Chapter 14 Genetic Engineering PP Notes

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14 Δεκ 2012 (πριν από 4 χρόνια και 6 μήνες)

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Chapter 14:
Genetic Engineering


-
Modification of the DNA of an organism to
produce new genes with new
characteristics

Biotechnology


Use of organisms to benefit humanity

Recombinant DNA technology


DNA from different organisms is spliced
together


Allows scientists to make many copies of any
DNA segment (clone)


Can introduce foreign DNA into cells of
microorganisms

Recombinant DNA technology


Restriction enzymes


cut DNA


Bacteria produce for defense against viruses


Vector


transports DNA into a cell


Ex: bacteriophage


Plasmid


separate, smaller circular DNA that
maybe be present and able to replicate inside
bacteria


Transformation


uptake of foreign DNA by
cells


How plasmids can get into bacteria

Bacterial
Conjugation
and
Recombination

Recombinant DNA technology


Palindromic sequences


reads the same as
complement, in opposite direction


AAGCTT


TTCGAA


Many restriction enzymes cut these sequences


Restriction enzymes cut on a stagger


sticky
ends

(can pair with complementary single
-
stranded end of other DNA cut with same
enzyme)


DNA Ligase


links 2 fragments


recombinant DNA

Fig. 20
-
3
-
3

Restriction site

DNA

Sticky end

Restriction enzyme

cuts sugar
-
phosphate

backbones.

5


3


3


5


1

One possible combination

Recombinant DNA molecule

DNA ligase

seals strands.

3

DNA fragment added

from another molecule

cut by same enzyme.

Base pairing occurs.

2

Restriction Enzymes

Steps of Creating a Recombinant
DNA Plasmid (Basic)


1. Plasmids and desired DNA cut by same
restriction enzyme


2. Mix 2 types of DNA so sticky ends pair


3. DNA ligase forms bonds between fragments

Fig. 20
-
2

DNA of

chromosome

Cell containing gene

of interest

Gene inserted into

plasmid

Plasmid put into

bacterial cell

Recombinant

DNA (
plasmid
)

Recombinant

bacterium

Bacterial

chromosome

Bacterium

Gene of

interest

Host cell grown in culture

to form a clone of cells

containing the “cloned”

gene of interest

Plasmid

Gene of

Interest

Protein expressed

by gene of interest

Basic research and

various applications

Copies of gene

Protein harvested

Basic

research

on gene

Basic

research

on protein

Gene for pest

resistance inserted

into plants


Gene used to alter

bacteria for cleaning

up toxic waste


Protein dissolves

blood clots in heart

attack therapy

Human growth hor
-

mone treats stunted

growth

2

4

1

3

Cloning a Gene

Fig. 20
-
4
-
4

Bacterial cell

Bacterial

plasmid

lacZ

gene

Hummingbird

cell

Gene of interest

Hummingbird

DNA fragments

Restriction

site

Sticky

ends

amp
R

gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant

plasmid

Bacteria carrying

plasmids

RESULTS

Colony carrying non
-

recombinant plasmid

with intact
lacZ

gene

One of many

bacterial

clones

Colony carrying recombinant

plasmid with disrupted
lacZ

gene

Cloning DNA


Genome


total DNA per cell


Genomic library


collection of DNA fragments
more or less representative of all DNA in
genome


Genetic Probe


single stranded DNA or RNA
that is radioactively labeled and can attach to
target sequence by base pairing rules


A probe can be synthesized that is complementary
to the gene of interest


For example, if the desired gene is






Then we would synthesize this probe

G

5


3






G

G

C

C

C

T

T

T

A

A

A

C

3


5


C

C

G

G

G

A

A

A

T

T

T

DNA Probe

Using a DNA
probe

Polymerase Chain Reaction (PCR)


Can amplify a small sample of DNA quickly


DNA replication in vitro


2 strands separated by heating so special
heat
-
resistant DNA polymerase called Taq
polymerase used (thermophile)


MAJOR BONUS: Only specific sequences can
be replicated


Study: crime scenes, archaeological remains

PCR

Gel Electrophoresis


Separates fragments like DNA, RNA or
polypeptides (they carry charge and can
migrate in an electrical field


RNA and DNA (
-
)
---

so they move to (+) pole


Smaller fragments go further


Compare sample to standard


Usually “blot”
-

transfer DNA from gel to
nitrocellulose filter for further analysis


DNA Fingerprinting

Gel Electrophoresis

Fig. 20
-
9a

Mixture of

DNA mol
-

ecules of

different

sizes

Power

source

Longer

molecules

Shorter

molecules

Gel

Anode

Cathode

TECHNIQUE

1

2

Power

source



+

+



Fig. 20
-
9b

RESULTS

DNA Fingerprint

Transgenic
Organisms


Plants and animals in which foreign genes
have been incorporated


Animals


Inject DNA into nucleus of egg or stem cell


Eggs implanted in uterus; stem cells injected into
blastocysts + then implanted into foster mother


Plants


Disease resistance


Pesticide resistance

Transgenics

Fig. 20
-
20

Cultured

stem cells

Early human embryo

at blastocyst stage

(mammalian equiva
-

lent of blastula)

Different

culture

conditions

Different

types of

differentiated

cells

Blood cells

Nerve cells

Liver cells

Cells generating

all embryonic

cell types

Adult stem cells

Cells generating

some cell types

Embryonic stem cells

From bone marrow

in this example

Fig. 20
-
18

TECHNIQUE

Mammary

cell donor

RESULTS

Surrogate

mother

Nucleus from

mammary cell

Cultured

mammary cells

Implanted

in uterus

of a third

sheep

Early embryo

Nucleus

removed

Egg cell

donor

Embryonic

development

Lamb (“Dolly”)

genetically identical to

mammary cell donor

Egg cell

from ovary

Cells fused

Grown in

culture

1

3

3

4

5

6

2

Fig. 20
-
19

Cloning Video

GE Plants

Application of GE


Human proteins


Insulin


Hormones
-

HGH


Human treatments for disease


Multiple sclerosis, certain cancers, heart attacks,
forms of anemia


Vaccines

Fig. 20
-
23

Fig. 20
-
24

This photo shows Earl

Washington just before

his release in 2001,

after 17 years in prison.

These and other STR data exonerated Washington and

led Tinsley to plead guilty to the murder.

(a)

Semen on victim

Earl Washington

Source of

sample

Kenneth Tinsley

STR

marker 1

STR

marker 2

STR

marker 3

(b)

17, 19


16, 18


17, 19


13, 16


12, 12


14, 15


11, 12


13, 16


12, 12


Forensics