Genetic Engineering (Chapter 10, Little Alberts)

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12 Δεκ 2012 (πριν από 4 χρόνια και 9 μήνες)

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Genetic Engineering

DNA Interactive:
http://www.dnai.org/


Using recombinant DNA technology, we can:


Sequence all the genes in an organism.


Amplify genes (e.g., for forensics or to find viruses).


Express rare proteins or construct new proteins by
splicing together pieces of genes, mutating genes, or
synthesizing genes.


Genetically alter animals (insert transgenes).


Diagnose genetic diseases.


Identify criminals or exonerate the innocent.


Isolating genes
--

a daunting problem until the
discovery of restriction nucleases


Genes are part of a larger DNA molecule. Even in a “simple”
case,
E. coli

(4.6 x 10
6

nucleotides), it would be hard to isolate
and study a single gene from a large chromosome.



Can break DNA into small pieces by mechanical shear, but
fragment containing single gene is one of >10
5

DNA fragments
obtained.



Solution:
Restriction nucleases

produce reproducible set of
specific DNA fragments from a genome.


A
nuclease

catalyzes hydrolysis of phosphodiester bond in nucleic
acid.


A
restriction

nuclease

cuts dsDNA in a sequence
-
specific manner.

Restriction nucleases

Figure 10
-
4, Little Alberts

Uniqueness / fragment lengths:

4
-
base hitter: 1 in 4
4

= 256

6
-
base hitter: 1 in 4
6

= 4096

8
-
base hitter: 1 in 4
8

= 65,536

Many restriction nucleases
generate “sticky ends” (short
single
-
stranded overlaps). Cut
DNA molecules can be joined
--

important for DNA cloning.

DNA molecules can be separated by size using gel electrophoresis

Figure 10
-
5A,B; Little Alberts

DNA fragments move through an agarose gel towards the positive electrode. Fragments
are separated by size.

A “restriction map” is a physical map of a region of DNA that shows the location of
each restriction enzyme site.

Which part of the structure of DNA is responsible for its
migration towards the positive electrode in gel
electrophoresis?


1) The bases


2) Ribose sugars


3) Hydrogen bonds between the bases


4) Phosphate groups


5) Fatty acids

Clicker question


A DNA fragment migrates through an agarose gel
according to its size.

_____ fragments migrate faster (closer to the _____
electrode) than ______ fragments.


1) Large, negative, small


2) Large, positive, small


3) Small, negative, large


4) Small, positive, large


Clicker question


A DNA fragment migrates through an agarose gel
according to its size.

_____ fragments migrate faster (closer to the _____
electrode) than ______ fragments.


1) Large, negative, small


2) Large, positive, small


3) Small, negative, large


4) Small, positive, large


Clicker question

See B
-
12 to B
-
13 at end of textbook for explanation of electrophoresis

Natural processes by which bacteria
exchange genes

Bacteria evolved to take up DNA from their surroundings. DNA can be
integrated into the genome or maintained independently as plasmid DNA*.


Figure 10
-
19; Little Alberts

*Plasmid DNA contains its own origin of replication so it can be propagated separately
from the chromosomal DNA.


Where do restriction nucleases come from?


Transfer of DNA between certain strains of bacteria
is “restricted”.



Restriction nucleases used by these bacteria to
degrade foreign DNA (e.g., from a virus).



Bacterial DNA is protected from digestion by
chemical modification (e.g., methylation).


Properties of restriction enzymes and their
targets



Target sequences are short (4
-
8 base pairs), so
will occur by chance.




A given restriction nuclease always cuts a given
DNA molecule at the same sites; always produces
same set of DNA fragments.




Hundreds of different restriction nucleases
now available commercially.

DNA cloning


Cleave DNA using restriction nucleases and separate fragments on a gel.


Join DNA fragments using DNA ligase, an enzyme that normally reseals
nicks in DNA backbone during DNA replication and repair.


Can recombine isolated DNA fragments to produce new DNA molecules
not normally found in nature.


New piece of DNA can be introduced into DNA of a host cell, then will be
replicated and transcribed.

