RESTRICTION ENZYMES

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14 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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Biotechnology

*History of Genetic Engineering


Before technology, humans were using the
process of
selective breeding

to produce
the type of organism they want.


Ex. Dog breeding

=

+

Lab + Poodle

Labradoodle

*Genetic Engineering


Scientists can now use their knowledge of
the structure of DNA and its chemical
properties to study and
change

DNA
molecules.

*Manipulating DNA


Techniques used to manipulate DNA:



DNA Extraction



Cut DNA in to smaller pieces



Identify base sequences



Make unlimited copies of DNA

Cutting DNA


DNA molecules are large; they must be cut
to be analyzed



RESTRICTION ENZYMES
(ENDONUCLEASES)
are proteins that act as “molecular scissors”


Restriction Enzymes


Restriction enzymes
are extremely precise



They only cut in one spot on the DNA molecule


called the
RECOGNITION SEQUENCE


Restriction Enzymes

Restriction Enzymes



Recognition sequences are usually
palindromes


Same backwards and forwards


Ex.
Eco R1
enzyme recognizes:

Restriction Enzymes


Cuts usually leave little single stranded
fragments called
STICKY ENDS

Restriction Enzymes


If the enzyme cuts right down the middle, the
ends are
BLUNT

Restriction Enzymes


Pieces can be glued back together using
LIGASE

Restriction Enzymes


http://highered.mcgraw
-
hill.com/olc/dl/120078/bio37.swf



http://www.dnalc.org/ddnalc/resources/restriction.html

Cutting DNA


Everyone has a unique DNA sequence


Rec. sequences are in different places


When a restriction enzyme cuts the DNA of two
different people, it will cut it into different sized
pieces

Suspect #1

Suspect #2

Analyzing DNA


The pieces are then
analyzed


Each piece has its own
unique weight and
shape


We use these
properties to perform a
technique called
DNA
fingerprinting

DNA fingerprinting


To get the genetic
“fingerprint” of an
organism, its DNA is
first cut using
restriction enzymes


The mixture of DNA
and enzymes are
then placed into a gel
to be separated by
gel electrophoresis


Gel Electrophoresis



The gel acts like a filter
by separating strands
of different sizes


It’s like a sponge made
of Jello


lots of small
holes and a similar
consistency

Gel Electrophoresis


An electric current is
applied to the gel to
get the DNA moving


Small molecules move
faster


Big molecules move
slower


What charge do DNA
molecules have?


Think of the backbone


DNA moving through gel

DNA Fingerprinting


Once separated, a
unique DNA pattern
can be seen for
every organism
tested



How might this be
used to find the
owner of a particular
blood sample?

DNA fingerprinting

How much DNA is needed?


To perform DNA fingerprinting, you need a
lot of DNA


Many copies of the DNA must be made
using a process called
polymerase chain
reaction

PCR


What was the function of the polymerase
enzyme?


PCR utilizes this enzyme to make multiple
copies of a DNA strand

PCR

DNA heated to
separate strands

PCR cycles

DNA copies

1

2

3

4

5 etc.

1

2

4

8

16 etc.

DNA polymerase adds
complementary strand

DNA fragment
to be copied

Utilizing Technology



What good is it to perform these techniques if it we can’t
use them in living cells?



Luckily we can, using a process known as
gene transfer


Gene Transfer


During
gene transfer,
a
gene from one organism is
placed into the DNA of
another organism



Resulting DNA is called
recombinant DNA.


Human insulin is produced
using
recombinant DNA.



Bacteria are commonly
used
-

but how do we get
the genes into them?




Bacterial

Recombinant

DNA

insulin

Bacterial Plasmids


Bacteria have small,
circular DNA segments
called
plasmids.



Plasmids can be used
as a
vector,
or a DNA
molecule that carries
foreign DNA into a host
cell

Creation of Recombinant
DNA


In a lab, plasmid is
extracted from bacteria



Insulin also extracted from
human DNA



Both gene for insulin and
plasmid are cut with same
restriction enzyme.


Have the same sticky ends

Sticky
ends

Insulin gene

(cut from chromosome)

Bacterial Plasmid

Transformation


The gene is inserted into
the plasmid by connecting
sticky ends.



Plasmid taken up by
bacteria through

transformation.



Bacteria grows in Petri dish
and replicates recombinant
DNA


(plasmid + insulin gene)

insulin

human insulin

Creation of Insulin


As the bacteria grow
and replicate, more
and more bacteria are
created with the
human insulin gene



The bacteria express
the gene and create
insulin for us to use

Transforming plant &
animal cells


Bacterial plasmids can
also be put into plant
and animal cells



The plasmid
incorporates into the
plant or animal cell’s
chromosome

Transformed bacteria

introduce plasmids

into plant/animal cells

Transgenic Organisms


Because the bacteria
now has DNA from two
species in it, it is
known as a
transgenic
organism.