Techniques You Should Know or learn on your own - DNA Sequencing - Polymerase Chain Reaction - DNA Footprinting/Nucelase protection - Restriction enzyme manipulation - Nucelase protection - cDNA Synthesis - Site-Directed Mutagenesis
What Does It Mean: “To Clone”? Clone: a collection of molecules or cells, all identical to an original molecule or cell • To "clone a gene" is to make many copies of it - for example, by replicating it in a culture of bacteria. • Cloned gene can be a normal copy of a gene (= “wild type”). • Cloned gene can be an altered version of a gene (= “mutant”). • Recombinant DNA technology makes manipulating genes possible.
Restriction Enzymes • Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”. • Cut up “foreign” DNA that invades the cell. • Type II and III restriction enzymes cleave DNA chains at selected sites. • Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage. • An enzyme that recognizes a 6-base sequence is called a "six-base cutter”.
Basics of type II Restriction Enzymes • No ATP requirement.