Restriction endonuclease
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Restriction endonucleases are enzymes produced by bacteria that recognize specific sequences in foreign DNA and cut the DNA at or near the recognition sites. They function as part of the bacterial immune system to degrade foreign DNA. Restriction endonucleases cut DNA into fragments that have either blunt or sticky ends, depending on where they cut the DNA strands. They are named based on the bacteria that produces them and are classified into three types depending on their cleavage patterns. Type II restriction endonucleases cut directly at their recognition sites and produce fragments with sticky or blunt ends, making them useful for genetic engineering applications.
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Restriction enzyme
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
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Restriction enzyme
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
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MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
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Alkaline Phosphatase
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Nucleases
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Isocaudomers
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Restriction endonucleases are enzymes found in bacteria that recognize specific nucleotide sequences in DNA and cut the DNA at those sites. This acts as a defense mechanism for bacteria by destroying foreign DNA, such as that from viruses. There are several types of restriction endonucleases, but type II are most useful for genetic engineering as they cut DNA at predictable, site-specific locations. The resulting DNA fragments can be joined back together via DNA ligases. Restriction endonucleases have four- to six-letter recognition sequences that are palindromic, and they produce sticky or blunt ends depending on whether cuts are offset or at the same position.
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Restriction Endonuclease (Cutting of DNA)
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Nucleases
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Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
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Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
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Restriction enzymes are proteins isolated from bacteria that cleave DNA into fragments at specific recognition sites. They recognize short palindromic nucleotide sequences, usually 4-8 bases long, and cut through both strands of the DNA double helix. There are four main types of restriction enzymes that differ in their recognition sites and cleavage activities. Restriction enzymes are indispensable tools in recombinant DNA technology, as they allow DNA fragments to be cut and recombined in new configurations.
Restriction enzymes
This document discusses restriction enzymes and DNA ligases. It explains that restriction enzymes cut DNA at specific recognition sequences, producing sticky or blunt ends. DNA ligases then join the cut DNA fragments back together by forming phosphodiester bonds between the 3' hydroxyl and 5' phosphate ends. Restriction enzymes are classified into types I, II, and III, with type II being the most useful for molecular biology. Common restriction enzymes and their recognition sequences are also listed. DNA ligases require a cofactor like NAD+ or ATP to catalyze the ligation reaction.
Genetic engineering
This document summarizes recombinant DNA (rDNA) technology and genetic engineering. It discusses how restriction enzymes and DNA ligases are used to cut and paste DNA from different sources to create recombinant DNA molecules. It also describes genetically modified organisms (GMOs) and provides examples of transgenic organisms. Vectors such as plasmids are used to carry and replicate foreign DNA fragments in bacteria. Selection techniques like antibiotic resistance allow identification of transformed bacteria.
Enzymes used in RDT
DNA manipulative enzymes can be grouped into four classes: nucleases cut DNA, ligases join DNA, polymerases copy DNA, and modifying enzymes add or remove chemical groups from DNA. Restriction endonucleases are nucleases that cut DNA at specific recognition sequences and are important for gene cloning. They produce either blunt or sticky ends. DNA ligase joins DNA fragments together by forming phosphodiester bonds. Polymerases like Klenow fragment and Taq polymerase copy DNA. Modifying enzymes alter DNA through addition or removal of chemical groups from the DNA backbone. Together, these enzymes enable the precise cutting and joining of DNA required for gene cloning experiments.
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Ligases
DNA ligases mediate the sealing of single stranded breaks or gaps in DNA molecules by catalyzing the formation of phosphodiester bonds between DNA fragments, acting as molecular glue to join DNA fragments together. There are two main types of DNA ligases, E. coli ligase which requires NAD as a cofactor and T4 DNA ligase which uses ATP as a cofactor.
Application of biotechnology in agriculture
This document discusses the application of biotechnology in agriculture, focusing on genetically modified crops. It provides examples of GM crops including Bt cotton, Bt brinjal, and golden rice. Bt crops are engineered to produce insecticidal Cry proteins from Bacillus thuringiensis to resist pests. Golden rice is genetically modified to produce beta-carotene and address vitamin A deficiency. The document also discusses the Flavr Savr tomato, the first commercially available GM food, which was engineered for longer shelf life by inhibiting the polygalacturonase enzyme. Finally, it introduces the concept of edible vaccines, where plants are engineered to produce vaccine proteins that are consumed orally.
Application of biotechnology in forensic
DNA fingerprinting involves isolating DNA from a sample, cutting it into fragments using restriction enzymes, separating the fragments by size through gel electrophoresis, and comparing the unique band patterns to identify individuals. It has various forensic and medical uses such as identifying crime suspects by comparing DNA profiles, determining paternity in legal cases, and diagnosing inherited disorders. The chances of any two individuals having the exact same DNA profile are extremely low, making DNA fingerprinting a powerful tool for individual identification.
