Transcript Restriction Enzyme Digestion

Restriction Enzyme
Digest Analysis
IMBB 2016
BecA-ILRI Hub, Nairobi
May 9 – 20, 2016
Eunice Machuka
DNA and its bonds
• DNA molecules are macromolecules that hold
the genetic information of living organisms.
• The covalent bond joining adjacent nucleotides
in DNA is called a phosphodiester bond.
• The phosphodiester bonds between nucleotides
in DNA molecules are very stable unless they are
physically stretched or exposed to enzymes
called nucleases.
Endo- and Exonucleases
• Nucleases are enzymes capable of breaking (hydrolyzing)
phosphodiester bonds in DNA molecules.
• Classified into two major groups:
• Exonucleases: If the enzyme digest nucleotides from the
ends of the DNA molecules.
• Endonucleases: If the enzyme digest nucleotides in the
interior of a DNA molecule.
• Restriction enzyme: A protein that recognizes a particular
sequence of DNA and cuts the DNA at that site (the
restriction site)
Restriction enzymes
• Isolated from bacteria as endogenous
“restrictors” of bacterial pathogens. Evolved
by bacteria to protect against viral DNA
infection
• Recognize short specific sequences, often
palindromes and cut DNA at highly specific
points
• Recognize specific sequences
• Four to seven bases
• Each is unique
• Consistent results
http://arbl.cvmbs.colostate.edu/
Different enzymes…different
sites…different cuts
http://www.phschool.com/science/
EcoRI
http://www.phschool.com/science/
Pst I
http://www.phschool.com/science/
Sma I
• These endonucleases cut double strands asymmetrically,
leaving protruding ends referred to as sticky ends a.k.a
compatible cohesive ends.
Digestion Procedure
• Digestion: the act of breaking down into pieces
Prepare Master Mix
Add buffer and enzyme in correct proportions.
Add restriction digest master mix to DNA
Mix thoroughly by flicking tube.
Incubate
Temperature and time depend on enzyme to be used.
View result by gel electrophoresis
Restriction Digest Analysis
• Aim: the digested fragments must be separated and identified.
• Fragments are separated by agarose gel electrophoresis.
[Agarose is a large polysaccharide].
• Gel electrophoresis: your DNA will move through spaces in
agarose against a current
• DNA has a negative charge and will migrate towards the
cathode
Restriction Digest Analysis
• Length=1650bp
• SaII yields two
fragments (1200bp and
450bp)
• KpnI cuts at 2 sites
giving 3 fragments
• SaII and KpnI cut 3X
yielding 4 fragments
Example
• Digestion of a gene and expected fragment sizes
Applications
• Cloning and library
preparation
http://www.kochi-u.ac.jp/~tatataa/tech2/gene/ligation.jpg
• DNA Fingerprinting, RFLP,
AFLP, TRFLP etc.
Examples
• Restriction enzyme digestion is
used for:
• DNA fingerprinting- Identifying
individuals
• Identifying species
(e.g. Phytophtora)
• Cloning (moving genes in and out
of plasmids)
http://www.nottingham.ac.uk/biology
http://www.bio.davidson.edu/courses/genomics/m
ethod/inducepromoter.gif
http://www.kochi-u.ac.jp/~tatataa/tech2/gene/ligation.jpg
Non-Examples
Restriction enzyme digestion is
NOT used for:
• Cutting RNA
• Sequencing DNA
Resources
http://www.phschool.com/science/biology_place/biocoach/red/intro.html
http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html
http://web.indstate.edu/thcme/mwking/molecularmedicine.html#restriction
http://www.fermentas.com
http://www.chemsoc.org/timeline/graphic/1984_dnaf.jpg
END
Quick Quiz 1
A restriction enzyme cuts:
A.
B.
C.
D.
Single-stranded DNA
RNA
Proteins
Double-stranded DNA
Quick Quiz 2
A restriction enzyme works by identifying a specific DNA sequence
and cleaving:
A.
B.
C.
D.
The sugar-phosphate backbone of one strand
The sugar-phosphate backbone of both strands
The nitrogenous bases from one strand
The nitrogenous bases from both strands
Quick Quiz 3
ITS PCR products must be cut by restriction
digestion to identify the different animal/plant
species because:
A. Both species have an ITS PCR product of the
same size
B. Both species have the same restriction sites
C. ITS PCR products can vary in size
D. Neither species gives good ITS PCR bands
Questions
• 1. What is a nuclease?
• DNA held by covalent bond joining adjacent nucleotides in DNA is called a phosphodiester
bond. The phosphodiester bond between nucleotide in DNA molecules are very stable
unless they are physically stretched or exposed to enzymes name nucleases.
• Enzyme capable of breaking (hydrolyzing) phosphodiester bonds in DNA molecules and
classified into exonuclease and endonuclease .
• 2. How does an endonuclease differ from an exonuclease?
• Endonuclease digest DNA by breaking phosphodiester bonds in the interior of DNA
molecule. Exonuclease enzyme digest nucleotides from the ends of the DNA molecule.
• 3. What is a restriction endonucleases? Write names of some restriction endouclease.
• Restriction endonucleases are a special class of Endonuclease from bacteria to cut DNA.
EcoRI & Hind III. These are enzymes that digest DNA by recognizing specific short
sequences of bases called palindromic.