Biotechnology Chapter 20 Gene technology Biotechnology Manipulation of

Biotechnology Chapter 20

Gene technology

Biotechnology • Manipulation of organisms to make useful products

Genetic engineering • • Manipulation of genes Gene cloning: Multiple copies of a single gene Produce a specific product

Fig. 20 -2 Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Cell containing gene of interest Gene of interest DNA of chromosome 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Basic Protein harvested 4 Basic research and various applications research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth

Recombinant DNA • • • 1970’s Combining genes from different sources Even different species Combined into single DNA Example: Bacteria & mammal

Recombinant DNA • • • Genetically modified bacteria Mass produce beneficial chemicals Insulin Growth hormone Cancer drugs Pesticides

Plasmid

Plasmid • • Small separate circular DNA Replicated same as main DNA Foreign DNA added to plasmid Replicated along with plasmid

Recombinant DNA • • • Nucleases: Enzymes that degrade DNA Restriction endonulceases: Restriction enzymes Cut DNA into fragments Specific points

Recombinant DNA • Restriction sites: • Places where DNA is cut • Short DNA sequence

• • • Recombinant DNA Restriction enzyme Recognizes short sequences in DNA Cuts at these sequences Staggered cut Leaves single-stranded ends Called “sticky ends”

Recombinant DNA D: Chapter_20A_Power. Point_Lectures20_Lecture_Prese ntation2003 Restriction. Enzymes. A. html

Recombinant DNA • • Sticky ends Enables insertion of other DNA fragments from other sources Match ends by base pairs (complementary sequences) • DNA ligase: • Enzyme combines ends • Forms a phosphodiester bond

Recombinant DNA (Process) • 1. Isolate gene of interest & bacterial plasmid • 2. Cut DNA & plasmid into fragments • 3. Mix DNA fragments with cut plasmid. • Fragment with gene of interest is inserted into the plasmid • 4. Recombinant plasmid is mixed with bacteria

Recombinant DNA (Process) • 5. Bacteria with recombinant DNA reproduce • 6. Isolate bacterial clones that contain gene of interest • Producing protein of interest • 7. Grow large quantities of bacteria that produce the protein

Recombinant DNA (Process) D: Chapter_20A_Power. Point_Lectures20_Lecture_P resentation2004 Cloning. AGene. A. html

Fig. 20 -4 -4 Hummingbird cell TECHNIQUE Bacterial cell amp. R gene lac. Z gene Bacterial plasmid Restriction site Sticky ends Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lac. Z gene Colony carrying recombinant plasmid with disrupted lac. Z gene One of many bacterial clones

Recombinant DNA • • • Vector: DNA molecule that carry foreign DNA Enters & replicates in the host Plasmids & phages are common vectors Phages are larger than plasmid Can handle inserts up to 40 kilobases

PCR • • Polymerase chain reaction Amplify DNA Makes large quantities of DNA 1985

PCR • • • Heated Denatured DNA primer Heat stable DNA polymerase Makes DNA

Fig. 20 -8 5 TECHNIQUE 3 Target sequence 3 Genomic DNA 1 Denaturation 5 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

Gel electrophoresis • • • Study DNA Polymer (gel) Restriction fragments Separates DNA based on charge & size Nucleic acids negative charge (Phosphates) • Migrate towards + end (red)

Fig. 20 -9 TECHNIQUE Mixture of DNA molecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 RESULTS Shorter molecules

Fig. 20 -10 Normal -globin allele 175 bp Dde. I Sickle-cell allele Large fragment 201 bp Dde. I Normal allele Dde. I Large fragment Sickle-cell mutant -globin allele 376 bp Dde. I 201 bp 175 bp Large fragment 376 bp Dde. I (a) Dde. I restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

Cloning • Multicellular organisms come from a single cell. • Offspring are identical

Cloning • • • 1950 Carrots Totipotent: Mature cells that undifferentiated Give rise to any type of cells Common in plants

Cloning • Nuclear transplantation • Nucleus of an unfertilized/fertilized egg is removed • Replaced with nucleus of differentiated cell • Direct development of cell into tissues etc.

Cloning • • Removed nuclei from an egg Mammary cells Fused with egg cells Dolly, 1997, identical to mammary cell donor • Died prematurely age 6 • Arthritis & lung disease

Fig. 20 -18 TECHNIQUE Mammary cell donor Egg cell donor 2 1 Egg cell from ovary 3 Cells fused Cultured mammary cells 3 4 Grown in Nucleus removed Nucleus from mammary cell culture Early embryo 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development RESULTS Lamb (“Dolly”) genetically identical to mammary cell donor

Fig. 20 -19

Cloning • • • Few develop normally Abnormalities Epigenetic changes to the chromatin More methylation of chromatin Reprogram chromatin of differentiated cell

Stem cells • • • Started 1998 at UW Early embryonic cells Potential to become any type of cell Master cell generates specialized cells Such as muscle cells, bone cells, or blood cells

Stem cells • • • Embryos Bone marrow Umbilical cord blood Blood stem cells ? ? Turn skin cells into embryonic stem cells • Therapeutic cloning

Fig. 20 -20 Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equivalent of blastula) From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Cultured stem cells Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells

Medical applications • • • Genetic markers Detect abnormal disease SNP Single nucleotide polymorphisms Single base pair site where variation is found • RFLP • Restriction fragment length polymorphisms

Fig. 20 -21 DNA T Normal allele SNP C Disease-causing allele

Medical applications • • Gene therapy Treat genetic defects Alters person’s genes 2 girls with rare blood disease CF (vectors are viruses) SCID (immune disorder) Injected viral DNA with normal gene

Fig. 20 -22 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient 4 Inject engineered cells into patient. Bone marrow

Medical applications • Transgenic animal • Gene from one animal is inserted into another • Goat milk protein anti-thrombin • Isolated from milk • “pharm” animals

Animals • Transgenic animals engineered for specific traits • Genetically create a racehorse • Not have to breed • Sheep with better wool? ?

Agricultural applications • • Manipulate tomatoes Do not ripen as fast “Flavr-Savr” Slows down ethylene production

Agricultural applications • • • Introduce genes to plants Enable them to “fix” nitrogen Convert N 2 to NH 3 Help eliminate use fertilizers Cut $$

Agricultural applications • Herbicide resistance • Plant genetically resists the herbicide • Insect resistance

Agricultural applications • Transgenic rice • “golden rice” • Rice with genes that code for better absorption of iron and beta carotene • First of many genetically engineered foods • Helps dietary deficiencies

Forensics • • Genetic profile: Individual genetic markers “DNA fingerprint” RFLP STR Short tandem repeats Occur in specific regions in genome Unique

Fig. 20 -24 (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Source of sample STR marker 1 STR marker 2 STR marker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

• • Concerns over genetic engineering Genetically modified foods Harmful? Genetically engineered gametes Blonde and blue eyes? ?