Biotechnology

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Dolly

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

Sticky Ends

Escherichia coli

Eco RI

sticky end

Blunt ends

Haemophilus aegyptius Hae III

G G C C

C C G G

G G C C

C C G G

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

papaya's ringspot disease gene was introduced to

control the plague

http://www2.dpi.qld.gov.au

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

Analyzing Gene Expression Analyze genomes of other important species

for genetic engineering

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?

Who should have right to examine someone’s genetic info?

How should that info be used? Should a person’s genome be a factor in

determining eligibility for a job or insurance

The End