Gene Technology.pptx - Rossmant

Gene Technology

At the beginning:

•
Studies using interferon:

–
Interferon increases human
resistance to viral infection and
scientists were interested in its
possible usefulness in cancer
therapy.

–
It was rare in the blood and
needed in large quantities for
study.

–
Interferon gene introduced into
bacteria genome.

–
Bacteria divide rapidly so began
to produce interferon in large
quantities.

At the beginning:

•
Studies using interferon:

–
Interferon increases human
resistance to viral infection and
scientists were interested in its
possible usefulness in cancer
therapy.

–
It was rare in the blood and
needed in large quantities for
study.

–
Interferon gene introduced into
bacteria genome.

–
Bacteria divide rapidly so began
to produce interferon in large
quantities.

A bacteria


Bacterial Reproduction


Genetic Engineering

•
The bacteria produced a line of
genetically altered cells from a
single altered cell = cloning.

•
Insulin gene can also be “grown”
this way.


•
This marked the beginning of
genetic engineering.

–
The ability to cut DNA into
recognizable pieces and
rearrange them in different ways.

How To:

•
Cut the source of the
DNA that carries
what you want and
insert into a plasmid
or infective virus.

A Virus


Getting In


Taking Over


In order to make it work:

•
MUST

•
1. cut the source DNA and the plasmid DNA so that the desired fragment
can be spliced.

–
Done by enzymes the recognize and cleave (cut) specific sequences of
nucleotides.

Restriction

Enzyme

Source

Recognition

Site

BAMHI

Bacillus
amyloliquefaciens

G

GATC C

C CTAG G

EcoRI

Eschirichia

Coli R factor

G AATT C

C TTAA G

HaeIII

Hemophilus

aegyptus

GG CC

CC GG

HindIII

Hemophilus

influenzae

A AGCT T

T TCGA A

TaqI

Thermus

aquaticus

T CG A

A GC T

Restriction
Endonucleases

•
Bacteria must defend themselves against
bacteriophages

(viruses that
specifically attack bacteria).

•
Through the
lytic

cycle the viruses can attack, kill, and take over many more
bacteria.

•
To defend themselves bacteria contain enzymes called restriction
endonucleases
.

•
These recognize specific nucleotide sequences in a DNA strand, bind to them,
and cleave them at the recognition sequence.

•
To keep these enzymes from attacking their own DNA bacteria add methyl
(using
mehylases
) to the recognition sites so the
endonucleases

can’t bind.

Restriction
Endonucleases

•
Bacteria must defend themselves against
bacteriophages

(viruses that
specifically attack bacteria).

•
Through the
lytic

cycle the viruses can attack, kill, and take over many more
bacteria.

•
To defend themselves bacteria contain enzymes called restriction
endonucleases
.

•
These recognize specific nucleotide sequences in a DNA strand, bind to
them, and cleave them at the recognition sequence.

•
To keep these enzymes from attacking their own DNA bacteria add methyl
(using
mehylases
) to the recognition sites so the
endonucleases

can’t bind.

Restriction
endonuclease

How To Cut the DNA

•
Find the palindromes.

–
Nucleotides at one end of the recognition
sequence are
comlementary

to those at the
other end so they have the same nucleotide
sequence running in opposite directions.

•
Allows it to cut across two strands

•
Since the cleave site isn’t directly centered each
fragment has a single stranded end a few nucleotides
long.

•
These fragments are complementary to each other
and can recombine using DNA
ligase

(the
spotwelder
).

–
These are considered “sticky” ends and will attract their
complementary base pair sets.

Cutting Activity


Restrictions
Endonucleases

Cut

•
Hundreds of
restiction

enzymes

–
Each with own recognition sequence

•
By chance the recognition site will arise within a given
sample.

–
The shorter the sequence the more often it will probably
occur.

–
Once two fragments attach, they can be joined by DNA
ligase

to reform the
phosphodiester

bonds of DNA.

–
ANY two fragments produced by the same restriction
endonuclease

can be joined together. Elephant DNA
cleaved by an
endonuclease

can be joined to ostrich DNA if
cleaved by the same
endonuclease
.

EcoRI

•
Cleaves
sequence
GAATTC
between the G
and the A.

•
Since they run in
opposite
directions, they
are
complementary
… sticky.