Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a

DNA molecule

at a particular place. They are essential tools for

recombinant DNA technology

. The enzyme “scans” a DNA molecule, looking for a particular sequence, usually of four to six nucleotides. Once it finds this recognition sequence, it stops and cuts the strands. This is known as enzyme digestion. On double stranded DNA the recognition sequence is on both strands, but runs in opposite directions. This allows the enzyme to cut both strands. Sometimes the cut is blunt, sometimes the cut is uneven with dangling nucleotides on one of the two strands. This uneven cut is known as sticky ends.

A blunt end may look like this:

A sticky end like this:

Most plasmids used for recombinant technology have recognition sequences for a number of restriction enzymes. This allows a scientist to choose from a number of places to cut the plasmid with a restriction enzyme. Ligation enzymes can then be used to sort of paste in new genomic sequences. These mutated, or recombined, plasmids can then be grown up in bacterial cells and used for a number of purposes, including the addition of genes to mammalian genomes.

You always want to read carefully the information sheet that comes with your enzymes as well as the catalogue information. The better you know your enzyme, the more likely you will be to have a successful digestion.  Most enzymes come in glycerol solution as a storage buffer, but enzymes don’t work well in the presence of high glycerol concentration. You want to be sure to dilute the glycerol content down to less than 5% to ensure proper enzymatic activity.

Problems with enzyme activity can occur under the following conditions:

• High glycerol concentration
• Enzyme-to-DNA ratio is too high
• pH is too high
• Organic solvents, particularly ethanol, interfere with your DNA

Some other helpful tips for working with enzymes include:

• Wear gloves. This protects you as well as protecting your sample from contamination from you.
• Keep the enzymes cold.
• Don’t reuse tips. Contamination will ruin your experiment.
• Know your enzyme. Know what makes it work and what causes problems with it. Know what buffers to use.

The following example of iron, sulphur and iron sulphide will make us understand the difference between mixtures and compounds.

Properties of Iron and Sulphur

The following experiment shows the difference in properties between the elements iron and sulphur (Fig.4.1).



Introduction to compounds and elements

Compounds:

The compound is defined as a pure substance containing two or more elements which are combined together in a fixed proportion by mass.

Elements:

An element is the simplest or basic form of a pure substance which cannot be broken into anything simpler than it by physical or chemical methods.  The pure substance which is made up of one kind of atoms only.  The common examples of elements are hydrogen, carbon, , sulphur, gold etc.

Types of Elements

Elements are further classified into three types.

1. Metals
2. Non-metal
3. Semi-metals

Metals

• Metals are solids at room temperature.
• Metals are generally quite hard.
• Good conductors.
• Naturally Malleable.
• Naturally Ductile.
• Metals have generally high melting and boiling points.

Non-metals

• Non-metals are either gases or solids at room temperature.
• Non-metal varies in colour.  Solids have generally dull surfaces.
• Non-metals are mostly poor conductor of heat and electricity.
• Most of the non-metals are quite soft and have smaller densities than metals.
• Non-metal are non-malleable and non-ductile in nature.
• Non-metals are also not sonorous in nature.
• As compared to the metals, the non-metals have very low melting and boiling points.

Semi-metals

There are few elements which possess the characteristics of both metals and non-metals.  These are actually border-line elements and are known as semi-metals.  Semi-metals are also called as metalloids.  A few common examples are:  Arsenic, Antimony and Bismuth.

Types of elements based on physical states:

Based on physical states, the elements have been classified as solids, liquids and gases.

Solid elements: Most of the elements are solids at room temperature.  For example, copper, silver, gold, potassium, carbon (diamond, graphite), iodine, phosphorous etc.

Liquid elements: Only mercury and bromine exist as liquid at room temperature.  Gallium and cesium become liquids at a temperature 302 K and 303 K respectively.  These are slightly higher than the room temperature (298 K).

Gaseous elements: Eleven elements exist in the gaseous state at room temperature.  These are hydrogen, oxygen, nitrogen, fluorine, chlorine, helium, neon, argon, krypton, xenon and radon.

Types of Compounds

A compound is also a pure substance like elements. But it represents a combination of two or more elements which are combined chemically.

Types of compounds

The compounds have been classified into two types

• Organic compounds
• Inorganic compounds

Organic compounds

Organic compounds are the compounds which are obtained from living beings (plant and animal).  It has been found that all the organic compounds contain carbon as their essential constituent.  The organic compounds are quite often known as carbon compounds.

Examples:  Methane, ethane, propane, alcohol, etc.

Inorganic compounds

Inorganic compounds have mostly obtained from non-living sources such as rocks and minerals.

Example:  Salt, marble, washing soda, baking soda, etc.

Characteristics of compounds:

• A pure compound is composed of the same elements combined in a fixed ratio by mass.
• A pure compound is homogeneous in nature.
• A chemical compound is formed as a result of chemical reaction between the constituent elements.
• Properties of the compound are altogether different from the elements from which it is formed.
• Constituents of a chemical compound cannot be separated mechanically.

(i) Low dissociation energies

All the halogens have very low dissociation energies. As a result, they can readily dissociate into atoms and react with other substances. As shown below, the dissociation energies of halogens are quite low in comparison to common molecules such as H2, O2 and N2.

(ii) High electron affinity

Halogens have very high electron affinity values and therefore, have very strong tendency to gain an electron. Thus halogens are very reactive elements due to their low dissociation energies and high electron affinity values. As clear from the values of bond dissociation energies, fluorine has the lowest bond dissociation energy. This is due to weak F-F bond because of the repulsion between the non-bonding electrons in the small molecule. Therefore, it is most reactive among the halogens.

Some of the important chemical reactions of halogens are discussed ahead.