Figure 10
-
19A,B; Little Alberts

Recombinant DNA movie

The animation opens with a view of a DNA plasmid loop. An EcoRI enzyme approaches and attaches
to the DNA's major groove. The enzyme then runs along the groove scanning the DNA for the
base sequence xGAATTCx. When it finds this sequence it breaks the sugar
-
phosphate bonds on
either side of DNA, splicing the plasmid. A gene (glowing DNA) with complementary 'sticky ends"
then attaches to the end of the plasmid. The enzyme DNA Ligase (looking like frozen peas in this
animation) then repairs the nicks in the sugar
-
phosphate backbone, joining the two DNA strands.

Molecular biologists developed ways to introduce DNA into
commonly
-
used laboratory strains of bacteria (
E. coli
)




Bacterial plasmids
can be used as
cloning
vectors
. Can
propagate plasmids in
bacteria, then purify
them, cut them, and
insert new DNA
fragments into them.

Figure 10
-
21, 10
-
22; Little Alberts

Plasmid DNA is introduced into
E. Coli

bacteria by the process of
transformation
.

Millions of copies
of recombinant
DNA can be
produced as
plasmids in
bacteria

*recombinant
DNA
--

combining
DNA from ≥2
sources

*

Isolating genes from eukaryotic cells is
easiest in a form without introns


Genes encoding small proteins can
be >100,000 bp because of introns.



Easiest to work with genes
containing only coding sequence.



Make complementary DNA (cDNA)
from messenger RNA (mRNA) using
reverse transcriptase (RT)
. (RT
enzymes come from retroviruses*).




Can sequence part of a protein,
synthesize DNA corresponding to
the protein sequence, then probe a
cDNA library to isolate the gene to
get its complete sequence.

Figure 10
-
26; Little Alberts

*
The retroviral RTs used for molecular biology have significantly lower error rates (~10
-
fold) than HIV RT.

Error rates for RTs from Promega: Moloney Murine Leukemia Virus RT: 1 in 30,000; Avian Myeloblastosis virus RT: 1 in 17,000.

Cloned DNA can be used to make rare or new proteins

Link pieces of different
genes

Figure 10
-
31, Little Alberts

Figure 10
-
33, Little Alberts

Produce a protein in
prokaryotic or eukaryotic cells

Many experiments in biology use proteins fused to

Green Fluorescent Protein (GFP)

Express

Gene for your favorite protein

Gene for GFP

Your favorite protein

These are made by linking the genes for GFP and the protein using recombinant DNA technology.

Your protein can now be
visualized using
fluorescence microscopy

GFP was discovered in the
jellyfish
Aequorea victoria.

http://www.conncoll.edu/ccacad/zimmer/GFP
-
ww/

Original site
-
directed mutagenesis protocol

http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/site.html

PCR

http://bio
-
rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

PCR movie

http://bio
-
rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

PCR has revolutionized molecular biology


Can clone any gene (genomic copy or cDNA copy) if beginning and end
sequences are known.


Can amplify rare sequences (useful for forensics, cloning ancient
animals/humans).


Can amplify variable regions of genome (useful for forensics, paternity
testing).


Can introduce restriction sites,
make site
-
directed mutants
, construct
genes (iterative PCR).


Can quantitate amounts of a message or gene (quantitative PCR).


Can detect pathogen infection at early stages
--

one test for HIV
infection is by RT
-
PCR.


Figure 10
-
29, Little Alberts

RT
-
PCR test for HIV
detects a 142 base
target sequence in a
highly conserved region
of the
gag

gene of
HIV.

PCR amplification of DNA

Figure 10
-
27, Little Alberts

Another potentially exciting (and potentially
dangerous) development in biotechnology:

Synthesis of DNA



A complete poliovirus genome (7500 bases of DNA,
then converted to RNA) was synthesized and
assembled with poliovirus proteins in the absence of
cells.*



Pathogen sequences are available on the web.



Synthesis of DNA is not yet regulated.

* Wimmer, E (2006) The test
-
tube synthesis of a chemical called poliovirus
--

The simple
synthesis of a virus has far
-
reaching societal implications. EMBO Reports 7: S3
-
S9.