Transcript Restriction Enzymes

Restriction Enzyme
Digestion
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
Enzymes are the Tools of DNA
Technology
The tools of DNA technology are the same enzymes used
by cells to modify their own DNA:
– DNA Polymerases
– DNA Ligase
One special class of enzyme is pivotal to the cloning of
DNA and many other techniques used in DNA
Technology
These enzymes are the restriction endonucleases
– Restriction - Because for the way they work, they restrict
bacteriophages to only one host bacterial strain. They are also
restricted to acting on only specific DNA sequences
– Endonuclease - They cut nucleic acids in the middle not just
the ends
©2000 Timothy G. Standish
Restriction Endonucleases
There are a number of different sub classes of
restriction endonucleases
– Type I - Recognize specific sequences and cut DNA at
a nonspecific site > than 1,000 bp away
– Type II - Recognize palindromic sequences and cut
within the palindrome
– Type III - Recognize specific 5-7 bp sequences and
cut 24-27 bp down stream of the site.
Type II restriction endonucleases are the most
useful class as they recognize specific palindomic
sequences in DNA and cut the sugar phosphate
backbone within the palindrome
©2000 Timothy G. Standish
What is a Palindrome?
A palindrome is anything that reads the same
forwards and backwards:
English palindromes:
Mom
Dad
Tarzan raised Desi Arnaz rat.
Able was I ere I saw Elba (supposedly said by
Napoleon)
Doc note I dissent, a fast never prevents a fatness,
I diet on cod.
©2000 Timothy G. Standish
DNA Palindromes
Because DNA is double stranded and the strands
run antiparallel, palindromes are defined as any
double stranded DNA in which reading 5’ to 3’
both are the same
Some examples:
The EcoRI cutting site:
– 5'-GAATTC-3'
– 3'-CTTAAG-5'
The HindIII cutting site:
– 5'-AAGCTT-3'
– 3'-TTCGAA-5'
©2000 Timothy G. Standish
Uses of Restriction
Endonucleases
Because restriction endonucleases cut specific
sequences they can be used to make “DNA
fingerprints” of different samples of DNA. As
long as the cutting site changes on the DNA or
the distance between cutting sites changes,
fragments of different sizes will be made.
Because Type II restriction endonucleases cut
only at palindromes, they leave “sticky ends” that
will base pair with any other fragment of DNA
cut with the same enzyme. This is useful in
cloning.
©2000 Timothy G. Standish
R. E.s and DNA Ligase
Can be used to make recombinant DNA
EcoRI
EcoRI
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
1 Digestion
AATTC
G
2 Annealing of sticky ends
Ligase
G AATTC
CTTAA G
3 Ligation
4 Recombinant DNA
G AATTC
CTTAA G
©2000 Timothy G. Standish
Gel Electrophoresis
Separates DNA (or RNA or Protein) fragments on the
basis of charge and size
Because DNA is an acid, it looses protons in basic
buffers, thus it has a negative charge that should be
uniform per unit length
Agarose (a polysaccharide) or other gel matrices are
difficult for large DNA fragments to move through
The larger the fragment, the more difficulty it has
moving through gels
By placing DNA in a gel, then applying a voltage accross
the gel, the negatively charged DNA will move toward
the positive pole
Large fragments lag behind while small fragments move
throght the gel relatively rapidly
©2000 Timothy G. Standish
Gel Electrophoresis
Wells
©2000 Timothy G. Standish
Gel Electrophoresis
Wells
©2000 Timothy G. Standish
Gel Electrophoresis
Wells
Large
Direction
of
DNA
Travel
Small
+
©2000 Timothy G. Standish
©2000 Timothy G. Standish