Molecular Techniques
How to analyze and manipulate
DNA
Fluorescent
dye
Single strand
of DNA
Strand broken
after A
Strand broken
after C
Strand broken
after G
Strand broken
after T
Power
source
Gel
DNA sequencing
Restriction enzymes
Recognition sequences
DNA sequence
Restriction enzyme
EcoR
I
cuts the DNA
into fragments.
Sticky end
Restriction Enzyme
Sequence Recognition and cleavage:
Eco
RI
GAATTC
G
AATTC
CTTAAG
CTTAA
G
Kpn
I
GGTACC
GGTAC
C
CCATGG
C
CATGG
Sma
I
CCCGGG
CCC
GGG
GGGCCC
GGG
CCC
Analyze this!
BamH1
Pst 1
BamH1
Pst 1
EcoR1
Pst 1
Hind III
Hind III
How many pieces would cutting with BamH1 produce?
Hind III?
Pst I?
How big would each piece be???
How big are the pieces of DNA?
•
DNA is measured in base pairs
–
usually in kilobases, or kB.
•
Use
gel electrophoresis
to sort the
pieces by size.
•
DNA is negatively charged, so electric
current will push DNA away from a
negative pole and toward a positive.
•
Shorter pieces move faster.
Gel Electrophoresis
Genetic engineering
•
Making
recombinant
DNA.
•
Moving a gene from one organism to
another.
–
Why do this?
•
Removing or replacing faulty genes.
–
We can’t just replace nucleotides, yet.
–
Can only fix existing cells, unless you
work on gametes.
Making recombinant DNA
•
Find the gene you want
–
must know the nucleotide sequence
•
Cut out the gene
–
Use
restriction enzymes
.
•
Open the target DNA
–
Use same restriction enzyme
•
Introduce DNA and ligate it
–
Ligase again!
The goal:
Recombinant DNA
Flanking sequences
match host
Host Cell DNA
Target gene
Recombinant DNA replaces
target gene
Modified Host Cell DNA
A common use…
•
Use bacteria as factories for producing
human products.
–
Insulin, human growth hormone
•
Modify
plasmids
, not the main
chromosome.
–
Plasmids are small extra DNA loops that
bacteria can send to other bacteria.
•
Remember transformation???
Making recombinant DNA and transforming
bacteria
Human Cell
Gene for human
growth hormone
Recombinant
DNA
Gene for human
growth hormone
Sticky
ends
DNA
recombination
DNA
insertion
Bacterial Cell
Plasmid
Bacterial
chromosome
Bacterial cell for
containing gene for
human growth hormone
animation
•
The resulting organism is
transgenic
.
–
Has genes from two or more organisms
•
Why bother?
–
Has properties of a different organism
•
Mice with human immune systems?
–
Makes new gene products
–
Pharm animals
Cloning
•
Making genetically identical organisms
–
Why bother?
–
Will they
really
be identical?
Cloning
A body cell is taken from a donor animal.
An egg cell is taken from a donor animal.
The fused cell begins dividing, becoming an embryo.
The nucleus is removed from the egg.
The body cell and egg are fused by electric shock.
The embryo is implanted into the uterus of a foster mother.
The embryo develops into a cloned animal.
Cloning Dolly
A donor cell is taken from
a sheep’s udder.
Donor
Nucleus
These two cells are fused
using an electric shock.
Fused Cell
The fused cell
begins dividing
normally.
Embryo
The embryo is placed
in the uterus of a foster
mother.
Foster
Mother
The embryo
develops normally
into a lamb
—
Dolly
Cloned Lamb
Egg Cell
An egg cell is taken
from an adult
female sheep.
The nucleus of the
egg cell is removed.
Next?
•
Stem cells may be manipulated to
produce replacement tissues or even
organs.
•
Correct a defect in an embryo?
–
How would this work?
•
Problems????
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