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