General Genetics. 1. Introduce the students to digest genomic DNA by restriction endonucleases. 2....

Lab # 7Restriction Enzymes

General Genetics

Objectives:

1. Introduce the students to digest genomic DNA by restriction endonucleases.

2. Observe the results of digestion on agarose gel electrophoresis.

Theoretical Basis Using Restriction Enzymes

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

1. PH2. Temperature3. Salt Concentration4. Ions

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

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

Restriction Endonucleases: Type II

BamH1

GGATCCCCTAGG

HaeIII

GGCCCCGG

Cohesive Ends(5´ Overhang)

Cohesive Ends(3´ Overhang)

KpnI

GGTACCCCATGG

Blunt Ends(No Overhang)

Restriction Enzymes

Hundreds of restriction enzymes have been identified.

Most recognize and cut palindromic sequences

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

Important for molecular biologists because restriction enzymes create unpaired "sticky ends" which anneal with any complementary sequence

Bacterial" 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)

Cont.

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 or counterclockwise 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 insufficient 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 Mapping

Two possible maps inferred from the observations

Restriction Enzyme Mapping

4.3 kb3.7 kb

2.3 kb1.9 kb

1.4 kb1.3 kb

0.7 kb

BamH1XhoIBamH1XhoI

PCR and Restriction enzymes