Gene cloning lecture notes 5 for 2010

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1. Gene CloningLecture 5 2. Recombinant DNA Technology- Recapã A set of techniques for recombining genesfrom different sources in vitro andtransferring the recombinant…
  • 1. Gene CloningLecture 5
  • 2. Recombinant DNA Technology- Recap• A set of techniques for recombining genesfrom different sources in vitro andtransferring the recombinant DNA into cellswhere it may be expressed.• Use of recombinant DNA techniques allowsmodern biotechnology to be more precise andsystematic than earlier research results.
  • 3. Recombinant DNA• It allows genes to be moved across speciesbarriers – hence a powerful tool• Our understanding of eukaryotic molecularbiology has been enhanced• It has been applied in the Human GenomeProject – to transcribe and translate the entirehuman genome in order to understand thehuman organism• The ultimate goal is the improvement of humanhealth
  • 4. Gene (DNA) Cloning• Recombinant DNA technology makes it possible to clonegenes for basic research and commercial applications• Recombinant DNA technology allows scientists to examinethe structure and function of the eukaryotic genomebecause it contains: Biochemical tools for the construction of recombinant DNA Methods for purifying DNA molecules and proteins of interest Vectors for carrying recombinant DNA into cells for replication Techniques for determination of nucleotide sequences of DNAmolecules
  • 5. Application of Recombinant DNA
  • 6. Restriction enzymes are used to makeRecombinant DNA• Restriction enzymes were first discovered in the 1960s• They occur naturally in bacteria where they protect thebacterium against foreign invading DNA from otherorganisms (e.g. Viruses or phages)• The foreign DNA is restricted by it being cut into smallsegments – thus restriction is the process of cutting offoreign DNA into small pieces.• Most restriction enzymes only recognise small shortsequences (4-8 nt) called recognition sites and only cutat specific points within these sequences
  • 7. Example of a recognition sequence
  • 8. Restriction enzymes• Recognition sequences are symmetric in that the same sequence ofnucleotides is found on both strands, but run in opposite directions• They usually cut phosphodiester bonds of both strands in a staggeredmanner, so that the resulting dsDNA fragments have single strandedends, referred to as sticky ends• The sticky ends form hydrogen-bonded base pairs with complementarysticky ends on other DNA molecules• These unions are temporary since they are only held by a few hydrogenbonds• The bonding is made permanent by DNA ligase, which catalyses theformation of covalent phosphodiester bonds• This result is the same as natural genetic recombination, the production ofrecombinant DNA – a DNA molecule carrying a new combination ofgenes
  • 9. Cloning Vectors• A Cloning Vector is a DNA molecule that cancarry foreign DNA into a cell and replicate inthe cell• The two most often used vectors arebacterial plasmids and viruses (phages)
  • 10. Cloning with Plasmid Vectors• Plasmids are circular, dsDNA molecules thatseparate from a cell’s chromosomal DNA.• They occur naturally in bacteria• Plasmids exist in parasitic or symbioticrelationship with their host cells. Their sizesrange from a few thousand bp to more that100kb.• They duplicate before every cell division.• During cell division, at least, one is segregatedinto each daughter cell.
  • 11. Cloning with Plasmid Vectors• Many plasmids also contain transfer genes coding forproteins that transfer a copy of the plasmid to otherhost cells of the same or related bacteria species, byconjugation (E. coli plasmids can be engineered for useas cloning vectors)• Plasmid most commonly used in recombinant DNAtechnology replicate in E. coli. These plasmids havebeen engineered to optimize their use as vectors inDNA cloning• Their length (circumference) is reduced to range form1.2 – 3 kb - much less than that occurring naturally in Ecoli plasmids
  • 12. Cloning with Plasmid Vectors• Most plasmid vectors contain little more than the essentialnucleotide sequences required for their use in DNA cloning.• They contain a replication origin (ORI), a drug-resistancegene, and a region in which exogenous DNA fragmentscan be inserted• The ORI is a specific DNA sequence of 50-100 bp that mustbe present in a plasmid for it to replicate.• Host enzymes bind to ORI, initiating replication of thecircular plasmid• Thus, any DNA sequence inserted into such a plasmid isreplicated along with the rest of the plasmid DNA, the basisof molecular DNA cloning.
