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Transfer of genetic information by DNA Gene technology & Molecular biology Lecture 4 Recombinant DNA:cloning Recombinant DNA Generation of a recombinant DNA molecule • Recombinant DNA technology provided scientists with the ability to isolate, sequence, and manipulate individual genes from any type of cell. • It has enabled detailed molecular studies of the structure and function of eukaryotic genes and genomes, and revolutionized our understanding of cell biology. A molecular tool box • Enzymes -restriction endonuclease -ligase -DNA polymerase -reverse transcriptase (RT) -kinase -polynucleotide transferase -phosphatase • PCR • In vitro mutagenesis • Vectors -plasmids -phages (M13, λ) -cosmides -YACs • Oligonucleotide synthesis -viruses • Host cells -primers -probes -bacteria -gene assembly -yeast/fungus/molds -plant cells • DNA sequencing -insect cells -Maxam-Gilbert -mammalian cells -Sanger -pyrosequencing A molecular tool box • Transformation • Enzymes -heat-shock -electroporation -”gene gun” -micro injection -transfecion -restriction endonuclease -ligase -DNA polymerase -reverse transcriptase (RT) -kinase -polynucleotide transferase -phosphatase • Selection methods -antibiotic resistance -genetic complementation • Screening methods -genotypic -phenotypic • PCR • In vitro mutagenesis • Vectors -plasmids -phages (M13, λ) -cosmides -YACs • Oligonucleotide synthesis -viruses • Host cells -primers -probes -bacteria -gene assembly -yeast/fungus/molds -plant cells • DNA sequencing -insect cells -Maxam-Gilbert -mammalian cells -Sanger -pyrosequencing • Transformation -heat-shock -electroporation -”gene gun” -micro injection -transfecion • Selection methods -antibiotic resistance -genetic complementation • Screening methods -genotypic -phenotypic 1 Restriction enzymes Kloning - Expression Enzymes • Specific DNA sequences (4-8 bp) are recognized and cleaved by restriction endonucleases (RE) • Recognition sequence (RS) is often palindromic (can be read from both ends) • Is part of the defense system against foreign DNA; > hundreds of different RE • Specific methylases methylates certain bases in the RS, whereby the bacterium’s own DNA becomes protected from cleavage EcoRV EcoRI PstI GATATC CTATAG GAATTC CTTAAG CTGCAG GACGTC GAT CTA ATC TAG ”blunt end” Recognition sites of common RE G AATTC CTGCA G G G ACGTC CTTAA ”sticky ends” Restriction map EcoRI recognizes the sequence GAATTC. This sequence is present at five sites in DNA of the bacteriophage λ, so EcoRI digests λ DNA into six fragments ranging from 3.6 to 21.2 kilobases long. Restriction maps of λ and adenovirus DNAs Restriction enzymes • For larger DNA molecules such as cellular genomes, restriction endonuclease digestion alone does not provide sufficient resolution. • For example, the human genome would yield more than 500,000 EcoRI fragments. 2 Restriction enzymes Ligase • Ligases recreate phosphodiesterbonds by joining the 5’-phosphate group to the free 3’-hydroxyl group. A common enzyme is the T4-DNA ligase. • Ligases are used to ”glue” DNA fragments to each other. G-OH P-GATCT CCTAG-P HO-A G-OH P-GATCC CCTAG-P HO-G + T4-DNA ligase, ATP + T4-DNA ligase, ATP G GATCT CCTAG A GGATCC CCTAGG can be re-cleaved by BamHI (5’-GˇGATCC-3’) Ligase reaction cannot be re-cleaved by BamHI (5’-GˇGATCC-3’) DNA polymerases • DNA polymerases incorporates deoxynucleotides (dNTPs) to a growing DNA chain; the dNTPs are added to the free 3’-OH group using the opposite strand as template. Some frequently used polymerases are DNA polymerase I, T7-DNA polymerase, and Taqpolymerase. • They are used for several things, for example: - generation of blunt-ends - in DNA sequencing reactions G CCATG Reverse transcription and retrovirus replication + dNTPs, polymerase GGTAC CCATG Reverse transcriptase (RT) • Reverse transcriptase (RT) synthesizes a complementary DNA chain from a RNA template. Retroviruses (e.g., HIV) use this mechanism to make a DNA copy of it’s RNA genome. • Can be used to our advantage: - in cDNA synthesis