Chapter on Biotechnology

lowlytoolboxBiotechnology

Oct 22, 2013 (3 years and 9 months ago)

73 views

Biotechnology

repairstemcell.files.wordpress.com

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 for quality


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 be

isolated

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

reverse

transcriptase

plus mRNAs

mRNAs

mRNA is degraded

by an enzyme

DNA polymerase

synthesizes the 2nd

strand

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 is


considered a "book"

phage

clones

recombinant

phage "book"

foreign genome


Bacterial Artificial Chromosome (BAC)

Library

Copies of
multiple


DNA fragments can

be stored in a bacterial

artificial chromosome

plasmid with many genes

BAC

clone

each clone

occupies one

well

Gene products

Product



Made in



Use



human insulin



E. coli




diabetes

human growth hormone


E. coli



growth defects

epidermal growth factor


E. coli



burns, ulcers

interleukin
-
2



E. coli



possibly cancer

bovine growth hormone


E. coli


improving weight gain

cellulase




E. coli


breakdown of cellulose

Taxol




E. coli



ovarian cancer

hepatitis B vaccine


S. cerevisiae


prevents hepatitis

erythropoietin



mammalian cells


anemia

factor VIII



mammalian cells


hemophilia

tissue plasminogen activator

mammalian cells


heart attacks


Other Examples


"golden rice"


genetically modified


rich in beta
-
carotene


prevents blindness

news.bbc.co.uk


papaya's ring spot 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


publicly 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