Recombinant DNA recombinant DNA – techniques in which genes from two different sources - often different species - are combined in vitro into the same molecule genetic engineering – the direct manipulation of genes for practical purposes DNA technology has resulted in biotechnology, the manipulation of organisms or their components to make useful products DNA technology is now applied in areas ranging from agriculture to criminal law

genetic engineering is possible because of restriction enzymes (restriction endonucleases): Very specific – recognize and then cut DNA molecules at specific base sequences called a restriction site (recognition sequence) –These are often a symmetrical series of four to eight bases on both strands running in opposite directions. If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3’. In nature, bacteria use restriction enzymes for protection to cut foreign DNA (from invading viruses)

Restriction enzymes cut the covalent bonds of both strands, often in a staggered way creating single-stranded sticky ends. –Sticky ends will form hydrogen-bonded base pairs with complementary sticky ends on other DNA molecules cut with the same restriction enzyme. DNA ligase bonds the complementary sticky ends together Restriction enzymes and DNA ligase are used to “cut and paste” DNA pieces together

DNA Fingerprinting Restriction Enzymes are also used for DNA fingerprinting (profiling) –Creating a pattern of DNA bands on a gel Because the restriction site (recognition sequence) usually occurs (by chance) many times on a long DNA molecule, a restriction enzyme will make many cuts Result: production of fragments of DNA of various lengths – Restriction Fragment Length Polymorphs (RFLPs) Since all individuals have unique sequences of DNA, restriction enzymes cut each individual’s DNA into different sized RFLPs

The RFLPs are then separated by gel electrophoresis resulting in a bar-like pattern Electrophoresis means “to carry with an electric current” Different sized RFLPs will be carried different distances by an electric current as they migrate through an agarose gel inside a gel box –Electricity is run through the gel box creating a positive end and a negative end Negatively charged DNA migrates from the negative end of the gel box through the pores in the gel to the positive end of the gel box Smaller RFLPs will migrate farther than larger pieces, spreading the RFLPs across the gel in a bar-like pattern Stain is used to make the DNA bands visible

SEM photo of a 1% LE Agarose gel at 22kX magnification

Uses for DNA fingerprinting Allows scientists to compare DNA from various organisms and identify a particular individual (DNA can be extracted from blood, saliva, hair roots, and skin) Crimework: rape and murder cases (forensics) Paternity suits Missing persons and unidentified bodies Immigration disputes Animal work - breeding