Chapter 4

Basic Tools and Techniques of DNA SciencePart 1

by Alvin Zhu

Cloning

In 1997, Dr. Ian Wilmut and his team of researchers from the Roslin Institute in Scotland successfully cloned a sheep.

WHO IS THIS SHEEP? HOW DID THEY CLONE HER?

Dolly's Cloning

Process of Dolly's cloning

1. Removed nucleus from an unfertilized egg

2. Fused the egg with the nucleus of an adult cell from a sheep's utter

3. Fused egg grown in culture and then transplanted into a surrogate mother

1. General Procedure for Cloning

1. Transplant the gene of interest into a carrier DNA (Vector)

2. Vector transferred into an appropriate host cell

3. Mitosis by the host cell

Restriction Endonucleases

Restriction Endonuclease- cuts DNA in a predictable and reproducible way

Early 1950s: Luria and Bertani (University of Illnois) and Weigle (California Institute of Technology, studied the immune system of bacteria

Observed that certain strains of E. coli were resistant to infections

This resistance seemed to be a property of the bacterial cell

It was able to restrict growth and replication of certain phages

1962: Arber (University of GEneva) found evidence of an enzyme system that recognizes and destroys invading DNA while modifying its own DNA to prevent self-destruction

Several years later, Arber and his associates isolated E. coli extracts that efficiently cleaved phages

Contained the first restriction endonuclease

Restriction endonuclease is part of a larger group of enzymes (nucleases), which break the phosphodiester bonds linking adjacent nucleotides

WHAT IS THE DIFFERENCE BETWEEN ENDONUCLEASES AND EXONUCLEASES?

• Endonucleases cut DNA within the DNA (in the middle)• Exonucleases cut DNA from the sides

Some endonucleases also protects host DNA from digestion

They add methyl groups to the restriction site of an enzyme, effectively blocking the enzyme from recognizing its restriction site.

Only one strand needs to be methylated (hemimethylated) but both strands are usually methylated. This is important because methylation does not come immediately after synthesis. The parent strand with the methyl group protects the newly created daughter strand.

1970: Smith and Wilcox (John Hopkins) isolated enzyme HindII, from Haemophilus influenzae. Their enzyme was much more useful than the

one discovered by Arber.

WHY?

• Arber's enzyme cut the DNA thousands of nucleotides away from its restriction site. Smith and Wilcox's cut it within its recognition sequence

• Smith and Wilcox's restriction enzyme did not modify cellular activity

Methylated DNA Diagram

Three Major Classes of Restriction Endonucleases

Type I AND type III

Restriction and modification activity

Cut DNA outside of their recognition sequences

Uses ATP to move from recognition site to cleavage site

Little use because of unpredictability and requirement of ATP

Type II

Restriction activity but not modification

Predictable cuts in sites within or next to recognition site

Require magnesium ion (Mg++)

Recognition sequences are usually 4-8 nucleotides long and palindromic

Decoding a Restriction Enzyme

EcoRI

E = genus Escherichia

co = strain coli

R = strain RY13

I = first endonuclease identified

Cuts of Type II restriction Enzymes

1. Straight through both strands (creating a blunt end)

2. Cleave 3' to the center of the two strands, leaving 2-4 nucleotides from the 5' end (sticky ends)

3. Cleave 5' to the center of the two strands, leaving 2-4 nucleotides from the 5' end (sticky ends)

The sticky ends are complementary to any strand cut by the same enzyme. It is important in cloning.

How EcoRI cuts DNA

It acts as two EcoRI molecules working together (homodimer) to simultaneously cut both strands of NDA

1. The two molecules align at the recognition site in opposite orientations

2. Amino acids within EcoRI form H bonds with the recognition sequence

3. Residuals within EcoRI catalyzes hydrolysis to break the phosphodiester linkage on each strand

4. DNA molecule cut in two (phosphate group at the 5' end and a hydroxyl group at the 3' end)

Theoretical Frequency of Cuts

Assuming the four nucleotides are distributed equally and randomly,

4-nucelotide sequences will occur once every 256 nucleotides

6-nucleotide sequences will occur once every 4096 nucleotides

8-nucleotide sequences will occur once every 65536 nucleotides

4^n where n=number of nucleotide sequences

IS THIS CALCULATION ACCURATE?

No because DNA sequences are not randomly arranged. For example, enzyme NotI recognizes a site every 1 million base pairs because its sequence is rich in G's, which are not very frequent

Origins Gel Electrophoresis

HOW DID SCIENTISTS SEPARATE DNA FRAGMENTS BEFORE GEL ELECTROPHORESIS?

Velocity-sedimentation ultracentrifugation DNA samples are centrifuged through salt or sugar,

separating the fragments by size (largest at the top, smallest at the bottom)

Origins of Electrophoresis

1970: Daniel Nathans first used polyacrylamide gel electrophoresis to separate DNA (up to 1000 nucleotides)

The thought behind why electrophoresis works is that DNA is negatively charged. Oxygen from the phosphate backbone radiates the negative charge. Therefore they are attracted to the positive pole (anode) and repelled from the negative pole (cathode)

Smaller molecules move through the gel more easily than the larger ones. Thus the distance moved and the molecular weight are inversely proportional.

Although it was easier to do than centrifugation, it was still hard work at that time .

The gel had to be cut into bands and the DNA had to be radioactively labeled.

Improvements for Gel Electrophoresis 1973: Sambrook and his team at the Cold Spring Harbor

Laboratory made important refinements:

1. Replaced polyacrylamide with agarose (highly purified agar). This can separate DNA from 100 nucleotides to 50,000 nucleotides).

A low concentration of agarose (.3%) is used to separate large fragments

A high concentration of agarose (2%) is used to separate smaller fragments

2. Replaced radioactively labeling with fluorescent dye to stain DNA. The fluorescent dye is extremely sensitive to UV light, so tiny (5 ng) amounts of DNA can be found.

Their changes are so effective that they are still used today

Basic Procedure for Gel Electrophoresis1. Molten agar is poured into a casting tray where plastic or a Plexiglas comb is

suspended.

2. Agarose cools to a jelly substance and immersed in buffer solution with ions needed to conduct electricity.

3. When the gel solidifies, the comb is removed.DNA is mixed with a loading solution (sucrose and dyes). The sucrose helps the DNA sink into the wells

4. Current is conducted through the agar, creating an electric field.

5. DNA moves into the gels from the wells. The stains, however, migrate independently from the DNA. For example, bromophenol blue migrates at a pace equivalent to 300 nucleotides in 1% gel.

6. Gel soaked in a dilute solution of ethidium bromide.

7. Stain diffuses and concentrates in regions where it binds to DNA (OR this can be done at the beginning, incorporated into the buffer)

8. Stained gel exposed to UV light, producing a glowing orange (590 nm) band. This band is not a single DNA but millions of identical DNA with the same length.