Chapter 13 – Genetic Engineering

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Dec 12, 2012 (4 years and 8 months ago)

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Biotechnology

Biology III

Bellringer 1/10/12


What are the 4 bases in DNA?


What is
Transcription?


What is
Translation?

What is Biotechnology?


Biotechnology:
is a field of applied biology
that involves the use of living organisms
and bioprocesses in engineering,
technology, medicine and other fields of
requiring bio products.

Selective Breeding


Choose organisms with the desired traits
and breed them, so the next generation also
has those traits


Nearly all domesticated animals and crops


Luther Burbank (1849
-
1926) developed >800
diff varieties of plants in his lifetime

Selective Breeding


For a long time, humans have selected the
best plants and animals to
breed


Why?


Examples?


Milk Cows


1947
-

produced 4,997 lbs... of milk/year


1997
-

produced 16,915 lbs.... of milk/year


Increasing the frequency of desired alleles
in a population is the essence of genetic
technology






Hybridization



The act or process of mating organisms of different varieties or
species to create a hybrid.


In plants


often results in better lines


hybrids are larger, stronger,
etc


In animals


hybrids produced may be weaker and sterile


Ex


wolf x dog
----

weak wolf
-
dog


Ex


horse x donkey
----

mule (sterile)


Inbreeding


Breeding two organisms that are


very similar to produce offspring


with the desired traits.


Ex


dog breeds



Risks



might bring together two
individuals that carry bad recessive genes


many purebred dogs have genetic disorders
that mutts don’t get.

Inbreeding


Mating between closely
related individuals


Why?


Done to make sure that
breeds consistently exhibit a
trait and to eliminate
undesired trait


Creates purebred lines


Can be bad also


Can bring out harmful,
recessive alleles in a “family”

Increasing Variation


Induce mutations


the ultimate source of
genetic variations among a group of
organisms


Mutagens used


radiation and chemicals


Some organisms are formed that have more
desirable variations.

Producing new kinds of bacteria


Can expose millions of bacteria at one time
to radiation


increases chances of
producing a successful mutant.


Ex


bacteria that can digest oil have been
produced this way







Producing new kinds of plants:


Drugs that prevent chromosomal separation
in meiosis have been used to create plants
that have more than two sets of
chromosomes (2n). These are called
polyploid plants.


Ex


bananas, citrus fruit, strawberries, many
ornamental flowers





Diploid corn

Tetraploid corn

What is Genetic Engineering?


Making changes in the genetic code of a
living organism.



-

Transferring of DNA/genes



from one organism to another


-

Also called recombinant DNA
technology or gene splicing.

What is Genetic Engineering?


Genetic engineering can take place:


Within a species (switching genes between
humans)


Or between species (switching genes between
humans and bacteria)


Why is this possible?

What is Genetic Engineering?


Gene:
holds the genetic information to
build and maintain an organism’s cells and
pass genetic traits to offspring.

What is Genetic Engineering?


Genome:
the entirety of an organism’s
heredity information.

How does genetic
engineering take place?

Manipulating DNA


tools of
the molecular biologist

1. DNA extraction


open the cells and
separate DNA from all the other cell parts.

Steps to DNA Extraction

1.
Break the cells open to expose DNA

2.
Remove membrane lipids by adding
detergent

3.
Precipitate DNA with an alcohol


usually
ethanol

or
isopropanol
.


Since DNA is insoluble in these alcohols, it will
aggregate together, giving a
pellet

upon
centrifugation. This step also removes alcohol
-
soluble salt.


2. Cutting DNA


Sequences of DNA are isolated

u
sing
restriction enzymes.


Use restriction enzymes




each one cuts DNA at a


specific sequence of nucleotides
.


(Usually 4
-
6 nucleotides)


This will make different


lengths of DNA

What is the role/function of
restriction enzymes in bacteria?


Many enzyme cut in palindromes


Ex: a protein only cuts at AATT, it will cut the two
fragments at different points
-

not across from each
other (called sticky ends)


Called sticky ends because they want to bond with things
due to their “open” end

Restriction Enzymes

Restriction Enzymes


These sticky ends are beneficial, because if the
same enzyme is used in both organisms, they will
have identical ends and will bond with each other.



Restriction Enzymes


The cut ends (because they are complementary)
can reattach or pair up with any other DNA
fragment or gene cut by the
same restriction
enzyme.


http://www.youtube.com/watch?v=8rXizmLjegI


Restriction Enzymes


Restriction enzymes are used to cut or cleave the
source DNA into fragments called:
RFLP’s
(Restriction Fragments Length Polymorphism)


Because the restriction enzyme’s recognition sequence
is likely to occur many times within the source DNA,
cutting will produce many fragments of different
lengths.


Different RFLP’s may be made by using different
restriction enzymes that recognize different DNA
sequences.



