1. Chapter 20 Notes: DNA Technology

2. Understanding & Manipulating Genomes 1995: sequencing of the first complete genome (bacteria) 2003: sequencing of the Human Genome mostly completed These accomplishments depended on new technology: –Recombinant DNA: DNA from 2 sources (often 2 species) are combined in vitro into the same DNA molecule Called Genetic engineering: direct manipulation of genes for practical purposes

3.  DNA technology has launched a revolution in the area of:  BIOTECHNOLOGY: the use of living organisms or their components to do practical tasks -microorganisms to make wine/cheese -selective breeding of livestock -production of antibiotics -agriculture -criminal law

4. **Practical goal of biotech = improvement of human health and food production

5. Ch 20 looks at: 1.Main techniques for manipulating DNA 2.How genomes are analyzed & compared at the DNA level 3.Practical applications of DNA technology (including social & ethical issues)

6. “Toolkit” for DNA technology involves: -DNA vectors -host organisms - restriction enzymes

7. VECTORS = carriers for moving DNA from test tubes back into cells -bacterial plasmids (small, circular DNA molecules that replicate within bacterial cells) -viruses

8. HOST ORGANISMS: bacteria are commonly used as hosts in genetic engineering because: 1) DNA can easily be isolated from & reintroduced into bacterial cells; 2) bacterial cultures grow quickly, rapidly replicating any foreign genes they carry.

9. RESTRICTION ENZYMES = enzymes that recognize and cut short, specific nucleotide sequences (called restriction sites) -in nature, these enzymes protect the bacterial cell from other organisms by cutting up their foreign DNA

10. Restriction Enzymes (cont.)…  most restriction sequences are symmetrical in that the same sequence of 4-8 nucleotides is found on both strands, but run in opposite directions  restriction enzymes usually cut phosphodiester bonds of both strands in a staggered manner producing single stranded “sticky ends”

12. Restriction Enzymes (cont.)…  “sticky ends” of restriction fragments are used in the lab to join DNA pieces from different sources (complementary base pairing) *  RECOMBINANT DNA  unions of different DNA sources can be made permanent by adding DNA ligase enzyme (form covalent bonds between bases)

14. DNA Technologies: 1) Cloning 2) DNA fingerprinting (profiling) 3) Microarray 4) Gene therapy

15. Steps Involved in Cloning a Human Gene: 1) Isolate human gene to clone (ex: insulin); 2) Isolate plasmid from bacterial cell; 3) cut both DNA samples with the same restriction enzyme to open up bacterial plasmid & create sticky ends on both samples; 4) Mix the cut plasmids and human DNA genes & seal with DNA ligase; plasmid Human gene

17. Cloning a Human Gene (cont.)… 5) Insert recombinant DNA plasmid back into bacterial cell; 6) As bacterial cell reproduces, it makes copies of the desired gene; -grow cells on a petri dish 7) Identify cell clones carrying the gene of interest. -HOW? Which ones took up the gene & are making insulin? *Add a 2 nd gene besides insulin; add one for antibiotic resistance & then grow bacteria on that antibiotic

18. LE 20-4_3 Isolate plasmid DNA and human DNA. Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone

19. Why can bacteria produce insulin through recombinant DNA technology? The genetic code is universal!!!!