Presentation on theme: "Chapter 20 Notes: DNA Technology. Understanding & Manipulating Genomes 1995: sequencing of the first complete genome (bacteria) 2003: sequencing of the."— Presentation transcript:
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
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
**Practical goal of biotech = improvement of human health and food production
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)
“Toolkit” for DNA technology involves: -DNA vectors -host organisms - restriction enzymes
VECTORS = carriers for moving DNA from test tubes back into cells -bacterial plasmids (small, circular DNA molecules that replicate within bacterial cells) -viruses
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.
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
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”
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)
DNA Technologies: 1) Cloning 2) DNA fingerprinting (profiling) 3) Microarray 4) Gene therapy
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
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
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
Why can bacteria produce insulin through recombinant DNA technology? The genetic code is universal!!!!