Application of biotechnology in medicine
This document discusses several applications of biotechnology in medicine, including the production of human insulin, human growth hormone, vaccines, and gene therapy. It provides details on how recombinant DNA technology is used to produce these therapeutic products. Human insulin is extracted from pancreas cells and inserted into bacterial plasmids to be mass produced. Vaccines like hepatitis B vaccine involve isolating antigen genes and expressing them in yeast or bacteria. Gene therapy approaches like ex vivo therapy aim to correct genetic disorders by isolating cells, adding functional genes, and reinserting the cells.
Methods of gene transfer
Gene transfer techniques can be direct or indirect. Direct techniques introduce foreign DNA into plant cells without a biological agent, using methods like microinjection, microprojectiles, protoplast fusion, electroporation, and polyethylene glycol treatment. Indirect gene transfer uses the bacterium Agrobacterium tumefaciens, which transfers DNA (T-DNA) from its tumor-inducing plasmid into the host plant genome, allowing genetic modification of plants. Techniques like bacterial transformation and transduction can also directly transfer genes between bacteria using viruses or naked DNA. Overall, a variety of methods have been developed to introduce foreign genes into organisms and achieve genetic modification.
Southern blotting
Southern blotting is a technique used to detect specific DNA sequences. It involves separating DNA fragments by size via gel electrophoresis, transferring them to a membrane, and using a labeled probe to hybridize to the target sequence. The probe binds only to complementary DNA fragments, allowing their detection and location on the membrane via autoradiography. Key steps include restriction enzyme digestion of DNA, gel electrophoresis, membrane transfer, probe hybridization, and x-ray film development to visualize hybridized fragments. Southern blotting is used for applications like mutation detection, gene rearrangement studies, and forensic analysis.
Polymerase chain reaction (pcr)
PCR is a technique used to amplify a specific segment of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA denatures, the primers anneal to the DNA, and the polymerase extends the primers to make copies of the DNA segment. This allows millions of copies of the target DNA sequence to be generated from a very small initial sample. PCR is widely used in applications like disease diagnosis, DNA fingerprinting, and molecular cloning.
Gene cloning
Gene cloning involves producing multiple copies of a gene using genetic engineering techniques. The main steps are: isolating the donor DNA fragment, inserting it into a vector, transforming host cells, and selecting recombinant cells containing the desired DNA insert. Gene cloning strategies include creating genomic DNA libraries, which contain all DNA from an organism's genome, and cDNA libraries, which contain only coding regions created from mRNA. Genomic libraries contain both coding and non-coding regions from genomes, while cDNA libraries contain only coding exons synthesized from mRNA using reverse transcriptase.
rDna technology
rDNA technology involves deliberately manipulating the genetic makeup of cells or organisms in a controlled way. Its major goals are to form gene libraries, isolate and amplify genes of interest, and alter genetic makeup and gene expression patterns. The key steps are isolating DNA from cells, fragmenting the DNA with restriction enzymes, amplifying the gene of interest with PCR, and inserting the recombinant DNA into a host with enzymes.
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Restriction endonuclease
- 1. BY MERIN ALICE RESTRICTION ENDONUCLEASES -MOLECULAR SCISSORS.
- 2. A restriction enzyme /restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Dna cutting enzymes/site specific bacterial endonucleases that recognize specific sequences of foreign Dna and cleave the Dna to fragments. Pioneer – Werner Arber; Discovered by Hamilton Smith & Daniel Nathans (1970) (1986 Nobel Prize in Physiology & Medicine)
- 3. Immune systems of prokaryotes’ Restriction enzymes are so called- they restrict or prevent the intrusion of foreign Dna from other species into genetic system of a cell. When foreign Dna gets to bacterial cells it disarms / degrade it recognize foreign Dna, bind with it slash it to pieces
- 4. Recognition sequences 4 to 6 bp long Recognized by Restriction endonucleases and produce a double-stranded cut in the DNA Palindromic – a region of nucleic acid which contain a pair of ‘inverted repeat’ sequences (nucleotide – pair sequences that read the same in both forward and back ward directions)
- 5. Naming of Restriction Endonucleases Consists of of three parts: 1. An abbreviation of the genus and species Use the first letter of the genus First two letters of the species that produces the enzymes (Eco for Escherichia coli). A letter, number or combination of the two to indicate the strain of relevant species. A Roman numeral to indicate the order in which different restriction modification systems were found in the same organism E Escherichia (Genus) co coli (species) R RY13 (strain) I First identified EcoRI
- 6. STICKY ENDS are staggered ends on a DNA molecule with short, single-stranded overhangs Produced by certain restriction endonucleases Can base pair with each other or with complementary sticky ends of other DNA fragments Eg: Cleavage of DNA with EcoRI G AATTC C TTAA G After cleavage, G AATTC C TTAA G
- 8. Blunt – ended Fragments Some restriction endonucleases cut both strands of DNA at the same place and produce blunt – ended fragments Eg: Recognition sequence and cleavage site of Pvu II CAG CTG GTC GAC After cleavage, CAG CTG GTC GAC
- 10. Types of Restriction Endonucleases TYPE I TYPEII TYPE III Random,cut- Several bases away from restriction site At restriction site cut the DNA at a specific point that is a measured distance (about 25 bp from the recognition sequence No site specific cleavage Site specific cleavage Not site specific Not suitable for genetic engineering Used Not used