Recombinant DNA & Biotechnology Con't

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?