Transcript Restriction Enzymes

Restriction Enzyme Digestion
of DNA
Experiment Goals
• Digestion of DNA by restriction enzyme
• Analyze digested DNA by electrophoresis
Restriction Enzymes
• Restriction enzymes are endonucleases (Endo (inside),
nuclease(cuts nucleic acid), which catalyze the cleavage
of the phosphodiester bonds within both strands of
DNA.
• They require Mg+2 for activity and generate a 5 prime
(5') phosphate and a 3 prime (3') hydroxyl group at the
point of cleavage.
• The distinguishing feature of restriction enzymes is
that they only cut at very specific sequences of bases.
This specific DNA sequence is called recognition
sequence.
Restriction enzymes are traditionally classified according
to the subunit composition, cleavage position,
sequence-specificity and cofactor requirements.
• A restriction enzyme requires a specific double stranded
recognition sequence of nucleotides to cut DNA.
• Recognition sites are usually 4 to 8 base pairs in length.
• Cleavage occurs within or near the site.
Naming of Restriction enzymes
• Restriction enzymes are named according to the
organism from which they are isolated.
• This is done by using the first letter of the genus
followed by the first two letters of the species and
additional letter or number represent the strain or
serotypes.
• Only certain strains or sub-strains of a particular
species may produce restriction enzymes.
Example of RE naming:
HindII: Restriction enzyme was isolated from
Haemophilus influenzae serotype d
• There are hundreds of different REs from different
microorganisms.
• Different restriction
recognition sequences.
enzymes
have
different
• This makes it possible to create a wide variety of
different gene fragments.
Examples:
Target sequence
Enzyme
Organism from which derived
(cut at *)
5' -->3'
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Eco RI
Escherichia coli RY 13
G* A A T T C
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Mbo I
Moraxella bovis
*G A T C
Pst I
Providencia stuartii
CTGCA*G
Sma I
Serratia marcescens
CCC*GGG
Taq I
Thermophilus aquaticus
T*CGA
Xma I
Xanthamonas malvacearum
C*CCGGG
Many recognition sequences are palindromic. For
example,
5’ GAATTC 3’
3’ CTTAAG 5’
palindromic: read the same in the opposite
direction
Sticky and Blunt end cutters
Not all restriction endonucleases cut symmetrically
and leave blunt ends.
Many endonucleases cleave the DNA backbones in
positions that are not directly opposite each other or
can make staggered cuts, which produce single
stranded “sticky-ends”
DNA from different sources can be spliced easily
because of these sticky-end overhangs.
Example of RE that produce sticky end cutters:
Some restriction enzymes cut DNA at opposite
base.
They leave blunt ended DNA fragments
These are called blunt end cutters
HaeIII
Restriction Enzyme Uses
1. Recombinant DNA technology
*Discovery of enzymes that cut and paste DNA make
genetic engineering possible.
*Restriction enzyme cuts DNA and generates fragments
*Ligase joins different DNA fragments
*DNA fragments from different species can be ligated
(joined) to create Recombinant DNA
2. Cloning
Replicates a sequence inserted into a host
cell
3. DNA restriction mapping
The location of the restriction enzyme cleavage
sites on the DNA molecule.
Biological Function
• Restriction enzyme is part of the cell’s
restriction-modification system in bacteria.
• The phenomenon of restriction modification in
bacteria is a small scale immune system for
protection from infection by foreign DNA.
• Bacteria can protect themselves only after foreign
DNA has entered their cytoplasm.
Unit Determination Assay
• One unit of restriction endonuclease is defined
as the amount of enzyme required to digest
one microgram of the appropriate substrate
DNA completely in 60 minutes under the
conditions specified for that enzyme.
Set up of a restriction enzyme reaction
• A RE reaction contains the DNA to be
analyzed,
• A restriction enzyme,
• A restriction enzyme buffer mix.
– contains a buffering agent to maintain
constant pH,
– and Mg++ (from MgCl2) as a necessary
cofactor for enzyme activity.
Digestion by Restriction Enzyme
• Measure the DNA concentration
– Use the Nano-drop spectrophotometer to measure
the concentration of DNA, this is used to determine
the amount of HinfI restriction enzyme to be used.
Digestion of DNA
• Mix the following components in a clean microtube.
• Mix gently and spin down for a few seconds.
• Incubate at 37°C for 16 hours.
Analysis of DNA digestion
• Analyze products on 2% agarose gel containing
ethidium bromide.
• Samples are prepared with loading dye and then
loaded on the gel.
• Visualize the PCR product on UV transilluminator.
Electrophoresis of Genomic DNA
Odd numbered lanes contain undigested genomic DNA
Even numbered lanes contain digested genomic DNA
Undigested DNA is represented by a sharp band near the wells of the gel, while
smearing indicates digested DNA sample.
Exercise
Q1 Restriction enzyme AluI which recognize
sequence
5’ AGCT 3'
3' TCGA 5‘
What are the double strand cut in DNA ( begin at G
from 5’)
Q2: PstI RE recognize
5’ CTGCA*G 3'
3' G*ACGTC 5’
What are the digestion products?