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Ioana D 2010Recombinant DNA technology IAWhat is biotechnologyB Recombinant DNA technologyrestriction enzymes cloning vectors and hosts C Polymerase chain reactions PCRs sequence amplification and gel electrophoresis D Restriction mapping Northern blotting and DNA sequencing and analysis F Applications of biotechnologygenetic engineering gene therapy genetic testing What is biotechnologyAnalyse DNA sequences Measure gene expression Construct molecules Produce transgenic organisms Produce medicines drugs beer milk Bioremediation biological weapons Genetic engineering Gene therapy Genetic testing Recombinant DNA technologyrecombinant DNA refers to molecules produced by artificially joining DNA obtained from different biological sources The basic procedure for producing recombinant DNA involves generating specific DNA fragments using enzymes that recognize and cut restriction enzymesDNA molecules at specific nucleotide sequencesFragments produced by restriction enzymes are joined to other DNA molecules that serve as vectors or carrier molecules A vector joined to a DNA fragment is a recombinant DNA moleculethe recombinant DNA molecule is transferred to a host cell and within it the recombinant molecule replicates producing dozens of identical copiesclones host cell replicates passing on the recombinant DNA to their progeny creating a population of host cells each carrying copies of the cloned DNA sequencethe cloned DNA is recovered from the host cells purified and analyzedthe cloned DNA can then be transcribed the mRNA translated and the encoded gene products isolatedRestriction enzymes Ioana D 2010usually produced by bacteria as a defense mechanism against infection by viruses ie they degrade the DNA of the invading viruses a restriction enzyme binds to the DNA and recognizes a particular nucleotide sequence aka recognition sequenceMost recognition sequences are palindromic and restriction enzymes often cleave these sequences in an offset manner the nucleotide sequence reads the same on both strands of the DNA when red in the 5 to 3 direction EcoR1 HindIII BamHI are all restriction enzymes found in different organisms ECOR1 recognizes GAATTCWhen the enzyme recognize the sequence it makes a cut between G and A This is the first step for gene cloning and recombinant DNAie the fragments produced upon cleavage with a restriction enzyme have single stranded tails sticky ends that can base pair with complementary single stranded tails on other DNA fragmentsEnds are sticky because single stranded DNA wants to find its complement We must cut both molecules ie both the gene of interest as well as the vector molecule with the same restriction enzyme such that the matching sites can stick to each other DNA ligase joins restriction fragments covalently to produce intact DNA molecules Annealing allows recombinant DNA molecules to form by complementary base pairing The 2 strands are not covalently bonded initially DNA ligase is the one that seals the gaps covalently bonding the fragments to form the recombinant DNA moleculeVectors They are carrier DNA molecules that can replicate the inserted DNA fragmentsVectors must be able to independently replicate themselves and the fragment they carry once inside a host cellshould have several restriction enzyme cleavage sites to allow insertion of a DNA fragmentsFirst we cut the vector with restriction enzyme and then mixed with a pool of DNA fragments produced by cutting with the same enzyme Recombinant vectorvector carrying an insert ie a recombinant DNA molecule produced by joining DNA from 2 diff sourcesTo distinguish the host cells that have taken up vectors from host cells that havent the vector should carry a marker gene like an antibiotic resistance gene Types of vectors plasmids phage vectors cosmid vectors bacterial artificial chromosomes BAC expression vectors The use of each depends on the host cell they are able to enter the size of the insert they can carry etcAll vectors have 1 common feature they all have a polylinker region ie a region with many different sites for restriction enzymes If we want to put an insert into that plasmid the plasmid needs to a be cut with the same restriction enzymePlasmids
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