Genetic Engineering: Direct manipulation of DNA

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14 Δεκ 2012 (πριν από 4 χρόνια και 7 μήνες)

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Today:
Biotechnology


Exam #2

Th 10/23 in class

http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?ORG=human&CHR=X&MAPS=i
deogr[Xpter:Xqter],genes[1.00:153692391.00]

Map of human chromosome 20

Your DNA

Over 600 recent transposon insertions were
identified by examining DNA from 36 genetically
diverse humans.

Tbl 1

Which transposable elements are active in the human genome? (2007) Ryan E. Mills et al. Trends in
Genetics 23: 183
-
191

DNA fingerprinting using RFLPs

Visualizing differences in DNA sequence
by using restriction enzymes

Sequence 1

Sequence 2

Restriction Enzymes

cut DNA at specific sequences

Fig 18.1

Examples of some restriction enzymes…

tbl 18.3

Visualizing differences in DNA sequence
by using restriction enzymes

Sequence 1

Sequence 2

Fig 20.5+.6

Separating DNA on a gel by size

Fig 20.6


Gel electrophoresis

Fig 24.21

The different sized bands can arise from different cut sites
and/or different number of nucleotides between the cut sites.

Fig 22.23

Sequence 1

Sequence 1

Sequence 2

Sequence 2

DNA fingerprinting

DNA fingerprinting

DNA fingerprinting

Can DNA be obtained from hair?

How can DNA be obtained from
such a small sample?

The inventor of PCR

Polymerase
Chain Reaction:

amplifying DNA

Fig 18.6

Polymerase Chain
Reaction

Fig 18.6

Polymerase
Chain Reaction:

Primers allow
specific regions
to be amplified.

Fig 18.6

The inventor of PCR

PCR animation
http://www.dnalc.org/ddnalc/resources/pcr.html


Areas of DNA from very small samples can be
amplified by PCR, and then cut with
restriction enzymes for RFLP analysis.

Genetic Engineering: Direct manipulation of DNA

Fig 18.2

Bacteria can be modified or serve as intermediates

Fig 18.2

a typical bacteria

Bacterial DNA

plasmid DNA

A typical
bacterial plasmid
used for genetic
engineering

tbl 18.2

Moving a gene into bacteria via a plasmid

Fig 18.2

Bacterial DNA

plasmid DNA

What problems exist for expressing eukaryotic
gene in bacteria?

Reverse
transcriptase
can be used to
obtain coding
regions without
introns.

Fig 18.4

After RT, PCR will
amplify the gene or DNA

Fig 18.6

Moving a gene into bacteria via a plasmid

RT and PCR

Fig 18.2

Restriction Enzymes

cut DNA at specific sequences

Fig 18.1

Restriction enzymes cut DNA at a specific
sequence

Fig 18.1

Cutting the
plasmid and insert
with the same
restriction enzyme
makes matching
sticky ends

Fig 18.1

A typical
bacterial plasmid
used for genetic
engineering

Using sticky ends to add DNA to a bacterial plasmid

Fig 18.1

If the same
restriction enzyme
is used for both
sides, the plasmid
is likely to religate
to itself.

Fig 18.1

The plasmid is
treated with
phosphatase to
remove the 5’
-
P,
preventing self
-
ligation

Fig 18.1

Transformation of bacteria can happen via
several different methods.

tbl 6.1

Bacteria can take up DNA from the environment

Fig 9.2

Tbl 6.1

Transformation of bacteria can happen via
several different methods all involving
perturbing the bacterial membrane:


Electroporation


Heat shock


Osmotic Stress

How can you know which bacteria have been
transformed, and whether they have the insert?

Fig 18.1

Resistance genes allow
bacteria with the
plasmid to be selected.

Bacteria with the resistance
gene will survive when
grown in the presence of
antibiotic

Fig 20.5

Is the insert present?

Plasmids with the MCS
in the lacZ gene can be
used for blue/white
screening…

Fig 18.1

A typical
bacterial plasmid
used for genetic
engineering

Intact lacZ makes a
blue color when
expressed and provided
X
-
galactose

When the lacZ gene is
disrupted, the bacteria
appear white

Blue/white
screening:

Transformed
bacteria plated on
antibiotic and X
-
gal plates.

Each colony
represents millions
of clones of one
transformed cell.

Fig 18.1

Successful transformation
will grow a colony of
genetically modified
bacteria

Fig 18.1

Inserting a gene into a
bacterial plasmid

RT and/or
PCR

Fig 18.1

Millions of Hectares

Texas =

70 ha

Bacteria can be used to transform plants

Global area planted
with GM crops

http://www.gmo
-
compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html

Agrobacterium infect plants, inserting their
plasmid DNA into the plants genome.

Fig 19.15b

Fig 19.15

Agrobacterium infect plants, inserting their
plasmid DNA into the plants genome.

By replacing the gall forming genes with other
DNA when the Agrobacterium infect a plant, it
will insert that DNA into the plant.

Fig 19.16

Fig 19.16

The generation of a transgenic plant

Grown on herbicide