mRNA 5’Cap AAAAA 3’ TTTTT 5’ reverse transcriptase + dNTPs mRNA 5’Cap cDNA 3’ AAAAA 3’ TTTTT 5’ 3 Terminal transferase Phosphatases och kinases • Terminal transferase adds nucleotides to the 3’-end of DNA chains. • Used, among other things, for generation of cohesive ends from blunt-end fragments and also at one step in cDNA synthesis 5’ 3’ + 3’ dATP 5’ dTTP terminal transferase 3’ AA…AA-OH 5’ HO-TT…TT 3’ 5’ • Phosphatases remove free 5’-phosphate groups from DNA - used to minimize the incidence of self-ligated vector in DNA cloning - commonly used phosphatases are the shrimp- and bovine alkaline phosphatase • Kinases adds phosphate groups to the 5’-end of DNA - used to phosphorylate synthetic DNA (e.g., linkers) before ligation - polynucleotidekinase (PNK) ligation 5’ AA…AA 3’ 3’ TT…TT 5’ Kloning - Expression Vectors Cloning vectors: desired properties • small • ”universal”; should work in different organisms • easy to isolate from the host organism • easy to detect and select • multiple copies (is usually advantageous) • several unique RE localized to a specific region (mcs) • convenient method for detection of cloned DNA Plasmids Plasmid preparation • Plasmids are circular extrachromosomal DNA • Used as vectors = carriers of ”foreign” DNA - used in cloning and recombinant protein expression - insert size ≤ 5 kb • Plasmids usually contain: - ori (origin of replication) - gene/s for selection (often antibiotic resistance genes; bla, cat) - multiple cloning site (mcs) - sometimes a gene that allows for screening (e.g., lacZ for blue-white screening) • Different plasmid-types have different ”copy nr” • Plasmids belong to different incompatibility groups; two plasmids belonging to the same group cannot be stably propagated simultaneously within a single cell. Figure 8-40 Molecular Biology of the Cell (© Garland Science 2008) 4 Bacteriophage λ Bacteriophage λ-life cycle Figure 5-78 Molecular Biology of the Cell (© Garland Science 2008) Bacteriophage λ In vitro packaging:bacteriophage λ • Bacteriophage λ is a virus that infects bacteria (E. coli). - a lytic and a lysogenic phase (prophage) in the life cycle. - genome size approx. 45 kb; a central region of 15 kb is not essential for replication. - have complementary single stranded cohesive ends (COS-sites); used by the phage to make concatamers of it’s genome when the DNA is packed into phage particles. • the λ genome has been modified to work better as a vector; unique sites that flank the central region have been introduced to simplify DNA cloning/replacement. - inserts must have a certain size (approx. 15 kb), or else no infectious phage particles can be formed. • λ used for cloning of genomic fragments, since plasmids are not suitable for cloning of larger DNA fragments. Cosmids • Cosmids are plasmid-phage λ hybrids. They contain: - ori - gene for antibiotic resistance (e.g., Tetr) - cos-sites - cloning sites • Cosmids are used for cloning of genomic DNA fragments larger than 15 kb (this is the limit of what phage λ can harbor). Cosmids can accommodate an insert size of approx. 45 kb. cos vector DNA (cosmid) Biotechnology: Applying the genetic revolution, Figure 3.17 Yeast artificial chromosome (YAC) • YACs contain: - autonomously replicating sequence (ARS) from yeast chromosome - centromer (CEN); ensures stable and even distribution of the YACs between mother and daughter cells - two telomers; constitute chromosome ends and enables replication of the YACs as small linear chromosomes. - selectable gene markers, e.g., LEU2 that complement Leu negative strains. - cloning sites • YACs used for cloning of very large genomic fragments (1002000 kb) antibiotic resistance gene genomic DNA telomer LEU2 ARS CEN jäst DNA telomer 50 kb 5 Various cloning vectors Vectors for cloning large DNA fragments Biotechnology: Applying the genetic revolution, Figure 3.16 Host cells Kloning - Expression Host cells Different vectors are used to introduce recombinant DNA in various types of host cells, for example: • Baceria • • • • Kloning - Expression - E. coli the most common - High expression levels - Relatively easy to scale-up the production process. Yeast Insect cells Eukaryotic cells Mammalian cells Plant cells Post-translational modifications (e.g., glycosylations) possible Heat shock transformation Transformation methods Biotechnology: Applying the genetic revolution, Figure 3.18 6 Electroporation Kloning - Expression Selection methods Acta Physiol Scand. 