 Genetic Engineering USES:  Cure Diseases  Treat Genetic Disorders  Improve Food Crops  Improve Human Lives

Figure 20.2 Bacterium Bacterial chromosome Plasmid 2134 Gene inserted into plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed from gene of interest Protein harvested Copies of gene Basic research and various applications Basic research on protein Basic 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 Human growth hormone treats stunted growth

 Bacterial enzymes that cut DNA into pieces  R.E. recognize specific nucleotide sequences

 Single chain “tails” of DNA that are created on each DNA segment  Sticky Ends readily bond to complementary chains of DNA.

Figure Recombinant DNA molecule One possible combination DNA ligase seals strands DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA Sticky end GAATTC CTTAAG CTTAA G AATTC G G G CTTAA G G G G AATT C C TTAA

 Restriction Enzymes can isolate a specific gene.  Can be transferred by a cloning vector to an organism PLASMID  Small ring of DNA found in bacteria that can serve as a cloning vector.

1. Restriction Enzymes cut the Plasmid open. 2. DONOR GENE is spliced in to the plasmid: Specific gene isolated from another organism 3. Plasmid is returned to the bacterium The gene is replicated as the bacterium is copied EACH PLASMID HAS A GENE CLONE – Exact copy of gene

Figure 20.4 Bacterial plasmid TECHNIQUE RESULTS amp R gene lacZ gene Restriction site Hummingbird cell Sticky ends Gene of interest Humming- bird DNA fragments Recombinant plasmidsNonrecombinant plasmid Bacteria carrying plasmids Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones

Figure 20.5 Foreign genome Cut with restriction enzymes into either small fragments large fragments or Recombinant plasmids Plasmid clone (a) Plasmid library (b) BAC clone Bacterial artificial chromosome (BAC) Large insert with many genes (c) Storing genome libraries

 Plasmids transfer a gene to a bacterium so it will produce a specific protein. EXAMPLE: Insulin production  Large quantities are produced by inserting a human gene for insulin into a bacterium.

 Isolate Human DNA and Plasmid from DNA  Use Restriction Enzyme to cut DNA  Splice the DNA into the plasmid to create a GENOMIC LIBRARY Thousands of DNA pieces from a genome that have been inserted into a cloning vector

 RECOMBINANT DNA : DNA from 2 or more sources.

DNA FINGERPRINTS  Pattern of bands made up of specific fragments from an individuals DNA.  Banding patterns can determine how closely related different organisms are.

 RFLP: Restriction Fragment Length Polymorphisms 1. Remove DNA and cut into fragments with restriction enzymes 2. Separate the fragments with Gel Electrophoresis  Procedure that separates nucleic acids based on size and charge.

Figure 20.9 Mixture of DNA mol- ecules of different sizes Power source Longer molecules Cathode Anode Wells Gel Shorter molecules TECHNIQUE RESULTS 1 2    

3. Make visible only the bands being compared. DNA fragments are blotted onto the filter paper. 4. Form PROBES:  Radioactive segments of DNA complementary to the segments being compared.  Form visible bands when exposed to photographic film.  Bands can be analyzed

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

Figure DNA  restriction enzyme TECHNIQUE I Normal  -globin allele II Sickle-cell allele III Heterozygote Restriction fragments Nitrocellulose membrane (blot) Heavy weight Gel Sponge Alkaline solution Paper towels II IIII II IIII II IIII Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting) Radioactively labeled probe for  -globin gene Nitrocellulose blot Probe base-pairs with fragments Fragment from sickle-cell  -globin allele Fragment from normal  - globin allele Film over blot Hybridization with labeled probe Probe detection 5

 Based on how unique the prints are  A complete DNA sequence is NOT USED, only a small portion.  VERY ACCURATE since they focus on unique regions – (non-coding areas)  They look for repeat patterns at 5 different sites.  Less than 1 in 1 million chance of non- twins having the same patterns

 Procedure for making many copies of the selected segments of the available DNA

What is needed and the procedures: 1. A sample of DNA 2. A supply of the 4 DNA nucleotides (A,T,C,G) 3. DNA Polymerase (Taq) 4. PRIMERS: › Artificially made single strand of DNA required to initiate replication

What is needed and the procedures: 5. Incubation (with all ingredients) 6. DNA will quickly double – Every 5 Minutes 7. New samples will make a DNA fingerprint 8. Only need about 20 blood cells to make a sample

