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Chapt. 4 Molecular Cloning Methods Learning outcomes : Explain major tools of recombinant DNA technology date from 1970s Expand understanding of new tools Impt. Figs: 1, 2, 3, 4, 5, 10, 11, 12, 15*, 16, 17, 18, 20, 23, 24, 25, 26; Table 1 Review Q: 2, 4*, 6, 7, 8, 9*, 10, 11, 13*, 14*; AQ 1 4-1 Slide 2 4-2 4.1 Gene Cloning in Bacteria Fig. 4 Link prokaryotic or eukaryotic genes to small bacterial DNAs (plasmids or phage) Insert recombinant molecules into bacterial hosts to replicate Can produce large quantities of these genes (or proteins) in pure form Slide 3 4-3 Type II Restriction Endonucleases Discovered late 60s Named for organism Recognize specific DNA sequence, cut ONLY at that sequence Recognize 4-bp to 8-bp sequences Frequency of cuts less when recognition sequence is longer: (1/4) n (size of piece larger ~4 n ) Slide 4 4-4 Restriction Endonucleases, DNA Ligase Many make staggered cuts in DNA strands Single-stranded overhangs, sticky ends, can base-pair together (5-PO 4, 3-OH ends) Makes joining 2 different DNA molecules easier DNA ligase needs 5-PO 4, 3-OH ends + ATP Recognition sequence usually symmetrical palindromes EcoRI: 5-GAATTC-3 3-CTTAAG-5 Slide 5 4-5 Restriction-Modification System Enzymes cut foreign DNA, protect cells What prevents enzymes from cutting up host DNA? paired methylases recognize, methylate, same site Form restriction-modification system, R-M system Methylation protects DNA; after replication, parental strand is partially methylated Fig. 1 Slide 6 4-6 Early Gene Cloning recombinant DNA Boyer & Cohen used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of host chromosome Each plasmid had site for EcoRI Linear DNA with same sticky ends Sticky ends base pair Some ends re-close Others join 2 pieces DNA ligase joins 2 pieces with covalent bonds Transform E. coli; select drug r Fig. 2 Slide 7 4-7 Vectors Vectors function as DNA carriers, permit replication of recombinant DNAs Typically 1 vector, 1 piece of foreign DNA Need vector for replication Foreign DNA has no origin of replication, (site where DNA replication begins) Prokaryotic vectors: Plasmids; Phages Selectable marker, Restriction sites for cloning Origin of replication pBR322 Slide 8 4-8 pBR322 Cloning Clone foreign DNA into PstI site of pBR322 Cut vector to generate sticky ends with PstI Cut foreign DNA with PstI Combine vector and foreign DNA: DNA ligase seals Transform E. coli (CaCl 2, electroporate) Select Tet r Fig. 4 Slide 9 4-9 Screening Transformants pBR322 Transformation yields bacteria : Religated plasmid Religated insert Recombinants Identify transformants, recombinants with antibiotic resistance In tetracycline, only cells with plasmid grow, not just foreign DNA pieces Test copies of original colonies with ampicillin (kills cells with plasmid including foreign DNA) Identify desired recombinants Fig. 4 Slide 10 4-10 Screen With Replica Plating Transfer copies of clones from original tetracycline plate to plate with ampicillin Transfer with sterile velvet Desired recombinant colonies do NOT grow on ampicillin plate Pick clone from original plate Fig. 5 Slide 11 4-11 pUC plasmid has -galactosidase fragment Ampicillin resistance gene MCS in lacZ (N-terminal -peptide of -galactosidase) Foreign DNA in MCS disrupts ability to make -gal - peptide: On X-gal media: clones producing functional -gal enzyme (from and host peptide) produce blue colonies; clones with insert are white. Fig. 6 Slide 12 4-12 Summary Basic Plasmid Cloning Vectors pBR322 and the pUC plasmids pBR322 has 2 antibiotic resistance genes Many unique restriction sites for inserting foreign DNA Most of these sites interrupt antibiotic resistance, making screening straightforward pUC has Ampicillin resistance gene MCS that interrupts the -galactosidase gene -peptide MCS also facilitates directional cloning, using pieces cut with 2 different restriction enzymes Slide 13 4-13 Phages As Vectors Bacteriophages: natural vectors transduce bacterial DNA from one cell to