2003 Apr;177(4):437-47 Issuses to consider: 1. Cell size 2. Temperature 3. Post-pulse manipulation 4. Composition of electrodes and pulsing medium Antibiotic resistance Commonly used antibiotics in molecular biology: - ampicillin Inhibits cell wall synthesis carbenicillin chloramphenicol Inhibits protein synthesis tetracyclin kanamycin Transform bacteria with ligated plasmid Kloning - Expression Cloned gene Gene for antibioticresistance Spread bacteria and grow on solid growth media (agar) supplemented with antibiotics Screening methods Plasmid Only bacteria containing the plasmid with the antibiotic resistance gene survive and form colonies Genotypic screening • Probe hybridization – known nucleotide sequence (gene sequence) • 15 nt long probe – unknown nucleotide sequence (gene sequence) • “guessmer”probe, approx. 50 nt long (unique) longer due to risk of mis-match • degenerate probe, approx. 18 nt long based on amino acid sequence (6 residues long) Phenotypic screening • Activity based – e.g., protease activity (subtilisin) • grow on casein media and a clear zone (“halo”) will appear around proteolytically active clones • Immuno based – antibody specific for the protein of interest 7 Blue-white screening Kloning - Expression EcoRI site LacZ AmpR Cloning of a DNA fragment in the EcoRI site in LacZ (encodes the enzyme β-galactosidase) An example of gene cloning - only the bacteria that harbor the plasmid are resistant to ampicillin and can grow Transform bacteria and grow on agar plates supplemented w ampicillin and X-gal - bacteria w/o cloned fragment have a functional β-galactosidase gene and convert the color-less substrate X-gal to a blue-colored product H N OH OH H OH O Br Cl H H H OH - in bacteria w a cloned fragment the β-galaktosidase gene is destroyed and therefore only give rise to white colonies O H X-gal Cloning of a prokaryotic gene • Example: Subtilisin • • • • from Bacillus subtilis protease gene size, approx. 1000 bp used in washing powders Create a gene library BamHI EcoRI BamHI Bacterial plasmid GATCC G G TAG CC G CTTAA C TT G AA create a gene library (genomic DNA) selection and genotypic screening phenotypic screening sequence verification clone into an expression vector activity studies/protein engineering EcoRI Chromosome • Sub tasks • • • • • • EcoRI DNA ligase Transformation Gene library • Chromosome digested w RE (“4-cutter”) • (4x4x4x4=256 bp) [e.g., Sau3AI, ^GATC^] • Partial digestion – Large randomly digested gene fragments (~2500 bp) • Plasmid cleaved w compatible RE (“6-cutter”) • Matching overlaps [e.g., BamHI, G^GATC^C] • Ligation (Phosphatase treated plasmid) • Transformation (CaCl2, Electroporation) • Selection • Colonies with recombinant plasmid Gene library Creating a DNA Library: Genomic DNA from the chosen organism is first partially digested with a restriction enzyme that recognizes a four base-pair sequence. Partial digestions are preferred because some of the restriction enzyme sites are not cut, and larger fragments are generated. If every recognition site were cut by the restriction enzyme, then the genomic DNA would not contain many whole genes. The genomic fragments are cloned into an appropriate vector, and transformed and maintained in bacteria. 8 Screening a recombinant library by hybridization Cloning of a eukaryotic gene • Often mRNA as source • Sub tasks • Create a gene library – mRNA purification – cDNA synthesis (S1-method, RNaseH-method) – Ligation into a vector (phage or plasmid) • • • • • Selection and genotypic screening Phenotypic screening Sequence verification Expression vector (choice of host!) Activity studies/protein engineering 9