Recombinant DNA recombinant DNA techniques in which genes

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 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 22 k. X 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