Restriction Enzymes. Theoretical Basis Using Restriction Enzymes The activity of restriction enzymes is dependent upon precise environmental condtions:

Restriction EnzymesRestriction Enzymes

Theoretical Basis Theoretical Basis Using Restriction EnzymesUsing Restriction Enzymes

The activity of restriction enzymes is dependent upon The activity of restriction enzymes is dependent upon precise environmental condtions:precise environmental condtions:

PHPHTemperatureTemperatureSalt ConcentrationSalt ConcentrationIonsIons

An An Enzymatic UnitEnzymatic Unit (u) (u) is defined as the amount of enzyme is defined as the amount of enzyme required to digest 1 ug of DNA under optimal conditions:required to digest 1 ug of DNA under optimal conditions:

3-5 u/ug of genomic DNA 3-5 u/ug of genomic DNA 1 u/ug of plasmid DNA1 u/ug of plasmid DNAStocks typically at 10 u/ulStocks typically at 10 u/ul

BamH1

GGATCCCCTAGG

HaeIII

GGCCCCGG

Cohesive Ends(5´ Overhang)

Cohesive Ends(3´ Overhang)

KpnI

GGTACCCCATGG

Blunt Ends(No Overhang)

Restriction Endonucleases: Restriction Endonucleases: Type IIType II

Restriction EnzymesRestriction Enzymes

Hundreds of restriction enzymes have been Hundreds of restriction enzymes have been identified. identified.

Most recognize and cut Most recognize and cut palindromic palindromic sequencessequences

Many leave staggered (sticky) endsMany leave staggered (sticky) ends by choosing correct enzymes can cut DNA by choosing correct enzymes can cut DNA

very preciselyvery precisely Important for molecular biologists because Important for molecular biologists because

restriction enzymes create unpaired restriction enzymes create unpaired "sticky "sticky ends" ends" which anneal with any which anneal with any complementary sequencecomplementary sequence

bacterial" immune system": destroy any "non-self" DNAbacterial" immune system": destroy any "non-self" DNA

methylase recognizes same sequence in host DNA and protects it by methylating it; restriction enzyme destroys unprotected = non-self DNA (restriction/modification systems)

As an example, consider a 5000 base pair, circular plasmid DNA containing single recognition sites for enzymes A, B, and C. Any one of these enzymes will cleave the DNA once to produce a linear molecule of 5000 base pairs.

Differently paired combinations of enzymes in the same reaction mixture (double-digests) will produce the following DNA fragments (sizes in base pairs):

Arbitrarily placing one of the cleavage sites at the top of a circle. This site acts as a reference point.

The closest cleavage site to this point can be placed in a clockwise orcounterclockwise direction.

The triple digest, A + B + C is a confirmatory test

Generally, a restriction enzyme map is constructed by first determining the number of fragments each individual enzyme produces. The size and number of fragments is determined by electrophoresis.

If a DNA molecule contains several recognition sites for a restriction enzyme, then under certain experimental conditions, it is possible that certain sites are cleaved but not others. These incompletely cleaved fragments of DNA are called partial digests(partials). Partials can arise if an insuffi cient amount of enzyme is used or the reaction is stopped after a short time (Figure 5). Reactions containing partials may also contain some molecules that have been completely cleaved.

Restriction Enzyme MappingRestriction Enzyme Mapping

Two possible maps inferred from the observations

Restriction Enzyme MappingRestriction Enzyme Mapping

4.3 kb3.7 kb

2.3 kb1.9 kb

1.4 kb1.3 kb

0.7 kb

BamH1 XhoIBamH1

XhoI

PCR and PCR and Restriction Restriction enzymesenzymes