Carbohydrates

Carbohydrates are used by organisms as a source of energy, as building materials, and as cell surface makers for cell-to-cell identification and communication 

• Carbohydrates contain carbon, hydrogen, and oxygen atoms in 1 : 2 : 1 ratio

• Carbohydrates can be classified into: 

 
monosaccharides: simples sugars with many _OH groups 

    

disaccharides: 2 monosaccharides covalently linked

    

polysaccharides: a few monosaccharides

    

oligosaccharides: chains of monosaccharides or disaccharide units

The term saccharide and the suffix -ose both refer to sugars.

• Glycosidic Bond ---> a bond between 2 monosaccharides to form disaccharides

• Condensation---> the process of putting together by removal of water

• Hydrolysis---> opposite of condensation, a large molecule is split into smaller sections by breaking a bond, adding a -H to one section and -OH the other. The products are simpler substances.

Monosaccharides                                                     

• Aldoses---> have aldehyde group at one end (RCHO), and ketoses---> have keto group usually at C2 (RC=OR)

• May be distinguished by the carbonyl group they posses_ aldehyde or ketone, and the number of atoms in their carbon backbone.

• Sugar with 5 carbons is called a pentose, one with 6 carbons a hexose

• Simplest monosaccharides are dihydroxyacetone and glyceraldehyde

• Glucose, galactose and fructose are isomers, they posses the same number and types of atoms but a different arrangement of those atoms

Disaccharides                                                           

• It's formed when two sugars are joined together and a molecule of water is removed. 

• Lactose is made from glucose and galactose 

• Cane sugar, sucrose is made from glucose and fructose

• Maltose is also a disaccharide

Polysaccharides                                                       

• Also known as complex carbohydrates are monosaccharide polymers composed of several hundred to several thousand monosaccharide subunits held together by Glycosidic Linkages.

• Some are in form of straight chains while others are branched 

• Serve 2 important functions in living cells: energy storage and structural support

• Starch and glycogen are storage polysaccharides while cellulose and chitin are structural.

Section of Glycogen

Oligosaccharides                                                 

• Sugars containing 2 or 3 simple sugars attached to one another by covalent bonds known as glycosidic linkages ----> these bonds form by condensation reactions in which the H atom comes from a hydroxyl group on one sugar and _OH group comes from hydroxyl group on another

• Lactose, maltose and sucrose are oligosaccharides consisting 2 simple sugars while raffinose is an oligosaccharide consisting of 3 simple sugars

• They are covalently attached to proteins or membrane lipids and may be linear or branched

• Have many functions; for example they are commonly found on the plasma membrane of animal cells where they play a role in cell-cell recognition

• Selectin is an integral protein that that protrudes on outer surface of mammalian cells

    -----> it participates in cell-cell recognition and binding

Raffinose

Biotechnological Tools and Techniques

Restriction Endonucleases

• Restriction endonucleases, also know as restriction enzymes, are molecular scissors that can cut double-stranded DNA at specific base-pair sequence;
• Each restriction enzyme recognizes a characteristic sequence of nucleotides that is known as its recognition site. Most recognition sites are 4 to 8 bases long and are characterized by complementary palindromic sequence;
• Fragment ends of DNA molecule with short single stranded overhangs, resulting from cleavage by restriction enzyme are called sticky ends;
• Fragment ends of a DNA molecule that are fully base paired, resulting from cleavage by restriction enzymes are known as blunt ends; and
• Restriction enzymes and DNA ligase make recombinant DNA (DNA ligase joins the blunt or sticky ends together).

Gel Electrophoresis

• DNA fragments can be separated using gel electrophoresis. Gel electrophoresis takes advantage of chemical and physical property of DNA;
• DNA is negatively charged. Gel electrophoresis takes advantage of DNA's negative charge. Solution that's containing different size fragments to be separated is placed in a well. A well is depression at one end of the gel;
• The gel is usually a square or rectangle slab and contains a buffer containing an electrolytes and agarose or polyacrylamide;
• Using direct current, negative charge is placed at one end of gel and positive charge is placed in the opposite end of gel;
• The negatively charged DNA will migrate towards the positively charged electrode. Shorter fragments migrate faster than the longer fragments, achieving separation; and 
• Once the gel electrophoresis is complete, the DNA fragments are made visible by staining the gel. Most commonly used stain is ethidium bromide.

Plasmids

• Plasmids are small, circular, double-stranded DNA molecules lacking protein coat that naturally exists in the cytoplasm of many strains of bacteria;
• Plasmids also possess a characteristic known as the copy number. The higher the copy number the higher the number of individual plasmid in ahost bacterial cell;
• Plasmids have the ability to enter and replicate in bacterial cells. Therefore they can be used as a vector to introduce new genes into bacterial cells;
• Region in plasmid that has been engineered to contain recognition sitesd of a nubmber restriction endonucleases is called a multiple cloning site; and
• The combination of the original plasmid DNA and the foreign DNA is known as the recombinant DNA.

Transformation

• Introduction of foreign DNA, usually by plasmid pr virus, into a bacterial cell is called transformation;
• Plasmids can be used as vectors to carry a desired gene into a host cell;
• If a bacterium readily takes up foreign DNA, it is known as a competent cell. Most bacteria are not naturally competent;
• Selective plating is a method that can be used to isolate the cells with recombinant DNA;
• The vectors used for cloning carry an antibiotic-resistance gene. If transformation is successful, the bacteria will grow in the media that contains the antibiotic. No growth ---> bacteria were not transformed and were eliminated by the antibiotic;
• Electroporators - chambers that subject the bacteria to electric shock are also used---> electric shock loosens the structure of cell walls and allows foreign DNA to enter; and
• Modern electrical "gene guns" are used to "shoot" DNA through cell membrane