DNA Technology and Genetic Engineering

kissimmeemisologistΒιοτεχνολογία

14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

167 εμφανίσεις

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DNA
Technology and Genetic
Engineering

Human protein

production

RFLP analysis

Human genome

project

Transgenic

organisms

Forensic analysis

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DNA Technology and Genetic
Engineering (making changes in DNA)


1: Production of human proteins


2: To identify people


3: To identify human diseases


4: To identify all human genes


5: To genetically engineer food


Supercoils

Coils

Nucleosomes

Histones

Nucleus

Chromosome

DNA

Cell

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Use 1: To produce human
proteins in bacteria (
E. coli
)

Example: Curing Pituitary Dwarfism

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What is Dwarfism?


Dwarfism is a recessive
disease that causes adults to
be no more than 4 feet tall


Little people produce little
or no growth hormone,
which is made by the
pituitary


Researchers studied families
with dwarfism and found that
people with dwarfism have
defective copies of the gene,
GH1



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Early attempts to treat Dwarfism


Attempts to inject growth hormone from pigs (a
strategy that worked previously for insulin) did not
work


only GH from humans would work (until
1982 source from human cadavers and up to 20,000
pituitaries were needed!)


Some of these pituitaries were contaminated with
prions, which cause degenerative brain disorders



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Solution: Use recombinant DNA to
produce GH in bacteria!


Recombinant DNA = DNA that
results from combining DNA from
different sources


ex. mouse + human DNA


human + bacterial DNA



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Plasmid

isolated

1

Bacterium

Bacterial

chromosome

Plasmid

2

DNA

isolated

Cell containing gene

of interest

DNA

Gene of

interest

3

Gene

inserted

into plasmid

Recombinant DNA

(plasmid)

4

Plasmid put into

bacterial cell

Recombinant

bacterium

5

Copies of gene

Copies of protein

Clones of cell

Gene for pest

resistance

inserted into

plants

Gene used to alter bacteria

for cleaning up toxic waste

Protein used to dissolve blood

clots in heart attack therapy

Protein used to

make snow form

at higher

temperature

Cell multiplies with

gene of interest

Recombinant DNA Overview

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How Do You Make Recombinant
DNA?


How do you make recombinant DNA? We need


A) To isolate genes with restriction enzymes


B) A vector


C) To combine the genes and the vector


What do we do with it?


D) Transfer the recombinant DNA to the host


E) Find the gene of interest (human growth
hormone)

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A) Isolate genes with restriction
enzymes (DNA scissors)


Occur naturally in
bacteria


why?


Bacteriophages

infect
bacteria


Cut up foreign DNA


Hundreds are purified
and available
commercially


Recognize and cut at
specific base sequences
in DNA (usually 4
-
8
bases long)

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Products generated by restriction
enzymes


A) Sticky
-
end cutters

Enzyme

Recognition site


DNA after cuts







B) Blunt
-
end cutters

Enzyme

Recognition site


DNA after cuts

5’...G

3’...CTTAA

AATTC
...3’


G
...5’

5’...CCC

3’...GGG

GGG
...3’

CCC
...5’

5’...CCC
GGG
...3’

3’...GGG
CCC
...5’

SmaI



EcoRI

5’...G
AATTC
...3’

3’...CTTAA
G
...5’



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A) Isolate genes with restriction
enzymes (DNA scissors)


Take genomic DNA (in this case, human cells) and cut
it with a particular restriction enzyme


MANY restriction fragments formed


ALL parts of the DNA are cut whenever there is a
restriction site


Now what? Put these fragments in a vector

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B) Vectors


Vector = something to carry the gene of
interest into the host (i.e. bacteria)


A. Mechanical


micropipettes or gene
guns


B. Biological


virus or plasmid


Plasmid



additional, free
-
floating
ring

of
DNA found only in bacteria


Can replicate within a cell (has origin of
replication)


Has antibiotic resistance gene


Cut both the plasmid and gene with the
same restriction enzyme and their ends will
hydrogen bond = gene splicing


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C) Combine
the gene of
interest and
the vector by
sealing ends
with DNA
ligase
… now
you have made
recombinant
DNA

DNA

1

Restriction enzyme

recognition sequence

Restriction enzyme

cuts the DNA into

fragments

Sticky end

2

3

4

5

Restriction enzyme

cuts the DNA into

fragments

Addition of a DNA

fragment from

another source

Two (or more)

fragments stick

together by

base
-
pairing

DNA ligase

pastes the strand

Recombinant DNA molecule

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C) Combine
the gene of
interest and
the vector


a
different

picture

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D) Transfer the Recombinant DNA
to the Host (Transformation)


Recombinant DNA is transferred to a host cell.