  • 13. Cloning with Plasmid Vectors• Steps of recombinant plasmid formationProduction of restriction fragmentsLigation of restriction fragments to plasmid DNATransformation of antibiotic-sensitive E. coli cellswith recombinant plasmids.• Other cloning vectors are bacterial artificialchromosomes (BACs) and yeast artificialchromosomes (YACs)
  • 14. Cloning with Plasmid Vectors
  • 15. Plasmid Cloning• Remember the Genetic code is the same in allOrganisms• Plasmids commonly used in recombinant DNAtechnology replicate in E.coli• These plasmids have been engineered tooptimise their use as vectors for DNA cloning• Their length range from 1.2 to 3.0 kb• They contain a replication origin (ORI), a drugresistance gene and a region for insertion ofexogenous DNA
  • 16. Transformation of the host cell• Transformation is genetic alteration of a cellcaused by the uptake and expression offoreign DNA regardless of the mechanisminvolved• Transformation permits plasmid vectors to beintroduced into and expressed in E. coli cells• A plasmid vector must contain a drug-resistance gene coding for an enzyme thatinactivates a specific antibiotic in order to beuseful in DNA cloning.
  • 17. Transformation of the host cell• The ability to select transformed cells iscritical to DNA cloning because thetransformation of E.coli with isolated plasmidDNA is inefficient• The cells must be exposed divalent cationssuch as Ca2+to make the cells permeable• E.coli are treated with CaCl2 and mixed withplasmid vecotrs• Frequently, only 1 cell out of 10 000 or morecells beomes competent to take up foreignDNA
  • 18. Transformation of the host cell• Each cell takes up a single recombinant plasmid DNAmolecule• The treated cells are plated on a Petri dish ofnutrient agar containing the antibiotic• Only the transformed cells containing the antibiotic –resistance gene on the plasmid vector will survive• Thus all the plasmids in such a colony of selectedtransformed cells are descended from a singleplasmid taken up by cells that form the colony
  • 19. Isolation of DNA Fragment From a Mixture• The initial fragment of DNA inserted into theparental plasmid is referred to as cloned DNA since itcan be isolated from the cloned cells• DNA cloning allows a particular nucleotide sequenceto be isolated from a complex mixture of fragmentswith many different sequences.• For example, assume we have 4 different types ofDNA fragments each with a unique sequence.• Each fragment type is inserted alone into a plasmidsvector.
  • 20. Isolation of DNA Fragment From a Mixture• The resulting mixture of recombinant plasmids isincubated with E. coli cells treated with CaCl2.• The cells are then cultured on antibiotic selectiveplates. (e.g. plates containing ampicillin).• Each colony that develops arises from a single cellthat took up one or the other recombinant plasmids.• All the cells in a given colony thus carry the sameDNA fragment.
  • 21. Isolation of DNA Fragment From a Mixture• Overnight incubation of E. coli at 37°C producesvisible colonies.• These colonies are isolated from each other on theculture plate. Hence, copies of the DNA fragmentsin the original mixture are separated in theindividual colonies.• Therefore, DNA cloning is a powerful but simplemethod for purifying a particular DNA fragmentfrom a complex mixture of fragments andproducing large numbers of the fragment ofinterest.• Each transformed cell contains multiple copies of a
  • 22. Production of recombinant Plasmids• To clone specific DNA fragments in a plasmid (or anyother) vector, the fragments must be produced andthen inserted into the DNA vector.• Restriction enzymes and DNA ligases are utilized toproduce such recombinant cloning vector.• Restriction enzymes are bacterial enzymes thatrecognize specific 4 to 8 bp sequences (restrictionsites), and then cleave both DNA strands at this site.• These enzymes cut within the DNA molecule hencethey are called endonucleases, to distinguish themfrom exonucleases, which digest nucleic acids froman end.
  • 23. Production of recombinant Plasmids• Many restriction sites are short inverted repeatsequences. That is, the restriction site sequence isthe same on each DNA strand when read in the 5′→3 direction.′• The DNA digest (fragments) produced by restrictionenzymes are called restriction fragments.• Modification enzymes protect bacterial DNA fromcleavage by restriction enzymes, by adding a methylgroup to one or two bases usually within therestriction site.
