We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manuf. DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute mol.

Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large nos.

Here, the authors demonstrate a simple soln. The authors test this hierarchical self-assembly concept with DNA mols. By controlling the flexibility and concn. The authors expect that this assembly strategy can be adapted to allow the fabrication of a range of relatively complex 3-dimensional structures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods.

The self-assembly of DNA mols. Here the author describe a simple method for folding long, single-stranded DNA mols. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over short oligonucleotide 'staple strands' to hold the scaffold in place.

Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly nm in diam. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces.

Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles which constitutes a megadalton mol. To systematically create complex yet reliable circuits, elec. The authors report the design and exptl. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement.

National Academy of Sciences. In the simplest version of this process, two stable species of DNA hairpins coexist in soln. The av.

Amplification of more diverse recognition events can be achieved by coupling HCR to aptamer triggers. This functionality allows DNA to act as an amplifying transducer for biosensing applications. Yurke, Bernard; Turberfield, Andrew J. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semi-conductor nanocrystals and metal wires.

As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possible - for example, a nanomech. Melting of chem. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as 'fuel'. The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addn.

Nature , , 73 DOI: DNA aptamers have been used to assemble DNA-functionalized gold nanoparticles to produce highly sensitive and selective colorimetric sensors with an instantaneous color response on addn. The general method has been shown for adenosine and cocaine, but should be applicable to any aptamer of choice.

A review on different recent approaches to tailor "smart" DNA nanostructures for autonomous activation of catalytic DNA cascades and their use for sensing, logic operations, and assembly of complex nanostructures.

Copper is a key metal ion both in environment monitoring and in biol. The substrate strand of the DNAzyme was labeled with a fluorophore on the 3'-end and a quencher on the 5'-end, and the enzyme strand was labeled with a second quencher on the 5'-end. Initially, the fluorescence was quenched. The DNAzyme catalytic beacon method demonstrated here can be applied to designing turn-on fluorescent sensors for other paramagnetic metal ions.

The authors designed rationally highly sensitive and selective beacon for Hg based on a uranium-specific DNAzyme. The optimal DNAzyme was labeled with fluorophores and quenchers to construct a catalytic beacon. The sensor has a detection limit of 2. It is also highly selective and is silent to any other metal ions with up to millimolar concn. The catalytic-beacon performance may be further improved by the incorporation of in vitro selections to optimize the allosteric interactions.

This work further demonstrated that DNAzymes are a great platform for metal sensing. A highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reported. It consists of a DNAzyme and its substrate that can hybridize to a 5'-thio-modified DNA attached to gold nanoparticles. The hybridization brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage, which prevents the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles.

Royal Society of Chemistry. The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer-DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials.

DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing. A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as mol. This hybridization ratio limits the gain of most approaches and thus their sensitivity.

Here the authors demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target mols. The exonuclease-amplified assay can detect target DNA at concns. DNA consists of two complementary strands of nucleotides that spiral around each other in a double helix.

Sma I is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end. Other restriction enzymes, like Eco RI , cut through the DNA strands at nucleotides that are not exactly opposite each other.

This creates DNA fragments with one nucleotide strand that overhangs at the end. This overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments. Add to collection. Go to full glossary Add 0 items to collection. Download 0 items.