Can use heat
-
shocking or electricity to get plasmid
into bacteria


How do we know which bacterial cells have our
plasmid?


Antibiotic resistance gene!


Resistance gene allows ONLY those bacteria with the
plasmid to grow in media that have an antibiotic on it


When the host cell copies its DNA (replicates), it also
makes a copy of the plasmid. Results in clones, which
are genetically identical copies.


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D) Transfer the Recombinant DNA
to the Host


We use bacteria because they reproduce very
quickly and have all the protein synthesis
machinery (enzymes,
ribosomes
)


Insulin for Type I diabetics


Blood factor VIII
-
hemophilia (clotting factor)


Antigens for vaccines (
Hep

B, flu, meningitis)


Cutting chromosomes in order to study
individual pieces


Host Cells Produce Protein Products


ex. GH and insulin

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E) Find the gene of interest



Each clone consists of
identical cells containing one
fragment (of many) of human
DNA


The collection of all the
cloned DNA fragments is
known as a genomic library


Each fragment represents a
“book” that is “shelved” in
plasmids inside bacterial
cells


Thus, it is a library of all the
organism’s genes

Figure 12.6

Genome cut up

with restriction

enzyme

Recombinant

plasmid

OR

Recombinant

phage DNA

Bacterial

clone

Phage

clone

Phage

library

Plasmid

library

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E) *Side note


libraries can also
be made from
cDNA



The enzyme reverse transcriptase can be used to make a
smaller library, called a
cDNA

library



This contains only the genes that are expressed
(transcribed) by a specific cell type (rather than ALL the
genes found in an organism)



These genes can then be digested and placed in vectors


Why is this useful?


Bacterial mRNA does not have
introns



doesn’t have the
machinery to splice eukaryotic genes


Can help a researcher study the genes responsible for
specialized functions of a certain cell type

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Transcription

1

CELL NUCLEUS

DNA of

eukaryotic

gene

RNA

transcript

mRNA

Exon

Intron

Exon

Intron

Exon

TEST TUBE

Reverse transcriptase

cDNA strand

cDNA of gene

(no introns)

RNA splicing

(removes introns)

2

Isolation of mRNA

from cell and addition

of reverse transcriptase;

synthesis of DNA strand

3

Breakdown of RNA

4

Synthesis of second

DNA strand

5

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E) Find the gene of interest



How do we find the right “shelf” in the library?


If we know at least part of the DNA sequence, we can
create a nucleic acid probe


Probe = radioactively labeled single
-
stranded DNA piece
that can base pair with a particular sequence


Ex: AT
CC
GA


The probe is mixed with clones that have been heated or
treated with a chemical to separate the DNA strands


The probe will “tag” the correct “shelf” or bacterial clone
that contains the gene of interest

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E) Find the gene of interest

Radioactive

probe (DNA)

Mix with single
-

stranded DNA from

various bacterial

(or phage) clones

Single
-
stranded

DNA

Base pairing

indicates the

gene of interest

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E) Find the
gene of
interest

Bacterial colonies containing

cloned segments of foreign DNA

Radioactive DNA

Transfer

cells to

filter

1

Solution

containing

probe

Treat cells

on filter to

separate

DNA

strands

2

Add probe

to filter

3

Probe

DNA

Gene of

interest

Single
-
stranded

DNA from cell

Hydrogen
-
bonding

Autoradiography

4

Developed film

Colonies of living

cells containing

gene of interest

Compare autoradiograph

with master plate

5

Master plate

Filter

paper

The bacterial
colony can
then be
grown and
the protein of
interest can
be isolated in
large
amounts

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E) Isolating
the gene of
interest

Isolate DNA

from two sources

1

E. coli

Cut both

DNAs with

the same


restriction


enzyme

2

Plasmid

Human cell

DNA

Gene
V

Sticky ends

Mix the DNAs; they join

by base
-
pairing

3

Add DNA ligase

to bond the DNA covalently

4

Recombinant DNA

plasmid

Gene
V

Put plasmid into bacterium

by transformation

5

Clone the bacterium

6

Bacterial clone carrying many

copies of the human gene

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Mass
-
Produced Genes