  • 24. Production of recombinant Plasmids• Many restriction enzymes generate fragmentsthat have a single stranded tail at both ends.• These tails are complementary to those on allother fragments generated by the samerestriction enzyme.• These tails are referred to as sticky ends (alsocalled cohesive ends) and can transiently basepair at room temperature with those on otherDNA fragments generated with the samerestriction enzyme, regardless of the source ofthe DNA molecules.
  • 25. Production of recombinant Plasmids• The base paring of sticky ends permits DNA fromwidely different species to be ligated, formingchimeric molecules• Purified DNA ligase is used to covalently join theends of restriction fragments in vitro.• DNA ligase can catalyze the formation of a 5 →3′ ′phosphodiester bond between 3 - OH end of one′restriction fragment strand and the 5 - PO′ 4 end ofanother restriction fragment strand during the timethat the sticky ends are transiently base-paired.
  • 26. Production of recombinant Plasmids• Plasmids vectors containing a polylinker (ormultiple cloning site sequence) are commonlyused to produce recombinant plasmidscarrying exogenous DNA fragments.• Polylinkers are chemically synthesized andthen introduced into the plasmid vector.
  • 27. Production of recombinant Plasmids• Since the polylinker contains several differentrestriction sites, one of the restrictionenzymes whose recognition sites is in thepolylinker is used to cut both the plasmidmolecules and genomic DNA.• This generates singly cut plasmids andrestriction fragments with complementarysticky ends.
  • 28. Production of recombinant Plasmids• In the presence of DNA ligase, DNA fragmentsproduced with the same restriction enzymewill be inserted into the plasmid.• The ratio of DNA fragments to be inserted tocut vectors and other reaction conditions arechosen to maximize the insertion of onerestriction fragment per plasmid vector.
  • 29. Production of recombinant Plasmids• The recombinant plasmids produced in in vitroligation reactions are then used to transformantibiotic sensitive E. coli cells.• All cells in each antibiotic-resistant clone thatremains after selection contain plasmids withthe same inserted DNA fragment, butdifferent clones carry different fragments.
  • 30. Steps of transformation• Production of restriction fragments• Ligation of restriction fragments to plasmidDNA• Transformation of antibiotic-sensitive E. colicells with recombinant plasmids
  • 31. Identification of clonesMembrane hybridizationExpression cloningSouthern blotting
  • 32. Cloning with Bacteriophage)λ-Phage• Most cloning done with E. coli plasmidbecause of the relative simplicity of theprocedure• However, the number of individual clones thatcan be obtained by this method is limited by:(1)the low efficiency of E. coli transformationand (2) the small number of individualcolonies that be detected on a Petri dish.
  • 33. Cloning with Bacteriophage)λ-Phage• The limitations make plasmid cloning of allgenomic DNA of higher organisms impractical• For example, 1.5 x105clones carry 25 kb DNAfragments are required to represent the totalhuman genome• Cloning vectors from bacteriopage λ haveproved to be a more practical means for obtainingthe required number of clones to represent largegenomes
  • 34. Cloning with Bacteriophage)λ-Phage• Such a collection of λ clones that includes allthe DNA sequences of a given species is calleda genomic library.• A genomic library can be screened for λclones containing a sequence of interest.• Bacteriophage λ can be modified for use as acloning vector and assembled in vitro.
  • 35. Cloning with Bacteriophage)λ-Phage• A λ-phage virion has a head region, whichcontains the viral DNA.• It also has a tail region, which enables the λ-phage to infect E. coli host cells.• Only the λ DNA enters the cell when a λ virioninfects a host cell.• The viral DNA then undergoes either lytic orlysogenic growth.
  • 36. Cloning with Bacteriophage)λ-Phage• In lytic growth, the viral DNA is replicated andassembled into more than 100 progeny virionsin each infected cell.• This kills the cell in the process and releasesthe replicated virions.• In lysogenic growth, the viral DNA inserts inthe bacterial chromosome where it ispassively replicated along with the host-cellchromosome as the cell grows and divides.
  • 37. Cloning with Bacteriophage)λ-Phage• In the Lysogenic Cycle:Viral DNA merges with Cell DNA anddoes not destroy the cell.The Virus does not produce progeny.There are no symptoms of viralinfection.Temperate viral replication takesplace.
