Download Insertion of gene into plasmid

Ch 12
Researchers can insert desired genes into plasmids,
creating recombinant DNA and insert those
plasmids into bacteria
Bacterium
Cell containing gene
of interest
1 Plasmid
isolated
2 DNA
isolated
3 Gene inserted
into plasmid
Plasmid
Bacterial
chromosome
Recombinant DNA
(plasmid)
DNA
Gene of
4 Plasmid put into interest
bacterial cell
Recombinant
bacterium
5 Cell multiplies with
gene of interest
Copies of gene
Gene for pest
resistance
inserted into
plants
Figure 12.1
Copies of protein
Clone of cells
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to
make snow form
at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy
Creating recombinant DNA using restriction enzymes and
DNA ligase
Restriction enzyme
recognition sequence
1
GAATTC
CTTAAG
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Addition of a DNA
fragment from
another source
3
Two (or more)
fragments stick
together by
base-pairing
4
G A AT T C
C T TA A G
G A AT T C
C T TA A G
DNA ligase
pastes the strand
5
Figure 12.2
Recombinant DNA molecule
Cloning a gene in a bacterial plasmid
E.coli
Human cell
Isolate DNA
1 from two sources
Cut both DNAs
2 with the same
restriction enzyme
Plasmid
DNA
Gene V
Sticky ends
3
Mix the DNAs;
they join by
base-pairing
4
Add DNA ligase
to bond the DNA covalently
Gene V
Recombinant DNA
plasmid
5
Put plasmid into bacterium
by transformation
6
Clone the bacterium
Recombinant
bacterium
Figure 12.3
Bacterial clone carrying many
copies of the human gene
Cell nucleus
Reverse transcriptase makes
single stranded DNA from
mRNA
cDNA
DNA of
eukaryotic
gene
Exon Intron
Exon
Intron Exon
1 Transcription
RNA
transcript
2 RNA splicing
mRNA
3 Isolation of mRNA
Reverse transcriptase
Test tube
cDNA strand
being synthesized
cDNA of gene
(no introns)
and addition of reverse
transcriptase; synthesis
of DNA strand
4 Breakdown of RNA
5 Synthesis of second
DNA strand
Recombinant cells and organisms can mass-produce
gene products
Table 12.6
Therapeutic hormones & vaccines
– In 1982, humulin, human insulin produced by
bacteria
•
Became the first recombinant drug approved by the
Food and Drug Administration
– Vaccines are harmless mutants or derivative of a
pathogen that stimulate the immune system
Figure 12.7A
• GM organisms
– Acquired genes by artificial means
– Transgenic organisms
Agrobacterium tumefaciens
Plant cell
DNA containing
gene for desired trait
1
Ti
plasmid
Insertion of gene
into plasmid
Recombinant
Ti plasmid
3
2
Introduction
into plant
cells
Regeneration
of plant
DNA carrying new gene
Restriction site
Plant with new trait
Gene therapy (or the alteration of an afflicted individual’s
genes) may someday help treat a variety of diseases
Cloned gene
(normal allele)
1 Insert normal gene
into virus
Viral nucleic
acid
Retrovirus
2 Infect bone marrow
cell with virus
3 Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
Figure 12.13
4 Inject cells
into patient
• DNA profiling
Crime scene
1 DNA isolated
2 DNA of selected
markers amplified
3 Amplified DNA
compared
Suspect 1
Suspect 2
PCR used to amplify target section of DNA
Cycle 1
yields 2 molecules
Genomic
DNA
3
1
3
5
3
Target
sequence
5
5
5
3
Cycle 2
yields 4 molecules
5
5
2 Cool to allow
3
Heat to
primers to form
separate
DNA strands hydrogen bonds
with ends of
target sequences
5
3
5
3
Primer
3
5
DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
3
New DNA
Cycle 3
yields 8 molecules
Gel electrophoresis sorts DNA molecules by
size
Mixture of DNA
molecules of
different sizes
–
–
Longer
molecules
Power
source
Gel
+
Shorter
molecules
+
Figure 12.10
Completed gel
– After digestion by restriction enzymes the
fragments are run through a gel
1
–
2
Longer
fragments
z
x
w
Shorter
fragments
Figure 12.11B
+
y
y
DNA fingerprinting can help solve crimes
Defendant’s
blood
Blood from
defendant’s clothes
Figure 12.12A
Victim’s
blood
Figure 12.12B
• STR
– Short sequences of DNA repeated many
times in a row
– STR analysis compared lengths of STR
sequences at specific sites on the genome
• Used in forensic investigations
STR site 1
STR site 2
Crime scene DNA
Number of short tandem Number of short tandem
repeats match
repeats do not match
Suspect’s DNA
How Restriction Fragments Reflect DNA Sequence
– Restriction fragment length polymorphisms (RFLPs)
reflect differences in the sequences of DNA samples
Crime scene
Suspect
w
Cut
C
G
C
G
G
C
G
C
z
A
T
C
G
G
C
G
C
C
G
C
G
G
C
G
C
x
Cut
y
C
G
C
G
G
C
G
C
Figure 12.11A
Cut
y
DNA from chromosomes