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