Recombinant
DNA &
Biotechnology
Con’t
DNA Ligase
Gene Insertion
•
Why create recombinant DNA?
–
To
the gene you want (get lots of copies)
–
Must
transfect
(insert into) a host cell that will then
produce desired product
–
Include a
gene
(with a distinct, easily
distinguished phenotype)
Problem:
•
Prokaryotes & eukaryotes differ
significantly in structure and gene
expression
–
Prokaryotes cannot remove introns
(no spliceosomes)
–
Little post
-
translation modification
•
Eukaryotes do not have
plasmids, so how to get
recombinant DNA into the
nucleus
AND
inserted into host
DNA?
Vectors
•
Need to add desired gene/DNA
and
get it to
replicate when the host divides
–
New section must have gene + origin site
(
replicon
or replication unit)
–
Could insert gene randomly & hope to add near
host origin
–
Or, could add to
:
•
Replicate independently of host (pro/euk ori’s differ)
•
Have a recognition sequence for restriction enzymes
•
Contain a reporter gene
•
Be than the host chromosome
Step 2: Integration
•
Reporter genes distinguish successful
insertion of recombinant DNA
Recombinant DNA Sources
•
Genes come from
1.
: chromosomes that have been
digested with restriction enzymes & all possible
fragments grown in colonies
2.
cDNA
(complementary DNA): collections of
tissue
-
specific genes obtained via reverse
transcriptase and mRNA
3.
: artificial DNA created using the
genetic code & known AA sequences
Applications
•
: aka
homologous recombination
= creating an organism with 2 nonfunctional
alleles for a gene (common in determining
role of genes, esp. in early development)
•
: using antisense RNA to block
translation of mRNA
•
DNA chips
(microarrays): multi
-
task (check
for multiple sequences simultaneously)
Biotechnology
•
Using living things to produce things useful to
people
–
Ancient forms include domestication, agriculture,
selective breeding programs
–
Modern forms based on use of some bacteria,
yeast, & others as manufacturers of desired
products
–
Major issue: how to get expression of desired
gene?
•
Historically collect naturally occurring product
•
Modern: collect using transgenics &
expression vectors
Expression Vectors
•
Vectors with normal characteristics plus
transgene
sequence(s) that will cause
desired gene to be expressed. How?
1.
Inducible promoters
2.
Tissue
-
specific promoters (local vs global)
3.
Signal sequence (to help the product go
where it should)
Medical Applications
•
Introduce product into a
patient naturally lacking
it:
–
Ex: growth hormone,
Factor VIII, insulin
•
Introduce it into a patient
temporarily requiring it:
–
Ex: tissue plasminogen
activator (TPA), platelet
-
derived growth factor
Pharming
•
An agricultural
practice to
generate larger
quantities of
desired
products
(produce
proteins in milk)
rather than
using bacteria
or yeast
Biotech & Cultivation
•
Create transgenic
plants &
economically
valuable animals
with improved
characters
–
Additional nutrients
(ie golden rice)
–
Environmental
resistance (ie
drought
-
tolerance,
disease
-
and pest
-
resistance)
Animals & Biotech
•
Cloning
–
Concept: maintain good traits in
stock
–
Problem: behavior is
environmentally influenced, not
just genetic
–
Non
-
agricultural cloning uses:
•
Organ transplanting
•
Service
-
animal production
•
Conservation efforts
Problems With Genetic
Modification
•
Public resistance
•
In practice, hard to get
rid of undesired traits
Can You…
•
Name the 3 major sources of vectors, AND
the 4 general characteristics necessary for a
good vector (what is the difference between a
vector and an expression vector)?
•
List the sources of DNA (genes) for cloning &
explain how each works?
•
Tell me what a knockout is? How antisense
RNA can act as a silencer?
•
Answer the question: what is biotechnology?
How is it utilized in medicine? Agriculture?
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