Biotech (20).pptx - kraffleck

twoeggfinnishBiotechnology

Dec 14, 2012 (4 years and 10 months ago)

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Chapter 20


DNA Technology and Genomics

DNA Cloning


making multiple copies


of a single gene


Uses bacterial plasmids


Insert gene of interest into plasmid to make
recombinant DNA
(DNA from two sources)

DNA Cloning

DNA Cloning

Various uses for
cloned gene

Making Recombinant DNA

Restriction Enzymes:
bacterial enzymes that cut DNA at specific
locations


Cuts at specific
restriction sites



Cut pieces called
restriction fragments



Leaves “sticky ends”


Restriction enzymes occur naturally only in bacterial cells. Used
as a defense against foreign (viral) DNA

Recombinant DNA

1.
DNA cut with restriction
enzyme


2.
DNA from a different source
also cut with the same
restriction enzyme


3.
Two DNA sources are mixed so
sticky ends will combine (base
pair) forming recombinant
DNA


4.
DNA ligase seals backbone



1

2

3

Recombinant DNA

4

DNA Cloning


The Full Picture


1.
Plasmid from bacterial cell containing
gene for antibiotic resistance isolated.


2.
Human DNA extracted.


3.
Both bacterial plasmid and human DNA
cut with same restriction enzyme.


4.
Bacterial and human DNA combine due
to base pairing of “sticky ends”


5.
Plasmid inserted into bacteria

6.
Plate and grow bacteria on antibiotic
medium


7.
Why is antibiotic resistance gene
important?


8.
Only bacteria
that have taken up
recombinant plasmids will grow





bacteria containing
all DNA fragments =
gene library

Questions:


How large is the human genome?


How many restriction sites are on the human genome?


How many DNA fragments can be made from one restriction


enzyme?

Nucleic acid hybridization:
technique to find gene of interest
in gene library

Nucleic Acid Hybridization

Filter paper
pressed
against
master plate
to collect
some cells.

Filter paper is
treated to
denature DNA
and mixed with
radioactive
probe.

Filter paper is
laid under
photographic
film.
Radioactivity
exposes film.

Location of
“spots” on
film show
colonies which
contain
desired gene.

DNA Cloning


The Full Picture

Missing Detail

Question:

Why can’t a human (eukaryotic)
gene be directly inserted into a
bacterial cell to yield a functional
protein?

cDNA

must be made from human
mRNA using
reverse transcriptase

A recombinant DNA molecule is one
that is

A.
Produced through the process of crossing
over that occurs in meiosis

B.
Constructed from DNA from different
sources

C.
Constructed from novel combination of
DNA from the same source

D.
Produced through mitotic cell division

A DNA library is

A.
An orderly array of all the genes within an
organism

B.
A collection of vectors

C.
The collection of plasmids found within a single
E. coli.

D.
A collection of DNA fragments representing the
entire genome of an organism

Polymerase Chain Reaction
-

PCR

Amplify (make lots of copies of) DNA outside of a cell
(in vitro)

PCR requires:

1.
Primers complementary to either end of gene of interest

2.
Nucleotides to build copies of DNA

3.
Polymerase (heat resistant)

4.
Genomic DNA to be copied




Polymerase Chain Reaction
-

PCR

Step 1: Denature

Heat DNA so stands separate


Step 2: Annealing

Cool so primers bond with target
gene


Step 3: Extension

DNA polymerase adds base pairs
making complementary strand

Polymerase Chain Reaction
-

PCR

Repeat cycle
many times


Amount of target
DNA double with
each cycle

Polymerase Chain Reaction
-

PCR

Initial

DNA

segment

Number of DNA molecules

2

4

8

Allows for large amount of
DNA to be made in a very
short time (3 to 24 hours)


DNA can be amplified from
a very small sample


Ancient DNA from fossils


Crime scene


Many applications


Gel Electrophoresis

Separating large molecules (DNA fragments or proteins) based
on size

Gel acts a maze impeding
movement of fragments


Separates fragments based
on length

Gel Electrophoresis

Result: Banding pattern of
fragments sorted by length

Medical
D
iagnosis

1.
PCR gene of interest
from tissue sample


2.
Sickle cell gene is
missing a restriction
site


3.
Normal gene shows
three bands while
mutant gene show 2
bands


Southern Blotting


isolating bands of interest

Because the human genome is so large, if we did a restriction
digest and ran a gel of the entire genome, resulting gel would be
a smear.


