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Chapter 9 DNA-Based Information Technologies

Chapter 9 DNA-Based Information Technologies . B. Hakan Atak Alperen Çağatay Serdaroğlu Zeynep Ulupınar Ece Makbule Dörtkardeşler Mehmet Dinçer İnan Nimet Yazıcı. BIOL - 306. CONTENTS. 9.1 DNA Cloning: The Basics 9.2 From Genes to Genomes 9.3 From Genomes to Proteomes

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Chapter 9 DNA-Based Information Technologies

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  1. Chapter 9DNA-Based Information Technologies B. Hakan Atak Alperen Çağatay Serdaroğlu Zeynep Ulupınar Ece Makbule Dörtkardeşler Mehmet Dinçer İnan Nimet Yazıcı BIOL - 306

  2. CONTENTS • 9.1 DNA Cloning: The Basics • 9.2 From Genes to Genomes • 9.3 From Genomes to Proteomes • 9.4 Genome Alterations and New Products of Biotechnology BIOL - 306

  3. DNA cloningtechnologythat is fundamentalto the advance of modern biological sciences, definingpresent and future biochemical frontiers and illustratingmany important principles of biochemistry. • The real implications of the biochemical journeybegun in the nineteenth century are found in the everincreasingpower to analyze and alter living systems. BIOL - 306

  4. To understand a complex biological process, a biochemistisolates and studies the individual componentsin vitro,then pieces together the parts to get a coherentpicture of the overall process. A major source of molecularinsights is the cell’s own information archive, itsDNA. The sheer size of chromosomes, however, presentsan enormous challenge: how does one find andstudy a particular gene among the tens of thousands ofgenes nested in the billions of base pairs of a mammaliangenome? Solutions began to emerge in the 1970s. BIOL - 306

  5. Paul Berg Herbert Boyer Stanley N. Cohen BIOL - 306

  6. Techniques for DNA cloning paved the wayto the modern fields of genomics andproteomics, thestudy of genes and proteins on the scale of whole cellsandorganisms. BIOL - 306

  7. 9.1 DNA Cloning : The Basics • DNA is the keeper of the all the informationneeded to recreate an organism. • A clone is an identical copy. • DNA cloning involves separating a specific gene or DNA segmentfrom a larger chromosome, attaching it to a smallmolecule of carrier DNA, and then replicating this modifiedDNA thousands or millions of times through bothan increase in cell number and the creation of multiplecopies of the cloned DNA in each cell. BIOL - 306

  8. 9.1 DNA Cloning : The Basics Cloning of DNA from any organism entails five generalprocedures: 1. Cutting DNA at precise locations.Sequence-specificendonucleases (restrictionendonucleases) providethenecessarymolecularscissors. 2. Selecting a small molecule of DNAcapable of self-replication. TheseDNAs are called cloning vectors (a vector is adelivery agent). They are typically plasmids orviralDNAs. BIOL - 306

  9. 9.1 DNA Cloning : The Basics 3. Joining two DNA fragments covalently. The enzyme DNA ligase links the cloning vector and DNA to be cloned. Composite DNA molecules comprising covalently linked segments from two or more sources are called recombinant DNAs. 4. Moving recombinant DNA from the test tube to a host cell that will provide the enzymatic machineryfor DNA replication. 5. Selecting or identifying host cells that contain recombinant DNA. BIOL - 306

  10. 9.1 DNA Cloning : The Basics The methods used to accomplish these and related tasksare collectively referred to as recombinant DNA technology or, more informally, genetic engineering. BIOL - 306

  11. 9.1 DNA Cloning : The Basics Escherichia coli the firstorganismused for recombinant DNA work and still themostcommonhostcell. Plasmidsand bacteriophages(bacterial viruses; also calledphages), are well characterized; and techniques areavailable for moving DNA expeditiously from one bacterialcelltoanother. BIOL - 306

  12. 9.1 DNA Cloning : The Basics Restriction Endonucleases and DNALigase Yield Recombinant DNA BIOL - 306

  13. 9.1 DNA Cloning : The Basics Restrictionendonucleasesrestriction enzymes • recognize andcleave DNA at specific DNA sequences to generate a set of smallerfragments. The DNA fragment to be cloned canbe joined to a suitable cloning vector by using DNA ligasesto link the DNA molecules together.The recombinant vector is then introduced into a host cell, whichamplifies the fragment in the course of many generations of celldivision. BIOL - 306

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  15. 9.1 DNA Cloning : The Basics Restriction endonucleases are found in a wide range of bacterial species. Werner Arber discovered in the early 1960s. They recognizeand cleave foreign DNA (the DNA of an infecting virus,for example); such DNA is said to be restricted. In thehost cell’s DNA, the sequence that would be recognizedby its own restriction endonuclease is protected fromdigestion by methylation of the DNA, catalyzed by a specificDNA methylase. The restriction endonuclease andthe corresponding methylase are sometimes referred to as a restriction-modificationsystem. BIOL - 306

