Genetic Technology

Genetic Engineering Deliberately altering the information content of DNA molecules Genes are isolated, modified, and inserted into an organism Made possible by recombinant technology –Cut DNA up and recombine pieces –Amplify modified pieces

Gene Therapy The transfer of one or more normal or modified genes into and individual’s body cells to correct a genetic defect or boost resistance to disease

Restriction Enzymes Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack –Discovered bacteria have an enzyme that chops up viral DNA Restriction enzymes cut DNA at a specific sequence Number of cuts made in DNA will depend on number of times the “target” sequence occurs Recombinant DNA can be formed: by splicing 2 different segments of DNA together to create a new strand

Making Recombinant DNA 5’ 3’ G C T T A A A A T T C G G C T T A AG 3’ 5’ one DNA fragmentanother DNA fragment 3’ 5’ In-text figure Page 254 Recombinant DNA can be formed: by splicing 2 different segments of DNA together to create a new strand

Making Recombinant DNA nick 5’ 3’ 5’ GA A T T C C T T A AG nick GA A T T C C T T A AG DNA ligase action In-text figure Page 254

Using Plasmids Plasmid is small circle of bacterial DNA Foreign DNA can be inserted into plasmid –Forms recombinant plasmids –Plasmid is a cloning vector –Can deliver DNA into another cell

Using Plasmids DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids Figure 16.4 Page 255

Making cDNA mRNA transcript mRNA–cDNA hybrid single-stranded cDNA double-stranded cDNA Figure 16.5 Page 255 cDNA is a DNA molecule copied from a mature mRNA transcript by reverse transcription

Amplifying DNA Ways to take small pieces of DNA and make them larger –Fragments can be inserted into fast-growing microorganisms –Polymerase chain reaction (PCR)

Polymerase Chain Reaction Sequence to be copied is heated Primers are added and bind to ends of single strands DNA polymerase uses free nucleotides to create complementary strands Doubles number of copies of DNA

Polymerase Chain Reaction Double-stranded DNA to copy DNA heated to 90°– 94°C Primers added to base-pair with ends Mixture cooled; base-pairing of primers and ends of DNA strands DNA polymerases assemble new DNA strands Figure 16.6 Page 256 Stepped Art

Polymerase Chain Reaction Figure 16.6 Page 256 Stepped Art Mixture heated again; makes all DNA fragments unwind Mixture cooled; base- pairing between primers and ends of single DNA strands DNA polymerase action again doubles number of identical DNA fragments

DNA Fingerprints Individuals have a unique array of DNA fragments Inherited from parents in Mendelian fashion Even full siblings can be distinguished from one another by this technique

Tandem Repeats Short regions of DNA that differ substantially among people Many sites in genome where tandem repeats occur Each person carries a unique combination of repeat numbers

RFLPs Restriction fragment length polymorphisms DNA from areas with tandem repeats is cut with restriction enzymes Because of the variation in the amount of repeated DNA, the restriction fragments vary in size Variation is detected by gel electrophoresis

Gel Electrophoresis DNA is placed at one end of a gel A current is applied to the gel DNA molecules are negatively charged and move toward positive end of gel Smaller molecules move faster than larger ones

Analyzing DNA Fingerprints DNA is stained or made visible by use of a radioactive probe Pattern of bands is used to: –Identify or rule out criminal suspects –Identify bodies –Determine paternity

Genome Sequencing Sequence of bacterium Haemophilus influenzae determined Automated DNA sequencing now main method Draft sequence of entire human genome determined in this way Human Genome Project

The Human Genome Initiative Goal - Map the entire human genome Initially thought by many to be a waste of resources Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes Sequencing was completed ahead of schedule in early 2001

Genomics Structural genomics: actual mapping and sequencing of genomes of individuals Comparative genomics: concerned with possible evolutionary relationships of groups of organisms

Gene Libraries Bacteria that contain different cloned DNA fragments –Genomic library –cDNA library

Using a Probe to Find a Gene You want to find which bacteria in a library contain a specific gene Need a probe for that gene –A radioisotope-labeled piece of DNA –Will base-pair with gene of interest Colonies on plate Cells adhere to filter Cells are lysed; DNA sticks to filter Probe is added dark spot on film, indicates colony with gene Location where probe binds forms

Cloning Making a genetically identical copy of DNA or of an organism Three different types –Recombinant DNA or DNA cloning (we already talked about) –Reproductive Cloning – making of an entire organism –Therapeutic Cloning – also called “embryo cloning”; stem cells

Cloning Dolly A sheep cloned from an adult cell –Nucleus from mammary gland cell was inserted into enucleated egg –Embryo implanted into surrogate mother –Sheep is genetic replica of animal from which mammary cell was taken

Engineered Proteins Bacteria can be used to grow medically valuable proteins –Insulin, interferon, blood-clotting factors –Vaccines

Cleaning Up the Environment Microorganisms normally break down organic wastes and cycle materials Some can be engineered to break down pollutants or to take up larger amounts of harmful materials

Engineered Plants Cotton plants that display resistance to herbicide Aspen plants that produce less lignin and more cellulose Tobacco plants that produce human proteins Mustard plant cells that produce biodegradable plastic

First Engineered Mammals Experimenters used mice with hormone deficiency that leads to dwarfism Fertilized mouse eggs were injected with gene for rat growth hormone Gene was integrated into mouse DNA Engineered mice were 1-1/2 times larger than unmodified littermates

Can Genetically Engineered Bacteria “Escape”? Genetically engineered bacteria are designed so that they cannot survive outside lab Genes are included that will be turned on in outside environment, triggering death

Ethical Issues Who decides what should be “corrected” through genetic engineering? Should animals be modified to provide organs for human transplants? Should humans be cloned?