Genetic engineering Making recombinant DNA. Moving a gene from ...

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????