Chapter 12 DNA Technology and Genomics
A. Plasmids
Bacterial origin: circular DNA sequence: not
associated with the bacterial chromosome
Recombinant DNA technology to give:
1) Therapeutic effect
2) provide “better” resistance to pests
3) clean up oil spills
The little factory that could …
1) isolate the plasmid from bacterium
2) Grab DNA with gene of interest from
host cell (e.g., human)
3) splice that gene with the plasmid ->
this will produce recombinant DNA
4) bacterial cell will take up this plasmid
(with the foreign) DNA through the process
of bacterial transformation = call this
bacterium: recombinant bacterium
5) recombinant bacterium will reproduce
gene products through transcription of
recombinant DNA and translation of the
resulting mRNA into a gene product
(polypeptide)
Genetic engineering
B. Enzymes to cut and paste DNA
-Restriction enzyme: recognize a particular
sequence
-And then cutting of DNA into fragments
Fragments will contain a sticking
end:TTAA
-Add another DNA fragment from another
source: “glue” together
-DNA ligase: “pastes” together the
fragments, the foreign DNA to produce the
recombinant DNA molecule
C. Genes can be cloned in recombinant
plasmids
1. isolate DNA from two sources
2. cut both DNAs with same restriction
enzyme
3. Mix DNAs together; let them base pair
and
4. add DNA ligase to bond the DNA
covalently
5. form recombinant DNA and then place
the plasmid into the bacterium via
transformation
6. clone the bacteria
7. isolate gene product
D. Cloned Genes can be stored in genomic
libraries
BIOINFORMATICS
Store these cloned genes in genomic library
Collections of phage clones (viruses) and of
bacterial clones
E. Reverse transcriptase helps make genes
for cloning
1. chosen cells transcribe their genes into
mRNA
2. researcher isolate the mRNA and make
single-stranded DNA, usng the enzyme
reverse transcriptase
3. these enzymes will break down mRNA
4. DNA polymerase to synthesis the second
strand
This procedure allows the synthesis of a
gene WITHOUT introns! This means we
have a gene that can be immediately
expressed.
In nature, DNA contains genes with Introns
and Exons
F. Recombinant cells and organisms can
mass-produce gene products
List of products made with recombinant
DNA technology
Human insulin
Human growth hormone
Epidermal growth factor
Interleukin-2
Bovine growth hormone
Cellulase
Taxol
Interferons (alpha and gamma)
Hepatitis B vaccine
Erythropoietin (EPO)
Factor VIII
Tissue plasminogen activator
G. DNA technology is changing the
pharmaceutical industry and medicine
Pharmaceuticals, vaccines, hormones
H. Nucleic acid probes identify clones
carrying specific genes
Nucleic acid probe will be able to recognize
certain nucleotide sequences
Trace genetic ancestry
I. DNA microarrays test for the expression
of many genes at once
DNA microarray test: special wells with
treated DNA that fluoresces
Used to test for toxicity, disease
mechanisms, patterns of gene expression in
tumors
J. Gel electrophoresis sorts DNA molecules
by size
Electrophoresis to separate DNA molecules
by their molecular weight
Long molecules: high molecule weight
Short molecules: lower molecular weight
Applying electricity to the molecule and the
molecule will move to one end of the device
based on its molecular weight
DNA fingerprinting
K. Restriction fragment length
polymorphisms can be used to detect
differences in DNA sequences
Number of restriction fragments and their
size reflect the specific sequence of
nucleotides in the starting DNA
Any differences in these restriction
fragments are called RFLPs (restriction
fragment length polymorphisms)
Take DNA from chromosomes – cut
them up with the restriction enzyme
And then compare the fragments (long ones
and the short ones) and check for
commonality
1) crime scene and the suspect
2) Check out for harmful alleles
Dominant
Recessive
Check out use of recombinant DNA
technology to “cure” sickle cell anemia!
L. DNA technology is used in courts of law
Can free prisoners based on DNA evidence
Can imprison defendents
M. Gene therapy may someday help treat a
variety of diseases
Bone marrow transplants – save people
who have leukemia
In vitro gene therapy
Cystic fibrosis – can use spray
Ex vivo gene therapy
N. PCR method is used to amplify DNA
sequences
PCR: Polymerase chain reaction
Allows for replication of DNA sequences
Used for DNA fingerprinting, especially
from very old samples and from
samples that are very small
O. Human Genome Project is an application
of DNA technology
1. genetic linkage mapping – get markers
2. physical mapping – number of base pairs
between markers
3. DNA sequencing
Map of the human genome
P. Most of the human genome does not
consist of genes???
Non-coding DNA versus coding DNA
Repetitive DNA: nucleotide sequences
present in many copies
Telomeres: repetitive DNA at
chromosome ends
If lose telomeric DNA -> can lead to
cell death
Transposons: DNA segments that can
move or be copied from one location to
another in a chromosome or between
chromosomes
Q. Science of genomics compares whole
genomes
Genomics: whole sets of genes
Humans: 2.9 billion base pairs and
25,000 genes
Proteomics: full protein sets
Catalog life on a global perspective?
R. Genetically modified (GM) organisms are
transforming agriculture
GM organisms: have acquired one or more
genes by artificial means rather than
through traditional breeding methods
Plasmid – bacterial DNA
Transgenic plants
Transgenic animals
S. Could GM organisms harm human health
or the environment?
Genetically Modified ….
Add genes for herbicide resistance??