Download Recombinant DNA

CHANGING THE LIVING
WORLD
How we change the living world…
Selective breeding: crossing organisms with desired traits to
produce the next generation.
How we change the living world…
Hybridization: crossing dissimilar organisms to get the
best of both.
How we change the living world…
Inbreeding: continually breeding individuals with similar
characteristics.
GENETIC
ENGINEERING
Genetic engineering vocab
– Recombinant DNA- nucleotide sequences from two
different sources to form a single DNA molecule.
– Transgenic organism – contains a gene from another
organism, typically a different species
– Genetically modified organisms (GMOs)- organisms
that have acquired one or more genes by artificial
means.
Figure 12.1
Genetic Engineering
Genetic engineering: The process of
manipulating genes for practical purposes.
Genetic engineering may involve building
recombinant DNA
DNA made from two or more
different organisms.
Steps in a Genetic Engineering Experiment
Step 1 Isolate Target DNA and plasmid and cut
with restriction enzymes
Step 2 Recombinant DNA is produced.
Step 3 Gene cloning: the process by which many copies of
the gene of interest are made each time the host cell reproduces.
Step 4 Cells undergo selection and then are
screened.
Steps in a Genetic Engineering Experiment
Step 1 The DNA from the organism containing the gene of
interest and the vector are cut by restriction enzymes.
A vector is an agent that is used to carry
the gene of interest into another cell
Commonly used vectors: viruses,
yeast, and plasmids.
circular bacterial DNA
Plasmids
Colorized TEM
Bacterial
chromosome
Remnant of
bacterium
Figure 12.7
Bacterial cell
Isolate
plasmids.
Plasmid
Figure 12.8-1
Isolate
DNA.
Bacterial cell
Cell containing
the gene of interest
Isolate
plasmids.
Plasmid
DNA
Figure 12.8-2
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Cell containing
the gene of interest
Isolate
plasmids.
Plasmid
DNA
Figure 12.8-3
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Plasmid
DNA
Figure 12.8-4
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Recombinant bacteria
Figure 12.8-5
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Bacterial clone
Recombinant bacteria
Clone the bacteria.
Figure 12.8-6
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Bacterial clone
Recombinant bacteria
Clone the bacteria.
Find the clone with
the gene of interest.
Figure 12.8-7
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Bacterial clone
Recombinant bacteria
Clone the bacteria.
Find the clone with
the gene of interest.
Some uses
of genes
Some uses
of proteins
Gene for pest
resistance
Protein for
dissolving
clots
Gene for
toxic-cleanup
bacteria
Genes may be
inserted into
other organisms.
The gene and protein
of interest are isolated
from the bacteria.
Harvested
proteins may be
used directly.
Protein for
“stone-washing”
jeans
Figure 12.8-8
RESTRICTION ENZYMES
molecular scissors
A Closer Look: Cutting and Pasting DNA with
Restriction Enzymes
– Recombinant DNA is produced by combining two
ingredients:
A bacterial plasmid
The gene of interest
How do we cut them?
• Using restriction enzymes: bacterial enzymes
which cut DNA at specific nucleotide sequences
•
produce pieces of DNA called restriction
fragments.
• Why do you think bacteria contain restriction
Restriction Enzymes are palindromes: the same
forward as backwards, like RACECAR.
Examples:
GAATTC
CTTAAG
CCCGGG
GGGCCC
AAGCTT
TTCGAA
G
AATTC
CTTAA
G
CCC GGG
GGG CCC
A
AGCTT
TTCGA
A
Sticky Ends
Blunt End
Recognition sequence
for a restriction enzyme
DNA
A restriction enzyme cuts
the DNA into fragments.
Restriction
enzyme
Figure 12.9-1
Recognition sequence
for a restriction enzyme
DNA
A restriction enzyme cuts
the DNA into fragments.
Restriction
enzyme
A DNA fragment is added
from another source.
Figure 12.9-2
Recognition sequence
for a restriction enzyme
DNA
A restriction enzyme cuts
the DNA into fragments.
Restriction
enzyme
A DNA fragment is added
from another source.
Fragments stick together by
base pairing.
Figure 12.9-3
DNA LIGASE
– DNA ligase connects the DNA fragments into one
continuous strand (DNA Glue or tape)
Recognition sequence
for a restriction enzyme
DNA
A restriction enzyme cuts
the DNA into fragments.
Restriction
enzyme
A DNA fragment is added
from another source.
Fragments stick together by
base pairing.
DNA ligase joins the
fragments into strands.
DNA
ligase
Recombinant DNA molecule
Figure 12.9-4
Recognition sequences
DNA
sequence
Restriction enzyme EcoRI
cuts the DNA into fragments.
Sticky end
Your turn to try!!
– Plasmids:
• Can easily incorporate foreign DNA
• Are readily taken up by bacterial cells
• Can act as vectors, DNA carriers that move genes from one
cell to another
• Are ideal for gene cloning, the production of multiple
identical copies of a gene-carrying piece of DNA
Bacterial cells don’t edit the RNA, so how
can they make the correct protein?
Genetic Engineers can eliminate the introns
from mRNA and reverse the process, producing a
DNA strand that is only the instructions for the
protein.
Use Reverse Transcriptase
Cell nucleus
DNA of
eukaryotic
gene
Exon Intron Exon Intron Exon
Transcription
Test tube
Figure 12.11-1
Cell nucleus
DNA of
eukaryotic
gene
Exon Intron Exon Intron Exon
Transcription
RNA
transcript
Introns removed and
exons spliced together
mRNA
Test tube
Figure 12.11-2
Cell nucleus
DNA of
eukaryotic
gene
Exon Intron Exon Intron Exon
Transcription
RNA
transcript
Introns removed and
exons spliced together
mRNA
Test tube
Isolation of mRNA from
cell and addition of
reverse transcriptase
Reverse
transcriptase
Figure 12.11-3
Cell nucleus
DNA of
eukaryotic
gene
Exon Intron Exon Intron Exon
Transcription
RNA
transcript
Introns removed and
exons spliced together
mRNA
Test tube
Isolation of mRNA from
cell and addition of
reverse transcriptase
Reverse
transcriptase
Synthesis of cDNA
strand
cDNA strand
being synthesized
Figure 12.11-4
Cell nucleus
Exon Intron Exon Intron Exon
DNA of
eukaryotic
gene
Transcription
RNA
transcript
Introns removed and
exons spliced together
mRNA
Test tube
Isolation of mRNA from
cell and addition of
reverse transcriptase
Reverse
transcriptase
cDNA strand
being synthesized
Synthesis of cDNA
strand
Synthesis of second DNA
strand by DNA polymerase
cDNA of gene
without introns
Figure 12.11-5