  • 38. Cloning with Bacteriophage)λ-Phage• In the Lytic Cycle:Viral DNA destroys Cell DNA, takes over cellfunctions and destroys the cell.The Virus replicates and produces progenyphages.There are symptoms of viral infection.Virulent viral infection takes place.
  • 39. Life cycles of Viruses
  • 40. Cloning with Bacteriophage)λ-Phage• The λ genes coding for the head and tailproteins as well as various proteins involved inthe lytic and lysogenic growth pathways areclustered in discrete regions of about 50kbviral genome.• The genes in the lysogenic pathway are notrelevant for use of bacteriophage λ as avector.
  • 41. Cloning with Bacteriophage)λ-PhageThe λ proteins, designated Nu1 and A, bind toCOS sites and direct insertion of the DNAbetween 2 adjacent COS sites into apreassembled head.
  • 42. Cloning with Bacteriophage)λ-Phage• Genes in the lysogenic pathway are notrelevant for use of bacteriophage λ as avector.• Hence, they are removed from the viral DNAand replaced with other DNA sequences ofinterest.• Up to 25 kb of foreign DNA then can be insertedinto λ genome, resulting in a recombinant DNA thatcan be packaged to form virions capable ofreplicating and forming plaques in E. coli host cells.
  • 43. Cloning with Bacteriophage)λ-Phage• The key to the high efficiency of λ-phage cloning isthe ability to assemble λ virions in vitro.• Viral heads and tails initially are assembledseparately from multiple copies of the variousproteins that compose these complex structures.• λ DNA replication in the host cell generatesconcatemers - multimeric DNA molecules thatconsist of multiple copies of the viral genome linkedend-to-end and separated at COS sites.
  • 44. COS sites are protein-bindingnucleotide sequences that occur oncein each copy of the λ genome.
  • 45. Cloning with Bacteriophage)λ-Phage• The host-cell chromosomal DNA is notinserted into the λ heads because it does notcontain copies of the COS sequence.• Only one λ DNA is inserted into apreassembled λ head.• After insertion of the λ DNA the preassembledtail is attached producing complete virions.
  • 46. • Preparation of recombinant infectious λvirions:The phage-assembly process is carried out invitro.E. coli cells are infected with a mutant defectivein A protein (one of the 2 proteins required forpackaging λ DNA into preassembled phage heads).These cells then accumulate Empty headsPreassembled tails also accumulate since theyonly attach to heads filled with DNA
  • 47. Cloning with Bacteriophage)λ-Phage• The cells are lysed experimentally and anextract containing high concentrations ofheads and tails is prepared.• When this extract is mixed with A protein andrecombinant λ DNA containing a COS site, theDNA is packaged into the empty heads.• The tails in the extract then combine with thefilled heads, yielding complete virions carryingthe recombinant λ DNA.
  • 48. Cloning with Bacteriophage)λ-Phage• The recombinant virions produced by thismethod are fully infectious and can efficientlyinfect E. coli cells.• Each virion particle binds to receptor on thesurface of a host cell and injects it packedrecombinant DNA into the cell.• This infection process is about 1000x moreefficient than transformation with plasmidvector..
  • 49. Cloning with Bacteriophage)λ-Phage• ≈106colonies per µg in plasmid transformationcompared to ≈ 109plaques representing λ-phage representing λ clones for λ-phagetransformation per µg of recombinant DNA• a collection of λ clones that includes all theDNA sequences of a given species is called agenomic library. A genomic library can bescreened for λ clones containing a sequenceof interest.
  • 50. Identification of a specific clone from a λ phage libraryby membrane hybridization to a radiolabeled probe• The position of the signal on the autoradiogramidentifies the desired plaque on the plate.• In practice, in the initial plating of a library theplaques are not allowed to develop to a visible size sothat up to 50,000 recombinants can be analyzed on asingle plate.• Phage particles from the identified region of the plateare isolated and replated at low density so that theplaques are well separated.• Then pure isolates can be obtained by repeating theplaque hybridization as shown in the figure.
  • 51. ANYQUESTIONS??????????ElseRead on Gene libraries
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