  16. 9.1 DNA Cloning : The Basics There are three types of restrictionendonucleases,designated I, II, and III. Types I and III are generally large,multisubunit complexes containing both the endonuclease and methylase activities. BIOL - 306

  17. 9.1 DNA Cloning : The Basics BIOL - 306

  18. 9.1 DNA Cloning : The Basics BIOL - 306

  19. 9.1 DNA Cloning : The Basics Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to four nucleotidesof one strand unpaired at each resulting end. These unpaired strands are referred to as sticky ends, because they can base-pair with each other or with complementary sticky ends of other DNA fragments.Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called bluntends. BIOL - 306

  20. 9.1 DNA Cloning : The Basics BIOL - 306

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  22. 9.1 DNA Cloning : The Basics Researchers can create new DNA sequencesby inserting synthetic DNA fragments whicharecalled linkersbetween the ends that are being ligated. Inserted DNAfragmentswithmultiplerecognitionsequencesforrestrictionenzymes arecalledpolylinkers. BIOL - 306

  23. 9.1 DNA Cloning : The Basics CloningVectorsAllowAmplification of Inserted DNA Segments BIOL - 306

  24. 9.1 DNA Cloning : The Basics Plasmids are circular DNA molecules thatreplicate separately from the host chromosome. Naturallyoccurring bacterial plasmids range in size from5,000 to 400,000 bp. They can be introduced into bacterialcells by a process called transformation. BIOL - 306

  25. 9.1 DNA Cloning : The Basics Investigators have developed many different plasmidvectors suitable for cloning bymodifying naturallyoccurring plasmids. The E. coli plasmid pBR322 offersa good example of the features useful in a cloning vector. BIOL - 306

  26. 9.1 DNA Cloning : The Basics BIOL - 306

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  30. 9.1 DNA Cloning : The Basics Bacteriophages Bacteriophage has a very efficientmechanism for delivering its 48,502 bp of DNA into abacterium, and it can be used as a vector to clone somewhatlarger DNA segments. Two key featurescontributetoitsutility: BIOL - 306

  31. 9.1 DNA Cloning : The Basics 1. About one-third of the genome is nonessentialand can be replaced with foreign DNA. 2. DNA is packaged into infectious phageparticlesonly if it is between 40,000 and 53,000 bp long, aconstraint that can be used to ensure packaging of recombinant DNA only. BIOL - 306

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  33. 9.1 DNA Cloning : The Basics BIOL - 306

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  36. 9.1 DNA Cloning : The Basics BIOL - 306

  37. 9.1 DNA Cloning : The Basics • Research work with large genomes and the associatedneed for high-capacity cloning vectors led to thedevelopment of yeast artificial chromosomes. YAC vectors contain all the elementsneeded to maintain a eukaryotic chromosome in theyeast nucleus: a yeast origin of replication, two selectablemarkers, and specialized sequences needed for stability andproper segregation of the chromosomes at cell division. BIOL - 306

  38. 9.1 DNA Cloning : The Basics Genomic fragments are then separated by pulsed field gel electrophoresis,a variation of gel electrophoresis thatallows the separation of very large DNA segments. BIOL - 306

  39. 9.1 DNA Cloning : The Basics Specific DNA Sequences Are Detectable byHybridization BIOL - 306

  40. 9.1 DNA Cloning : The Basics DNA hybridizationis the most commonsequence-based processfor detecting aparticular gene or segment of nucleicacid.There are many variations of the basic method, most making use of a labeled (such asradioactive) DNAor RNA fragment, known as a probe, complementary tothe DNA beingsought. BIOL - 306

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  42. 9.1 DNA Cloning : The Basics A common limiting step in detecting and cloning agene is the generation of a complementary strand ofnucleic acid to use as a probe. The origin of a probe dependson what is known about the gene under investigation.Sometimes a homologous gene cloned fromanother species makes a suitable probe. Or, if the proteinproduct of a gene has been purified, probes can bedesigned and synthesized by working backward from theamino acid sequence, deducing the DNA sequence thatwould code for it. Now, researchers typicallyobtain the necessary DNA sequence information fromsequence databases that detail the structure of millionsof genes from a wide range of organisms. BIOL - 306

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  45. 9.1 DNA Cloning : The Basics Expression of Cloned Genes Produces LargeQuantities of Protein BIOL - 306

  46. 9.1 DNA Cloning : The Basics Most eukaryotic genes lack the DNA sequenceelements—such as promoters, sequences that instruct RNApolymerase where to bind—required for their expressionin E. coli cells, so bacterial regulatory sequences fortranscription and translation must be inserted at appropriatepositions relative to the eukaryotic gene in thevector DNA. BIOL - 306

  47. 9.1 DNA Cloning : The Basics • Cloning vectors with the transcription and translationsignals needed for the regulated expression of acloned gene are often called expression vectors.The rate of expression of the cloned gene is controlled byreplacing the gene’s own promoter and regulatorysequenceswith more efficient and convenient versions supplied by the vector. BIOL - 306

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  49. 9.1 DNA Cloning : The Basics Alterations in Cloned Genes Produce ModifiedProteins BIOL - 306

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