Technical Aspects of Recombinant DNA and Gene Cloning Strategies for obtaining genes in an isolated state DNA removed from cells, separated into fragments, inserted into a vector, and cloned; then undergo Southern blotting and probed Gene can be synthesized from isolated mRNA transcripts Gene can be amplified using PCR Once isolated, genes can be maintained in a cloning host and vector (genomic library)

Characteristics of Cloning Vectors Capable of carrying a significant piece of the donor DNA Readily accepted by the cloning host Must have a promoter in front of the cloned gene Vectors (such as plasmids and bacteriophages) should have three important attributes: An origin of replication somewhere on the vector Must accept DNA of the desired size Contain a gene that confers drug resistance to their cloning host

Figure 10.9

Characteristics of Cloning Hosts

Construction of a Recombinant, Insertion into a Cloning Host, and Genetic Expression Figure 10.10

Figure 10.11

Synthetic Biology: Engineering New Genetic Capabilities Scientists are attempting to create microbes that produce hydrogen as fuel Can use recombinant techniques mentioned previously

Biochemical Products of Recombinant DNA Technology

Genetically Modified Organisms Transgenic or genetically modified organisms (GMOs): recombinant organisms produced through the introduction of foreign genes These organisms can be patented

Recombinant Microbes: Modified Bacteria and Viruses Genetically altered strain of Pseudomonas syringae Can prevent ice crystals from forming Frostban to stop frost damage in crops Strain of Pseudomonas fluorescens Engineered with a gene from Bacillus thuringiensis Codes for an insecticide Drug therapy Bioremediation

Transgenic Plants: Improving Crops and Foods Agrobacterium can transfect host cells This idea can be used to engineer plants

Figure 10.12

Transgenic Animals: Engineering Embryos Several hundred strains have been introduced Can express human genes in organs and organ systems Most effective way is to use viruses

Figure 10.13

Genetic Treatments: Introducing DNA into the Body Gene Therapy For certain diseases, the phenotype is due to the lack of a protein Correct or repair a faulty gene permanently so it can make the protein Two strategies ex vivo in vivo

Figure 10.14

in vivo Skips the intermediate step of incubating excised patient tissue Instead the naked DNA or a virus vector is directly introduced into the patient’s tissues

DNA Technology as Genetic Medicine Some diseases result from the inappropriate expression of a protein Prevent transcription or translation of a gene

Antisense DNA and RNA: Targeting Messenger RNA Antisense RNA: bases complementary to the sense strand of mRNA in the area surrounding the initiation site When it binds to the mRNA, the dsRNA is inaccessible to the ribosome Translation cannot occur Single-stranded dNA usually used as the antisense agent (easier to manufacture) For some genes, once the antisense strand bound to the mRNA, the hybrid RNA was not able to leave the nucleus Antisense DNA: when delivered into the cytoplasm and nucleus, it binds to specific sites on any mRNAs that are the targets of therapy

Figure 10.15

Genome Analysis: Maps, Fingerprints, and Family Trees Possession of a particular sequence of DNA may indicate an increased risk of a genetic disease Genome Mapping and Screening: An Atlas of the Genome Locus: the exact position of a particular gene on a chromosome Alleles: sites that vary from one individual to another; the types and numbers are important to genetic engineers Mapping: the process of determining location of loci and other qualities of genomic DNA Linkage maps: show the relative proximity and order of genes on a chromosome Physical maps: more detailed arrays that also give the numerical size of sections in base pairs Sequence maps: produced by DNA sequencers Genomics and bioinformatics: managing mapping data

DNA Fingerprinting: A Unique Picture of a Genome DNA fingerprinting: tool of forensic science Uses methods such as restriction endonucleases, PCR, electrophoresis, hybridization probes, and Southern blot technique