Recombinant DNA & Biotechnology Con't

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22 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

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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?