Technical Aspects of Recombinant DNA and Gene Cloning

1. 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)

2. 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

3. Figure 10.9

4. Characteristics of Cloning Hosts

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

6. Figure 10.11

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

8. Biochemical Products of Recombinant DNA Technology

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

10. 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

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

12. Figure 10.12

14. 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

15. Figure 10.13

17. 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

18. Figure 10.14

19. 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

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

21. 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

22. Figure 10.15

23. 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

24. 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