another Phage vectors infect cells much more efficiently than plasmids transform cells Clones are not colonies of cells, but rather plaques, clearing of bacterial lawn due to phage infect, kill bacteria in area lambda Slide 14 4-14 Lambda ( Phage Vectors Replacement vectors: Removed middle region (needed only for lysogeny) Retained genes for phage replication Replace removed phage genes with foreign DNA Phage vectors hold larger amounts of foreign DNA Accept up to 20-kb of DNA Traditional plasmid vectors usually less Lambda phage vectors require minimum size DNA (12 kb) insert to package into phage particle Slide 15 4-15 Cloning Using a Lambda Phage Vector Fig. 8 Join DNA in vitro Add packaging proteins from cell extract Infect cells Slide 16 4-16 Genomic Libraries Large number of clones: all genes from species genome Restriction fragments can be packaged into phage, about 16 20 kb per fragment ( often partial digests ) Need thousands of clones for library: ex. Yeast genome 10 7 bp/ 10 4 bp/clone -> ____ clones ex. Human genome 3 x 10 9 bp /10 4 bp/clone -> ___ Once library is established, search for gene of interest ( BL 415 experiment with plasmid library ) Slide 17 4-17 Plaque Hybridization Search genomic library with probe to identify clone containing desired gene Ideal probe labeled nucleic acid with sequence matching the gene of interest Fig. 9 Slide 18 4-18 Cosmids Cosmids designed to clone large DNA fragments Behave as plasmid and phage Contain cos sites, cohesive ends of phage DNA, allow DNA to be packaged into phage head Plasmid origin of replication permits replication as a plasmid in bacteria Most genome removed so hold large inserts (40-50 kb) So little phage DNA, cant replicate like phage; infectious, carry recombinant DNA into bacterial cell, then plasmid Slide 19 4-19 M13 Phage Vectors Long, thin, filamentous phage M13 Normal size 6.4 kb (10 genes) Modified vector contains: Gene fragment with -galactosidase Multiple cloning site like pUC plasmid Advantages Can hold large DNA; length of phage increases Phage genome is single-stranded DNA Fragments cloned (into ds intracellular form) are recovered in single-stranded form Phage does not kill cells; extrudes particles Useful for sequencing DNA, mutagenesis Slide 20 4-20 M13 Cloning permits recovery of Single- stranded DNA After infecting E. coli, single- stranded phage DNA was converted to double-stranded replicative form (RF, plasmid-like) Purify replicative form to clone foreign DNA into MCS; ligate vitro Transform recombinant DNA into host cells, results in plasmid-like RF molecule in cells Phage particles, contain one single- stranded DNA (+ strand), secreted from transformed cells, can be collected from media Fig. 10 Slide 21 4-21 Phagemids are versatile Vectors: pBluescript (pBSK) Fig. 11 Aspects of both phages and plasmids : Amp r for selection of clones MCS inserted into lacZ gene permit blue/white screening Origin of replication of single-stranded phage f1 permits recovery of single-stranded recombinant DNA (like M13) MCS has phage RNA polymerase promoters, 1 on each side of MCS; permits specific RNA transcripts in vitro Slide 22 4-22 Eukaryotic Vectors, High Capacity vectors Plasmids and Viruses clone genes in eukaryotic cells Plasmid shuttle vectors (origins of replication for both bacterial and eukaryotic host cell; ex. Yeast YEP24 Selectable markers in both hosts; MCS sites Yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) clone huge pieces Vectors based on Ti plasmid for plant cells Slide 23 4-23 Identifying specific clone with specific probe Probes can identify a desired clone from among thousands of irrelevant ones: Two types widely used: Recognize gene or mRNA with Oligonucleotides: Short RNA or DNA sequences that hybridize Can use homologous gene from another organism If amino acid sequence is known, deduce possible nucleotide sequences to code for these amino acids Recognize specific protein product with Antibodies Slide 24 4-24 cDNA Cloning from Eukaryotes into Bacteria cDNA: complementary