CHAPTER 8 RECOMBINANT DNA and GENETIC ENGINEERING

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1 CHAPTER 8 RECOMBINANT DNA and GENETIC ENGINEERING Questions to be addressed: How are recombinant DNA molecules generated in vitro? How is recombinant DNA amplified? What analytical techniques are used on recombinant DNA? How is recombinant DNA used in genetic engineering? Obtaining Donor DNA 1. Genomic DNA - cells are lysed, and DNA separated from other molecules (carbohydrates, lipids, proteins, RNA) 2. cdna - complementary DNA is a double-stranded DNA copy of mrna a) mrna isolated from cells (Figure 8-4) b) reverse transcriptase from retrovirus makes single-stranded DNA from mrna c) ssdna used as a template to make ds DNA 3. Chemically synthesized DNA - automated techni ques can generate short DNA sequences Cutting DNA randomly broken through physical shearing cut in specific places by restriction endonucleases (RE) RE are produced by bacteria to protect against viral infection - any viral genome entering from another bacterial species is cut into fragments because it is recognized as "non-self" RE are named based on species name of bacteria e.g. EcoR1 is from Escherichia coli (Figure 8-6)

2 RE cut at specific short sequences, that are usually palindromic (rotationally symmetrical sequences) May cut at sites directly opposite from one another to generate blunt ends (e.g. HaeIII, SmaI) May cut at staggered sites to generate sticky ends (e.g. EcoR1, HindIII) Sticky ends will hybridize through complementary base pairing with sticky ends formed by the same RE Can join DNA from different sources to generate a recombinant DNA molecule (Figure 8-5) DNA ligase makes the phosphodiester bond at the sticky end joints DNA may be placed into a vector (e.g. a plasmid) that allows the piece of DNA to be replicated and/or transcribed in a host cell (Figure 8-7) Amplifying DNA In vitro amplification - polymerase chain reaction (pcr) (Figure 8-8) Primers specific to opposite ends of a DNA molecule are used to initiate DNA synthesis DNA is separated by heat Temperature is lowered and primers hybridize to DNA Heat stable Taq polymerase extends primers using DNA as template Process is repeated many time, eventually producing large numbers of copies of DNA between the primers Large amount of DNA can be made from tiny amounts of template Limited to short regions (< 2kB)

3 In vivo amplification DNA is amplified through cloning in an appropriate host cell Insert DNA is contained within a cloning vector Small, therefore efficient to manipulate Able to be replicated in the host Contain restriction sites that allow DNA to be easily inserted and excised Selectable marker allow easy identification Types of cloning vectors: 1. Plasmids (Figure 8-10) selectable marker = drug resistance (e.g. amp r ) allow selection of cells containing the plasmid Insertion of insert results in inactivation of another gene (e.g. tet r or lacz) allowing selection of cells with plasmid containing insert Multiple copies per cell result in rapid amplification 2. Bacteriophage vectors (Figure 8-11) Parts of phage genome not necessary for replication or infection are removed, leaving essential "arms" DNA insert is ligated to arms and replaces removed portion Only arms with insert are the correct size to form packagable and infective phage particles, therefore only phage with inserts are recovered 3. Cosmid vectors Combine characteristics of phage and plasmid, and allow much large inserts Infectivity is conferred by phage sequences (cos sites)

4 Replication is conferred by plasmid sequences Vectors must confer the following capabilities: Entry into bacteria (Figure 8-13) Through transformation (plasmids) or transduction (phage or cosmids) Replication Requires origin of replication Recovery a) Phage can be isolated from infected then lysed bacteria b) Plasmids can be isolated from chromosomal DNA because of their smaller size Analysis of recombinant molecules Libraries (Figure 8-14a) Collections of cloned DNA that include all parts of the genome (genomic library) or represent all mrna expressed by the organism (cdna library) Multiple predicted copies of each segment ensure that all segments are present at least once in a genomic library Completeness of cdna library depends on type of tissue/ condition from which its extracted Genomic libraries are larger than cdna libraries, but contain entire genes including regulatory sequences Identifying gene of interest Using a probe Probing a library with a DNA probe complementary to gene of interest (Figure 8-14b)

5 Membrane placed on plate, colonies or plaques lifted from plate and lysed DNA denatured (single-stranded) Membrane incubated with labeled probe Probe binds to complementary DNA, identifying colony/plaque containing DNA of interest Probe generated based on expected similarity to known gene or based on protein sequence (Figure 8-15) Probing a library with an antibody to protein product of the gene (Figure 8-16) cdna are cloned into an expression library, which has signals for transcription and translation in E. coli nitrocellulose membrane lifts colonies producing proteins proteins stick to membrane membrane incubated with antibody antibody binds to protein of interest, identifying encoding gene Probing a mixture of DN A with a DNA probe complementary to gene of interest (Figure 8-18) Fragments of DNA are separated by differential migration in an electric field (gel electrophoresis, Figure 8-17) DNA fragments are visualized by staining with ethidium bromide, and size determined by comparing to migration of fragments of known size DNA is transferred to a filter, denatured, and hybridized to complementary probe