Some Commonly Used

Restriction Enzymes




Eco RI 5'
-
G
|

AATTC



Eco RV 5'
-
GAT
|

ATC



Hin D III 5'
-
A
|

AGCTT



Sac I 5'
-
GAGCT
|

C



Sma I 5'
-
CCC
|

GGG



Xma I 5'
-
C
|

CCGGG




Bam HI I 5'
-
G
|

GATCC




Pst I I 5'
-
CTGCA
|
G



Plasmids


Plasmids:

is a DNA molecule that is
separate from and can replicate
independently of the
chromosomal DNA

Examples of the insertion of
genes

Bellringer 1/19/11


What does restriction enzymes do?


How are restriction enzymes important for
genetic engineering?


How has biotechnology affected you today?


(Give at least one example)

3. Separating DNA


RFLP can be separated (Based on their size)
by
electrophoresis


Since a 3
-
billion base sequence of the 4 DNA
nucleotides can produce more varied
combinations than there are humans, each of us
should have a unique DNA sequence.

Separating DNA


Gel Electrophoresis


1.
Place fragments at one end of a porous gel


we use agarose gel

2.
Apply an electric current


The DNA is
negatively charged and will travel toward the
positive end of the gel.

3.
The larger pieces of DNA move slower, the
smaller ones faster.

4.
Used to compare genomes of different
organisms or different individuals.

5.
Also used to locate and identify one particular
gene out of an individual’s genome.

Gel Electrophoresis

gslc

Click here for animation about
gel electrophoresis

Using the DNA Sequence


Sequence can be read, studied, and changed.


Techniques used to study DNA sequences:


Use DNA polymerase and the 4 DNA bases to
produce a new DNA strand complementary to
unknown strand


some of the bases are dyed.


Dye
-
labeled strands are then separated using gel
electrophoresis and the order of the bands tells the
DNA sequence of the unknown strand.


Once the DNA of interest is isolated it is
recombined with another organisms’ DNA


Cell Transformation:
A cell takes in DNA
from outside the cell.


The external DNA becomes a component of
the cell’s DNA

3. Recombinant DNA

Bacteria Transformation using
Recombinant DNA


Cut a gene with a restriction enzyme out of a
human cell (ex


gene for insulin or growth
hormone work well)


Cut a bacterial plasmid using the same restriction
enzyme (DNA ends will be complementary)


Insert Human gene into bacterial plasmid


Insert plasmid back into bacterial cell


Bacteria will multiply, and all offspring will have
that gene


these bacteria will then follow the
directions of the human gene and make the protein
coded for (insulin or human growth hormone)


Transforming Cells




Use bacterial plasmid to insert desired gene
into DNA


Foreign DNA is first joined to a small, circular
DNA molecule known as a
plasmid
.


Plasmids are found naturally in some bacteria and
have been very useful for DNA transfer.


Transforming Animal Cells


Directly inject DNA into the nucleus of an egg


it will become part of the chromosomes.


Has been used to replace specific genes.







Glowing mouse cells in
embryos that were made
from sperm given the gene
for bioluminescence from
jellyfish


now all the cells
glow!

Making Recombinant DNA


Step 1: To “recombine” or insert genes form
one organism


Must first cut out the desired gene using the
restriction enzyme


With the same restriction enzyme, cut out a
segment of DNA from a plasmid or virus.

Making Recombinant DNA


Step 2: Because the two different sources of
DNA (human and bacteria) were cut with
the same restriction enzyme, the “sticky
ends will allow their DNA to recombine.

Making Recombinant DNA


Step 3: Insert Human gene into bacterial
plasmid


Insert plasmid back into bacterial cell


Making Recombinant DNA


Step 4: Bacteria will
multiply, and all
offspring will have that
gene


these bacteria will then
follow the directions of
the human gene and make
the protein coded

Checking for recombinant cells


Recombinant molecules must be separated
from molecules consisting of just donor
DNA or plasmid DNA.



The experimenter designs the process so
that the plasmid contains two genes that
each enable a cell to grow in the presence of
a different antibiotic drug.

Applications of Genetic
Engineering


Gene for luciferase was isolated from
fireflies and inserted into tobacco plants


they glowed!


Transgenic organisms


contain genes from
other species

A transgenic mouse,
which carries

a jellyfish gene,
glows green under

fluorescent light.




Transgenic Organisms


Bacteria
-

Make human proteins like insulin


Plants


52% of soybeans, 25% of corn in
US in year 2000. Some produce natural
insecticide, some resist weed
-
killers, may
soon be used to produce human antibodies;
rice with vitamin A.



Animals


mice
with immune
systems like
humans; farm
animals that grow
faster and larger
with extra copies of
growth hormone
genes; animals with
leaner meat;
chickens resistant to
bacterial infections.





Cloning


Clone



member of a population


of genetically identical cells


produced from a single cell.




1996


Dolly

cloned




1
st

mammal (sheep) cloned.


She got arthritis several years


earlier than most sheep


Died in 2003