DNA or copy DNA made from mRNA A cDNA library: set of clones representing many mRNAs from cell type or tissue or time Library can contain thousands of different clones * Need cDNA clones because genomic clones have introns, arent expressed in bacteria Slide 25 4-25 Making cDNA Library from eukaryotic mRNA Fig. 12 Reverse Transcriptase (RT) mRNA -> DNA Primer oligo(dT) to polyA at 3 end RNAse H degrades RNA DNA polymerase Pol I finishes Terminal transferase to add C tails and G tails for cloning Slide 26 4-26 Vector choice for cDNA cloning Purpose and method to detect positive clones If cloning gene and small fragment, plasmid or phagemid like pUC or pBS used, with colony hybridization and labeled DNA probe If large fragments, use cosmids, or phage or YAC, BAC For expression of gene product, use cDNA of eukaryote cloned in pUC, phagemids of lambda under control of lac promoter for transcription and translation of gene; use hybridization, antibody screening (more later) Slide 27 4-27 Summary clone cDNA into Bacteria Make cDNA library with synthesis of cDNAs one strand at a time Use mRNAs from a cell as templates for 1 st strands, then 1 st strand as template for 2 nd Reverse transcriptase generates 1 st strand DNA polymerase I generates 2 nd strand Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector Use recombinant DNAs to transform bacteria Detect clones with: Colony hybridization using labeled probes Antibodies if gene product is translated Slide 28 4-28 Polymerase chain reaction (PCR): amplifies DNA exponentially; yields DNA fragments for cloning, forensics, diagnostics Fig. 15 Slide 29 4-29 Standard PCR Invented by Kary Mullis in 1980s DNA polymerase copies selected region of DNA Short DNA primers hybridize to DNA on either side of piece of interest initiate DNA synthesis through that area, X Selected region DNA doubles in amount each cycle Special heat-stable polymerases work after high temperatures that separate (denature) strands simplifies process ex. Taq polymerase Real-time PCR uses fluorescent dyes to quantify amplification, reflect amount of mRNA or DNA in cells (Fig. 17) Slide 30 4-30 Reverse transcriptase PCR (RT-PCR) to clone specific cDNA Start with mRNA, not dsDNA Convert mRNA to DNA with primer specific to gene Use forward primer to convert ssDNA to dsDNA Then standard PCR With primer choice, restriction enzyme sites added to cDNA 2 sticky ends permit directional cloning good for protein expression Fig. 16 Slide 31 4-31 4.3 Expressing Cloned Genes Cloned gene permits production of large quantities of particular DNA sequence Expression vectors yield protein products Strong promoter, ribosome binding site near ATG codon Make as pure protein, or as fusion to bacterial peptide Regulated expression: Inducible expression vectors keep cloned gene repressed until time to express: Large quantities of eukaryotic protein can be toxic Slide 32 4-32 Fusion proteins: Oligo-histidine (His-tagged) Expression Vector Sequence upstream of MCS encodes 6 His which get added to protein: Oligo-histidine high affinity for divalent metal ions (Ni 2+ ) Purify protein by nickel affinity chromatography His tag removed using enzyme enterokinase if needed Fig. 20 Slide 33 4-33 Fusion Proteins with lacZ Cloning vectors (pUC and pBS) are expression vectors using lac promoter with lacZ -peptide If inserted DNA in same reading frame as interrupted gene, fusion protein results Partial -galactosidase at N- end Inserted protein at C-end Fusions often more stable Fusions also assist detection, purification Fig. 18 Slide 34 4-34 Regulation: lac Promoter/ operator lac promoter inducible with lactose or synthetic IPTG; binds repressor and removes it from O Transcription stays off until induced Slide 35 4-35 Fusion Proteins also in gt11 vector Phage contains lac control region and lacZ gene ( - peptide) Products of gene correctly inserted are fusion proteins with - galactosidase peptide Use blue-white screening on host with -peptide Fusion reduces degradation of foreign protein Fig. 21 Slide 36 