Download DNA tech - May 16

Biotechnology
1. Making microbes work for you!
2. Base pairing between nucleic acids
is really useful
What if we wanted a lot of human insulin?
Fig. 20-2a
Bacterium
1 Gene inserted into
Cell containing gene
of interest
plasmid
Bacterial
chromosome
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
2
2 Plasmid put into
bacterial cell
Recombinant
bacterium
DNA of
chromosome
pET11c: an example of a plasmid
Restriction enzymes:
-Enzymes that break the sugar-phosphate backbone of
DNA (“restrict” the DNA) at specific sequences
-Many leave short, single-stranded “sticky ends” after
they cut DNA
Table of restriction enzymes
Fig. 20-3-1
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
Fig. 20-3-2
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
Fig. 20-3-3
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
3
DNA ligase
seals strands.
Recombinant DNA molecule
Fig. 20-4-1
Hummingbird
cell
TECHNIQUE
Bacterial cell
Restriction
site
ampR gene
Bacterial
plasmid
Sticky
ends
Gene of interest
Hummingbird
DNA fragments
Fig. 20-4-2
Hummingbird
cell
TECHNIQUE
Bacterial cell
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Fig. 20-4-3
Hummingbird
cell
TECHNIQUE
Bacterial cell
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
Fig. 20-4-4
Hummingbird
cell
TECHNIQUE
Bacterial cell
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
RESULTS
One of many
bacterial
clones
Fig. 20-4-4
Hummingbird
cell
TECHNIQUE
Bacterial cell
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
RESULTS
How do we know which plasmid
has our gene of interest (i.e.
insulin)?
One of many
bacterial
clones
Fig. 20-7
Base pairing (“hybridization”) between nucleic acids is very powerful
TECHNIQUE
Radioactively
labeled probe
molecules
Multiwell plates
holding library
clones
Probe
DNA
Gene of
interest
Single-stranded
DNA from cell
Film
•
Nylon membrane
Nylon
Location of
membrane
DNA with the
complementary
sequence
pET11c: an example of a plasmid
Will E. coli be able to express
human insulin?
Fig. 20-6-5
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
Reverse transcriptase allows RNA to be
copied (“reverse transcribed”) into
cDNA
Reverse
transcriptase
Poly-A tail
DNA Primer
strand
Degraded
mRNA
DNA
polymerase
cDNA
What if we wanted to introduce a functional insulin gene into a patient?
Fig. 20-22
Cloned
gene
Not yet feasible
1
Insert RNA version of normal allele
into retrovirus.
Viral RNA
2
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4
Inject engineered
cells into patient.
Bone
marrow
Fig. 20-11
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II
III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal
-globin
allele
II Sickle-cell
allele
III Heterozygote
1 Preparation of restriction fragments
Paper
towels
Alkaline
solution
2 Gel electrophoresis
3 DNA transfer (blotting)
Radioactively labeled
probe for -globin gene
I
II III
Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele
Nitrocellulose blot
4 Hybridization with radioactive probe
Fragment from
normal -globin
allele
I
II III
Film
over
blot
5 Probe detection
Fig. 20-13
TECHNIQUE
1 cDNA synthesis
mRNAs
cDNAs
2 PCR amplification
Primers
-globin
gene
3 Gel electrophoresis
RESULTS
Embryonic stages
1 2 3 4 5
6
Fig. 20-14
50 µm
Fig. 20-15
TECHNIQUE
1 Isolate mRNA.
2 Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
3 Apply the cDNA mixture to a
microarray, a different gene in
each spot. The cDNA hybridizes
with any complementary DNA on
the microarray.
Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA fragments
representing
specific genes
DNA microarray
4 Rinse off excess cDNA; scan
microarray for fluorescence.
Each fluorescent spot represents a
gene expressed in the tissue sample.
DNA microarray
with 2,400
human genes