6 Positional cloning (Figure 8-19) Gene is identified based on interesting mutant phenotype Gene is mapped to a chromosome Map using markers that are cloned These cloned DNA pieces are then used as baits to fish genomic fragments from a library Fished out fragments are used to generate new mapping markers to establish whether fragment is a step closer to the gene of interest Fragment closest to gene of interest is used as new bait to fish in the library for nearer fragments Functional complementation You have an organism carrying a mutation in the gene of interest Sequence of gene unknown Make a library containing wild type genomic DNA Transform the mutant organism with DNA from individual library clones If DNA causes transformed mutant to revert to wild type phenotype ("complementation"), the DNA contains the gene of interest Gene tagging (Figure 8-21) Use mobile DNA of known sequence to perform insertional mutagenesis (e.g. transposons such as Mu, En, P or TDNA) Any mutation created will be "tagged" with the known sequence, which can then be used as a starting point for cloning Tag is used to screen a genomic library made from the mutant line or to initiate degenerate pcr

7 Sequencing DNA (Figure 8-22) Once DNA is cloned and identified as being a gene of interest, DNA sequence is determined DNA sequence can be used to deduce protein sequence, and may suggest a function for the gene product Most common method is dideoxy or Sanger sequencing Dideoxy nucleotides have no 3' OH, therefore cannot provide OH for DNA polymerization (Figure 8-23) Cloned DNA is denatured in vitro, and a primer, complementary to a known sequence within the DNA (often in the vector) is added DNA polymerase, deoxynucleotides (dntps) for each of A, T, G and C are added to the cocktail Separated into 4 reactions, and one dideoxynucleotide (ddntp - A or G or T or C) is added to each Synthesis proceeds until a ddntp is incorporated Each tube has a set of synthesized DNA molecules with same starting point (primer) and different end points, all with the same nucleotide in a single tube (A, C G or T) Fragments are separated on an electrophoresis gel Fragments can be visualized because ddntp is radioactive, position of fragment marked by exposure of x-ray film Or, in automated sequencers, each ddntp has a different florescent tag and all four reactions are done in the same tube computer generates a single readout (Figure 8-24) DNA sequence is scanned for open reading frames (ORFs - Figure 8-25)

8 Detection of Disease-causing mutations a gene is cloned which, when mutated, causes a disease if mutant alleles that cause disease phenotype are identified, one can devise screens for the alleles that allow early detection (e.g. amniocentesis) a) restriction fragment length polymorphism (RFLP - Figure 8-27) mutation changes site recognized by restriction enzyme digest DNA from normal individuals and those suspected of carrying the mutant allele southern hybridization using gene as probe see different number of bands in mutant vs. wild type b) sequence differences probes designed that bind to wild type but not mutant DNA differences detected through Southern blotting c) PCR based differences Useful if mutation is the result of an insertion or deletion Amplify DNA using gene specific primers Different size products result in wild type vs. mutant Genetic Engineering Genes are manipulated Manipulated genes (transgenes) are introduced into an organism (transgenic organism) Transgene may replace resident gene or (more usually) be inserted ectopically (in some other location) Genetic Engineering in Fungi Yeast artificial chromosomes (YAC)

9 Any DNA with a centromere, replication orgins and chromosome ends (telomeres) Will segregate as cells divide along with other chromosomes Useful for cloning large pieces of DNA Genetic Engineering in Plants Agrobacterium tumefaciens (Ti plasmid) Naturally occurring soil bacterium that causes crown gall disease Ti plasmid is required for the disease, and operates by inserting a part of itself (T-DNA) into the plant genome (Figures 8-31, 8-32) Genes inserted cause proliferation of plant cells Plasmids have been generated that retain ability to insert T-DNA into genome, do not cause disease symptoms (disarmed plasmid) and can also propagate in E.coli (Shuttle vector) Plasmids also have a selectable marker (antibiotic resistance) in bacteria (e.g. spectinomycin) and plants (kanamycin) (Figure 8-33) Insertion by the "gene gun" (Figure 8-28) Genetic Engineering in Animals P-element mediated insertion in Drosophila (Figure 8-36) P-element = mobile DNA (transposon) Require P-element ends that are recognized for integration into genome and enzyme transposase, which cut and pastes DNA 2 different vectors injected into Drosophila embryo, one carrying transposase, one carrying gene of interest flanked by P-element ends gene of interest inserted into genome

10 Transgenesis in mouse 2 approaches: I) ectopic insertions; ii) gene replacement ectopic insertion- DNA injected into early embryo (Figure 8-37) gene replacement - gene inactivated by insertion of drug resistance gene is injected into embryonic stem cells (ES) where it replaces normal gene through homologous recombination (Figure 8-38) ES cells are injected into a mouse embyro to produce a chimeric mouse (Figure 8-39) Human gene therapy Germ line therapy (Figure 8-41a) Transgenic cells inserted into the germ line Trait is inherited Somatic gene therapy (Figure 8-41b) Some somatic cells are altered Only works if disease affects an isolated tissue (e.g. cystic fibrosis, immune deficiency)