4-36 Antibody screening with gt11 (or plasmids) Lambda phages with cDNA inserts on E. coli Proteins released are blotted onto membrane Probe with specific 1 o antibody to protein Antibody bound to protein from plaque is detected with labeled 2 o antibody Fig. 22 Slide 37 4-37 Summary Expression in Bacteria Expression vectors can produce fusion proteins One part from coding sequences in vector Other part from sequences in cloned gene Many fusion proteins can be simply purified by affinity chromatography (His-tag; Flag-tag; TAP-tag) Fusion proteins can be detected with antibodies after blotting of colonies or plaques: Slide 38 4-38 Bacterial Expression System Shortcomings Bacteria recognize proteins as foreign and destroy Posttranslational modifications (glycosylation, phosphorylation) are not done correctly Bacterial cytoplasm not permit correct protein folding High levels of cloned eukaryotic proteins can be expressed in useless, insoluble (inclusion) form Slide 39 4-39 Use Eukaryotic Expression Systems Initial cloning in E. coli using shuttle vector, that replicates in both bacterial and eukaryotic cells Ex. Yeast Saccharomyces cerevisiae well-suited : Rapid growth, ease of culture Multicopy plasmids (from natural 2 plasmids) Eukaryote, so more appropriate posttranslational modifications to expressed proteins Can secrete protein for easy purification Ex. Plasmid YEP24 shown earlier Slide 40 4-40 Ex. Baculovirus expression vectors; transform (transfect) insect cells in culture Fig. 23 Large circular DNA genome; Major viral structural protein made in huge amounts Strong promoter for polyhedrin protein Produce lots of recombinant protein per liter of medium Non-recombinant viral DNA do not replicate because lack essential gene Slide 41 4-41 Methods for Transfection of animal cells with vectors for clones or protein production [Transformation for animal cells used for cancer cells] Calcium phosphate Mix cells with DNA in phosphate buffer Solution of calcium salt added to form precipitate Cells take up calcium phosphate crystals, includes DNA Electroporation Electric shock for DNA to enter cells Liposomes DNA mixed with lipid to form liposomes, small vesicles Liposomes fuse with cell membrane, carry DNA inside Slide 42 4-42 Summary Foreign genes can be cloned and expressed in eukaryotic cells Eukaryotic systems have some advantages: Proteins tend to fold properly, are soluble rather than aggregated into insoluble inclusion bodies Posttranslational modifications (phosphorylation, glycosylation) made in eukaryotic manner Can use transient clones for tests gene function Select stable clones expressing transgene (as for construction transgenic organisms) Slide 43 Review problems 2. Why does one need to attach DNAs to vectors to clone them? 4.Diagram the process of cloning a DNA fragment into BamHI and PstI sites of vector pUC18. How would you screen for clones that contain an insert? Explain the blue/white lacZ detection system 7. You want to make a genomic library with DNA fragments averaging about 45 kb in length. Which vector discussed in this chapter would be most appropriate? Why? 4-43 Slide 44 Review problems 11. Diagram a method for creating a cDNA library. 13.Outline the polymerase chain reaction (PCR) method for amplifying a given stretch of DNA. 14.What is the difference between reverse transcriptase PCR (RT-PCR) and standard PCR? For what purpose would you use RT-PCR? For what purpose might you want cDNA vs genomic library? 4-44 Slide 45 Review problems AQ 1. Given an amino acid sequence of part of a protein from a hypothetical gene you want to clone: Pro-arg-tyr-met-cys-trp-ile-leu-met-ser Look at the code chart a.What sequence of 5 aa would give a 14-mer probe with the least degeneracy for probing a library to find gene of interest? b.How many different 14-mers would you need to make to be sure probe matches sequence in cloned gene perfectly? c.lf you started your probe one aa to the left of the one you chose in (a), how many different 14-mers would you have to make? 4-45