Transcript Restriction Enzymes

Today:
Biotechnology
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
Fig 18.1
Restriction Enzymes cut DNA at specific sequences
tbl 18.3
Examples of some restriction enzymes…
Recognition
Enzyme Sequence
EcoRI 5'GAATT C
3'CTTAAG
BamHI 5'GGAT CC
3'CCTAGG
HindIII 5'AAGCTT
3'TTCGAA
TaqI
5'TCGA
3'AGCT
AluI
5'AGCT
3'TCGA
Cut
5'---G AATT C---3'
3'---CTTAA G--- 5'
5'---G GAT CC---3'
3'---CCTAG G---5'
5'---A AG CTT---3'
3'---TTCGA A--- 5'
5'---T CGA---3'
3'---AGC T---5'
5'---AG CT---3'
3'---TC GA---5'
Fig 20.5+.6
Visualizing differences in DNA sequence
by using restriction enzymes
Sequence 1
Sequence 2
Fig 20.6
Separating DNA on a gel by size
• 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.
Sequence 1
Sequence 2
Sequence 1
Sequence 2
Fig 22.23
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
Fig 18.6
Polymerase
Chain Reaction:
amplifying DNA
Polymerase Chain
Reaction
Fig 18.6
Fig 18.6
Polymerase
Chain Reaction:
Primers allow
specific regions
to be amplified.
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
tbl 18.2
A typical
bacterial plasmid
used for genetic
engineering
Fig 18.2
Moving a gene into bacteria via a plasmid
What problems exist for expressing eukaryotic
gene in bacteria?
Bacterial DNA
plasmid DNA
Fig 18.4
Reverse
transcriptase
can be used to
obtain coding
regions without
introns.
Fig 18.6
After RT, PCR will
amplify the gene or DNA
Fig 18.2
Moving a gene into bacteria via a plasmid
RT and PCR
Fig 18.1
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
A typical
bacterial plasmid
used for genetic
engineering
Using sticky ends to add DNA to a bacterial plasmid
Fig 18.1
tbl 6.1
Transformation of bacteria can happen via
several different methods.
Bacteria can take up DNA from the environment
Fig 9.2
Transformation of bacteria can happen via
several different methods all involving
perturbing the bacterial membrane:
Tbl 6.1
•Electroporation
•Heat shock
•Osmotic Stress
Fig 18.1
How can you know which bacteria have been
transformed, and whether they have the insert?
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 18.1
Fig 20.5
Is the insert present?
Plasmids with the MCS
in the lacZ gene can be
used for blue/white
screening…
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
Fig 18.1
Blue/white
screening:
Transformed
bacteria plated on
antibiotic and Xgal plates.
Each colony
represents millions
of clones of one
transformed cell.
Fig 18.1
Successful transformation
will grow a colony of
genetically modified
bacteria
RT and/or
PCR
Fig 18.1
Inserting a gene into a
bacterial plasmid
Millions of Hectares
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
Texas =
70 ha