Figure 20.8 Genomic DNA Target sequence Denaturation Annealing Extension Primers New nucleotides Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence TECHNIQUE

THE START OF THE PROJECT: In 1990, the National Institutes of Health (NIH) and the Department of Energy joined with international partners in a quest to sequence all 3 billion base pairs, in the human genome  In 1990, the National Institutes of Health (NIH) and the Department of Energy joined with international partners in a quest to sequence all 3 billion base pairs, in the human genome  Projected to take 15 years to complete

The Completion of the Project:  In April 2003, researchers successfully completed the Human Genome Project  Under budget and more than two years ahead of schedule.

Figure Cytogenetic map Genes located by FISH Chromosome bands Linkage mapping Genetic markers 1 Physical mapping 2 Overlapping fragments DNA sequencing3

Figure DNA (template strand) TECHNIQUE 5 3 C C C C T T T G G A A A A G T T T DNA polymerase Primer 5 3 P P P OH G dATP dCTP dTTP dGTP Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) P P P H G ddATP ddCTP ddTTP ddGTP 5 3 C C C C T T T G G A A A A DNA (template strand) Labeled strands ShortestLongest 5 3 ddC ddG ddA ddG ddT ddC G T T T G T T T C G T T T C T T G G T T T C T G A G T T T C T G A A G T T T C T G A A G G T T T C T G A A G T G T T T C T G A A G T C G T T T C T G A A G T C A Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand G G G A A A C C T

Cut the DNA into overlapping frag- ments short enough for sequencing. 1 Clone the fragments in plasmid or phage vectors. 2 Sequence each fragment. 3 Order the sequences into one overall sequence with computer software. 4 Figure

What have we achieved with the HGP:  Fueled the discovery of more than 1,800 disease genes.  There are more than 1,000 genetic tests for human conditions.

The Future:  Completion of the HapMap (a catalog of common genetic variation, or haplotypes) HapMap  Genetic factors for many common diseases, such as heart disease, diabetes, and mental illness, will be found in the next few years.

 Having one’s complete genome sequence will make it easier to diagnose, manage and treat many diseases.

 Powerful form of preventive, personalized, and preemptive medicine.  Tailoring recommendations to each person’s DNA, health care professionals will be able to work with individuals to focus efforts on the specific strategies Examples:  Diet and high-tech medical surveillance

 Technique that uses genes to treat or prevent disease.  Treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. EXAMPLES:  Replacing a mutated gene that causes disease with a healthy copy of the gene.  Inactivating, or “knocking out,” a mutated gene that is functioning improperly.  Introducing a new gene into the body to help fight a disease.

 Nasal sprays for CF patients Nasal sprays for CF patients

Gene Therapy has had limited success  It poses one of the greatest technical challenges in modern medicine 1. Corrected gene must be delivered to several million cells 2. Genes must be activated 3. Concern that the genes may go to the wrong cells. 4. Concern that germ cells (sex cells) would get the genes and be passed to offspring

5. Immune response – body fight off the vector as a foreign invader. 6. Gene gets “stitched” into a wrong space and knocks out an important gene Patients treated for SCID’s developed Leukemia – It was found that new gene interfered with a gene that controls the rate of cell division.

 Altering GERM-LINE (sex cells)  Genetic enhancement  Concerns with past practices of EUGENICS – Adolf Hitler

Gene Therapy and Vision GENE THERAPY VIDEO #1

 Can be produced more inexpensively INSULIN: produced in bulk by bacteria

 VACCINE: Harmless version of a virus or bacterium  DNA technology may produce safer vaccines

 Can insert genes into plants to make them resistant to pests  Crops that don’t need fertilizer  EX: Genetically enhanced tomatoes that ripen without becoming soft

 FDA requires scientific evidence that allergy-inducing properties have not been introduced into the food.  If a food contains a new protein, carbohydrate or fat it must be approved by the FDA for sale.  Concerns that they could spread  “SUPERWEEDS”

Figure Mammary cell donor TECHNIQUE RESULTS Cultured mammary cells Egg cell from ovary Egg cell donor Nucleus removed Cells fused Grown in culture Implanted in uterus of a third sheep Embryonic development Nucleus from mammary cell Early embryo Surrogate mother Lamb (“Dolly”) genetically identical to mammary cell donor

Figure 20.20