Potential Uses for Cloned Genes to produce a protein product
plasminogen activator
to endow an organism with a metabolic capability engineered bacteria that degrade oil spills
create more copies of the gene for further study
Genetic Engineering Stages
1. Isolate gene cleavage of plasmid 2. Recombinant-DNA 3. Clone * Screen 4. Harvest
Gene Protein
1. Isolate Gene
Isolate the desired gene from the genome
the desired gene will be cut with restriction endonucleases (enzymes)
desired gene to beisolated
Cleavage
cut DNA of host with restriction endonucleases (enzymes)
several hundred RE’s exist discovered 1960s
produce sticky ends or blunt ends
2. Recombinant DNA
splice desired gene into host DNA, DNA ligase seals the strands
Vector, generally plasmid of bacteria (prok) or yeast (euk) viral DNA (not for harvesting protein)
plasmid with recombinant DNA
3. Clone
produce a cell line in which all members have identical copies of a particular gene
Screen Choose cells that carry desired gene & eliminate those cells that do not carry desired gene
4. Harvest (or Isolate)
harvest protein harvest gene
genetic harvesting protein harvesting
pest resistant gene oil eating bacteria dissolving clot protein human growth hormone
copies of the gene
protein molecules
Cloning
Directly from an organism complementary DNA
made from mRNA template through
reverse transcription (cDNA) Reverse transcriptase can be used
to make smaller cDNA libraries These contain only the genes that are
transcribed by a particular type of cell recognized by the addition of a RE
recognition sequence to it
reversetranscriptase plus mRNAs
mRNAs
mRNA is degradedby an enzyme
DNA polymerasesynthetizes the 2ndstrand
cDNA
Genomic Libraries
“Book,” a clone containing a foreign DNA
Plasmid library (bacterial, yeast) Phage library (virus) Bacterial Artificial Chromosome library
Plasmid Library
Copies of DNA fragments can be stored in a cloned bacterial plasmid
Each one of these is considered a "book"
recombinant plasmid
bacterial clones
foreign genome
Phage Library
DNA fragments can be stored in a cloned phage
each phage type isconsidered a "book"
phage clones
recombinant phage "book"
foreign genome
Bacterial Artificial Chromosome (BAC) LibraryCopies of multiple
DNA fragments can
be stored in a bacterial
artificial chromosome plasmid with many genes
BACclone
each clone occupies one well
Gene products Product Made in Use
human insulin E. coli diabeteshuman growth hormone E. coli growth defectsepidermal growth factor E. coli burns, ulcersinterleukin-2 E. coli possibly cancerbovine growth hormone E. coli improving weight gaincellulase E. coli breakdown of celluloseTaxol E. coli ovarian cancerhepatitis B vaccine S. cerevisiae prevents hepatitiserythropoietin mammalian cells
anemiafactor VIII mammalian cells hemophiliatissue plasminogen activator mammalian cells heart
attacks
Other Examples
"golden rice" genetically modified rich in beta-carotene prevents blindness
news.bbc.co.uk
Human Genome Project
Collaborative effort to map and sequence entire human genome
Began 1990 4 goals
genetic (linkage) mapping physical mapping sequencing analyzing the genomes of other species
Genetic Mapping of the Human Genome to locate genetic markers spaced evenly
throughout the chromosomes
to make it easier to find other loci
Physical Mapping of the Human Genome cutting chromosomes into identifiable
fragments then determining their order on the chromosome
Sequencing the Human Genome determining the exact nucleotide pairs haploid set of human chromosomes contains
approximately 3 billion nucleotide pairs Genbank
Database where DNA sequences have been deposited publically available via the Internet
final draft, 2004 (over 99% of genome was determined)
remain a few 100 gaps of unknown sequences that require special methods to figure out
Stem Cells
unspecialized blastula cells pluripotent
adult stem cells gives rise to specific types of cells bone marrow blood cells
Applications Medical
Diagnosis Human Gene therapy Pharmaceutical products
animal and plant application gold rice salinity resistant gene
Environmental biofuel oil cleaning bacteria
Forensic evidence The Innocence Project conviction of guilty
Genomes of other species and H. sapiens Bacteria
H. influenzae 1,700 1995 E. coli 4,400 1997
Fungi S. cerevisiae 6,200 1996
Plants Oryza sativa (rice) 60,000 2002
Animals D. melanogaster 13,700 2000 Mus musculus 22,000 2001 Rattus norvegius 25,000 2004 H. sapiens 21,000 2003
Ethical Issues
Should we engineer new genotypes for individuals with anomalies? diabetes, CF, immune deficiencies, MD, stunted
growth, sickle-cell disease myopia, altering personalities, increase length of
life
Should we engineer human germ cells? If they are carrying abnormal genes
eugenics - deliberate effort to control the genetic makeup of human populations color of eyes color of skin color of hair
We have technology to test for diseases for which there is no cure and sometimes no treatment. (Ex. Huntington’s disease, breast cancer)
Would you want to be tested?
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