©2000 Timothy G. Standish Restriction Enzyme Digestion Timothy G. Standish, Ph. D.

©2000 Timothy G. Standish

Restriction Enzyme Restriction Enzyme DigestionDigestion

Timothy G. Standish, Ph. D.


Enzymes are the Tools of Enzymes are the Tools of DNA TechnologyDNA 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


Restriction EndonucleasesRestriction Endonucleases There are a number of different subclasses 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

downstream of the site. Type II restriction endonucleases are the most useful class as

they recognize specific palindromic sequences in DNA and cut the sugar phosphate backbone within the palindrome


What is a Palindrome?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.


DNA PalindromesDNA 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'


Uses of Restriction Uses of Restriction EndonucleasesEndonucleases

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.


G

CTTAA

AATTC

G

1 Digestion

2 Annealing of sticky ends

3 Ligation

Ligase

G

CTTAA

AATTC

G

EcoRIEcoRI

R. E.s and DNA Ligase R. E.s and DNA Ligase Can be used to make recombinant DNACan be used to make recombinant DNA

GAATTC

CTTAAG

GAATTC

CTTAAG

G

CTTAA

AATTC

G

4 Recombinant DNA


Gel ElectrophoresisGel Electrophoresis Separates DNA (or RNA or Protein) fragments on the basis of charge

and size Because DNA is an acid, it loses 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 across the gel, the

negatively charged DNA will move toward the positive pole Large fragments lag behind while small fragments move through the

gel relatively rapidly


Gel ElectrophoresisGel Electrophoresis

Wells


Gel ElectrophoresisGel Electrophoresis

+

-

Direction of

DNATravel

Wells

Small

Large

©2000 Timothy G. Standish Restriction Enzyme Digestion Timothy G. Standish, Ph. D.

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Transcript of ©2000 Timothy G. Standish Restriction Enzyme Digestion Timothy G. Standish, Ph. D.