Restriction Endonucleases- enzymes that __ cleave ds DNA into

							Restriction Endonucleases - enzymes that cleave its DNA into fragments at specific sequences; also known
as restriction enzymes.

Recognition site: a specific sequence within a DNA, usually palindromic, and consisting of 4-8 nucleotides,
that a restriction endonuclease recognizes and cleaves.

These enzymes are obtained from bacterial species that have developed these enzymes to protect themselves
from viruses.

                                   Naming of Restriction Endonucleases:

                                         Escherichia coli (EcoRI)
                                    Haemophilus parainfluenzae (HindIII)


Different restriction enzymes can have the same recognition site - such enzymes are called isoschizomers:
Look at the recognition sites for Sacl and Sstl - they are identical. In some cases isoschizomers cut identically
within their recognition site, but sometimes they do not.




One other point to notice from the table above is that most recognition sequences are palindromes - they read
the same forward (5' to 3' on the top strand) and backward (5' to 3' on the bottom strand).


                        Patterns of DNA Cutting by Restriction Enzymes
Restriction enzymes hydrolyze the backbone of DNA between deoxyribose and phosphate groups. This

leaves a phosphate group on the 5' ends and a hydroxyl on the 3' ends of both strands. A few restriction

enzymes will cleave single stranded DNA, although usually at low efficiency.
The restriction enzymes most used in molecular biology labs cut within their recognition sites and generate

one of three different types of ends. In the diagrams below, the recognition site is boxed in and the cut sites

indicated by red triangles.

    •       5’ overhangs: the enzyme cuts asymmetrically within the recognition site such that a short single-
            stranded segment extends from the 5' ends. BamHI cuts in this mariner.




        •      3' overhangs: again, we see asymmetrical cutting within the recognition site, but the result is a
                       single-stranded overhang from the two 3' ends. KpnI cuts in this manner.




        •   Blunts: enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends
            without overhangs. SmaI is an example of an enzyme that generates blunt ends.




The 5'or 3'overhangs generated by enzymes that cut asymmetrically are called sticky ends or cohesive ends,

because they will readily stick or anneal with their partner by base pairing.


Types of digestion:

Sticky ends: both fragments have DNA nucleotides that are now lacking their complementary base. ie.
EcoRl

Blunt ends: ends of the DNA molecule fragments are fully paired.

Methylases

RE need to distinguish between foreign DNA and host DNA. Methylases in prokaryotes modify recognition
sites of a RE by placing a methyl group on one of the bases preventing the RE from cutting the DNA into
fragments.
Eg EcoRl methylase

GAATTC/CTTAAG (adds methyl at 2nd A)

DNA Ligase

When DNA is cut by RE, they need to be joined back together: DNA Ligase is the enzyme responsible for
joining nucleotides together.

DNA Ligases are enzymes responsible for reestablishing the phosphodiester bonds on the backbone of DNA.

Gel Electrophoresis

A method of separating charged molecules (ie DNA, proteins) on the basis of size by sorting through a gel
meshwork.

Recall DNA- sugar phosphate base (phosphate gives DNA -ve charge).

Types of gels

Agarose - gel forming polysaccharide derive4 from seaweed (often used to separate DNA).
Polyacrylamide - artificial polymer often used to separate protein.

Once DNA is separated using gel electrophoresis, the band patterns can be visualized by staining.

Ethidium bromide is often added to gel to help visualize the bands. This molecule acts by inserting between
the nucleotides and can be seen when examined under UV light.

Plasmids

Plasmids are small circular pieces of DNA that naturally exist in many bacteria.
Plasmids are small circular pieces of DNA that can exit and enter a bacterial cell. Often carrying an
important gene that imparts resistance to antibiotic or to toxic heavy metals.
Plasmids often have specific characteristics such as copy number. Copy number determines the number of
individual plasmids in a host bacterial cell. If more plasmids are present, this will result in more protein being
synthesized.

Because a Plasmids # ultimately reflects the amount of protein present, researchers have exploited in cloning.

Multiple Cloning Site - region in plasmid that has been engineered to contain recognition sites of a number
of RE.