Download “Ins and Outs” of Restrictions Enzymes

The “Ins and Outs” of
Restrictions Enzymes
DNA Enzymology History
• 1953: molecular structure of DNA described
• 1955: DNA polymerase
• 1966: DNA ligase
• 1968: 1st sequence specific restriction
nuclease identified (HindII)
• mid 1970’s: companies began to search for
more restriction nucleases
Nucleases
• Enzymes (phosphodiesterases) that catalyze
the hydrolysis of nucleic acids in all
organisms
• Processes under the control of nucleases
include:
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Protection against invading DNA
DNA repair
DNA recombination
DNA synthesis and packaging
Nucleases
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Work on ssDNA, dsDNA or both
Work in the 3’-5’ or 5’-3’ direction or both
Most recognize palindromic sequences
Some work on methylated DNA
Some require helper proteins or coenzymes
• Classified based on their specificity
– EXOnucleases
– ENDOnucleases
Endonuclease Classifications
• Type I
– Combination restriction-and-modification enzymes
– Cut at random outside of recognition site
– Produce indiscrete fragments
• Type II
– Cut at defined positions within their recognition
site
– Produce discrete fragments
– Wide variety of protein types which are divided
into subgroups
Endonuclease Classifications
• Type III
– Combination restriction-and-modification
enzymes
– Cleave outside the recognition sequence
– Rarely give complete digests
Naming Restriction Enzymes
• Name reflects their origin
– First letter from the genus
– Second two letters from the species
– Numbers indicate the order from which they were
isolated from single strains
• > 3600 restriction enzymes (most are
isoschizomers)
• > 250 sequence specificities
• > 230 strains of bacteria
Products of Cleavage
• Blunt End DNA Fragments
– Cuts are made immediately opposite one another
• Sticky Ends
– Cuts are staggered resulting in 5’ protruding ends
(commercial enzymes)
– Can pair with complementary sequence
Restriction Enzymes as Tools
• Restriction Mapping
– Obtaining structural information on a piece of DNA
• Cut large pieces of DNA into smaller pieces for
sequencing
• Compare DNA fragments without having information on
the nucleotide sequence
– Programs available to make maps
• www.restrictionmapper.org
• Webcutter 2.0
• NEBcutter V2.0
Restriction Enzymes as Tools
• Restriction Fragment
Length Polymorphism
(RFLP)
– Organisms can be
differentiated by analysis
of patterns derived from
cleavage of their DNA by
restriction enzymes
– When used with a
nucleic acid probe they
can be used as genetic
markers (t-RFLP)
V. Grüntzig, B. Stres, H. L. Ayala del Río, and J. M. Tiedje, Center for Microbial Ecology, Michigan State University,
East Lansing, Michigan, 48824
Restriction Enzymes as Tools
• DNA Cloning
– DNA fragments (created by RE cuts) from
donor are inserted into vector
– Vector is inserted into host cell and
replicated
– DNA fragment or gene product can be
detected
Factors that Influence
Restriction Enzyme Activity
• pH: 7.2-8.5*
• Cofactors: Mg2+; 5-30 mM*
• Salt concentration: NaCl, KCl, acetate
or chloride anions; 50-150 mM*
• Stabilizer: BSA; 0.1 mg/ml
• Temperature
Ref: RE Resource, www.promega.com
Factors that Influence
Restriction Enzyme Activity
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Detergents
Glycerol: 5-10%*
Reaction volume: 10-50 uL
DNA: 1 ug/10-50 uL
Ref: RE Resource, www.promega.com
Multiple Restriction Enzyme
Digests
• Choose a buffer that provides 75% or
greater activity for both enzymes
• Use optimal buffer for one enzyme and
adjust enzyme concentration for the
second enzyme*
• Choose an isochizomer
Multiple Restriction Enzyme
Digests
• Perform a single digest, stop reaction,
adjust concentrations, perform second
digest
• Perform each digest sequentially
(requires a precipitation or purification
step)
Experimental Controls
• Untreated DNA: integrity of DNA
• No enzyme: changes independent of
enzyme
• Control DNA (usually lambda):confirms
enzyme activity
Star Activity
• Occurs under non-optimal conditions
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High pH
High glycerol concentrations
High enzyme concentrations
Organic solvents
Low ionic strength
• Decrease in enzyme fidelity= non-specific cutting
• More bands than expected
Methylation
• Some RE’s don’t recognize methylated
sites
• Methylase recognition site overlaps the
RE recognition site
Resources
• REBASE (www.rebase.neb.com)
– NEB resource
– Restriction enzymes and related proteins
• InBase
– NEB resource
– Inteins and their properties
• Supplier Catalogues
– NEB
– Promega