Too many fragments

Southern blotting is a technique to highlight only the DNA
fragments that contain areas (genes) of interest

Southern Blotting


isolating bands of interest

Gel is placed on blotting
paper that pulls up and
denatures DNA

Southern Blotting


isolating bands of interest

Blotting paper is mixed with a probe
that binds only to gene fragments of
interest

Probe is analyzed

Individual III is a
heterozygote for the
sickle cell gene

What is the basis of separation of different
DNA fragments by gel electrophoresis?

A.
The negative charge on DNA

B.
The size of the DNA fragment

C.
The sequence of the DNA fragment

D.
The presence of a dye

[Default]

[MC Any]

[MC All]

Molecular hybridization is used to

A.
g
enerate
cDNA

from mRNA

B.
i
ntroduce a vector into a bacterial cell

C.
f
ind the gene of interest in a DNA library

D.
i
ntroduce mutations into genes

[Default]

[MC Any]

[MC All]


Individuals
have mutations (changes in
nucleotide sequences) called
single
nucleotide polymorphisms (SNPs)



Some
SNPs alter restriction sites and
cause
RFLPs (
Restriction Fragment
Length Polymorphisms)



RFLPs are inherited in a
Mendelian

fashion

Allele 1

Allele 2

w

x

y

Cut

Cut

Cut

z

y

DNA from chromosomes

DNA Fingerprinting
-

RFLP

Everyone’s DNA will cut in a
unique manner

Radioactive probes are used
identify certain sequences

Restriction fragment

preparation

1

Restriction

fragments

Gel electrophoresis

2

Blotting

3

Filter paper

Probe

Radioactive probe

4

Detection of radioactivity

(autoradiography)

5

Film

Radioactive

probe (DNA)

Mix with single
-

stranded DNA from

various bacterial

(or phage) clones

Single
-
stranded

DNA

Base pairing

indicates the

gene of interest

DNA Fingerprinting

Which of the
suspects were at
the crime scene?

Which of the following statements is
accurate for DNA replication in your cells,
but not PCR?

A.
Primers are required

B.
DNA polymerase is stable at high
temperatures

C.
Ligase is essential

D.
Free DNA nucleotides are necessary

[Default]

[MC Any]

[MC All]


Genome (n) = 23 chromosomes, ~ 3.2 billion nucleotide pairs


about
20
-
25,000

genes and a huge amount of
noncoding

DNA



ONLY 1
-
1.5% OF THE HUMAN GENOME IS MADE OF GENES!


Proteome = about 100,000 different proteins via alternative
splicing




genes






~
1 %



introns






24
%




heterochromatin (
centromeres

& telomeres)

20
%



simple sequence repeats





3
%



duplicated sequences





7
%



transposable elements




45
% !!

THE HUMAN GENOME

A Scientific Revolution


Genetic engineering

is the process of moving
genes from one organism to another


This technology has many useful applications


The
production of vaccines, cancer
drugs, and pesticides


Engineered bacteria that can clean up
toxic wastes


Having a major impact on agriculture &
medicine


Increasing yields

Curing disease

Producing insulin

In 1982, the world’s first

genetically engineered

pharmaceutical
product


Has only one extra gene: HGH


Herbicide resistance


Crop plants have
been created that
are resistant to
glyphosate


Herbicide resistance offers two main advantages


1
. Lowers the cost of producing crops


2. Reduces plowing and conserves the top soil

Petunias

Glyphosate
-
sensitive plants

Glyphosate
-
resistant plants

Genetically Modified
Organisms (GMO)

July 2000

Golden Rice


Genetically modified with beta
carotene to combat vitamin B
deficiency


Met with opposition and never
widely used


Several U.S. regulatory agencies evaluate biotechnology projects for potential risks:


Department of Agriculture


Food and Drug Administration


Environmental Protection Agency


National Institutes of Health

Transgenic Crops in U.S. (1996
-
2003)

Potential Risks of Genetically Modified (GM) Crops



2 Are GM foods safe to eat?


So far every ‘
serious’

scientific
investigation has concluded
that GM foods are safe


However, people have a right
to know what is in their food



Introduced proteins may cause
allergies in humans



Genetic engineering holds promise


but, it has generated considerable controversy and protest





1.
Are genetic engineers “playing God” by tampering with


the genetic material?




all agriculture is the result of selective breeding


3. Are GM foods safe for the environment?

Three legitimate concerns are raised


a. Harm to other organisms



Will other organisms be harmed
unintentionally?


b. Resistance


Will pests become resistant to
pesticides?


c. Gene flow


What if introduced genes will pass
from GM crops to their wild or
weedy relatives?


d. Decreased genetic variability


Potential Risks of Genetically Modified (GM) Crops