Biotechnology Packet #12 Chapter #9

Introduction Since the 1970’s, humans have been attempted to manipulate and modify genes in a way that was somewhat predictable.

Introduction II Scientists would select a gene to be inserted into an organism Cut two DNA molecules into fragments using restriction enzymes Splice the fragments together into the desired combination Producing recombinant DNA Introduce the new DNA into a living cell for replication

Introduction III Recombinant DNA technology isolates and amplifies specific sequences of DNA by incorporating them into vector DNA molecules.

The Players Involved in the Making of Recombinant DNA

Introduction Recombinant DNA is made by splicing a DNA fragment of interest into a small quickly dividing replicating molecule (plasmid).

Donor & Transgenic Organisms The organism providing the DNA is called the donor. After recombination, an organism that contains an artificially inserted, foreign piece of DNA, is called a transgenic organism.

The Importance of Transgenic Organisms Transgenic organisms allow gene targeting and mutagenesis screening that help identify the function of a gene and its protein product.

Vectors Vectors, normally in the form of plasmids, is a genome into which the DNA fragments, removed from the donor, are inserted.

Restriction Enzymes Enzymes that are used to cut DNA into specific fragments. Each restriction enzyme recognizes and cuts DNA at a highly specific base sequence.

The Making of Transgenic Organisms

The Making of a Transgenic Organism The DNA of interest is excised, from the donor, using “scissors” known as a restriction enzyme. The excised DNA is called a DNA fragment.

The Making of a Transgenic Organism The DNA fragment is inserted into the vector via one of multiple methods.

The Making of a Transgenic Organism Once inserted, DNA ligase is used to join the DNA fragment together with the vector’s genome.

The Making of a Transgenic Organism The new transgenic organism is duplicated.

Vectors Currently Under Study

Vectors Under Study… Vectors currently under study include Retroviruses Adenoviruses Herpes simplex virus Rhinovirus Human Immunodeficiency Virus (HIV)

Genomics & Genetic Libraries

Genomic Library & cDNA Library Genomic Library DNA library containing an organism’s complete genome In the form of thousands of DNA fragments cDNA Library DNA library made up of “DNA clones” reconstructed using reverse transcriptase Must be made from mRNA Genomics Sub-discipline in genetics of characterizing the entire genomes of organisms.

Homework Assignment What are some of the advantages, and disadvantages, of having a cDNA library?

Genetic Probes

Genetic probes are radioactively labeled DNA or RNA sequence that enables geneticists to identify complementary nucleic acid sequences. If used to identify a DNA strand, the DNA molecule will have to be separated into into two strands via artificial denaturation— heat.

The Making of Genetic Probes Southern Blot Technique DNA fragments, produced using restriction enzymes, are separated via gel electrophoresis. Fragments are blotted onto a nitrocellulose or nylon membrane. The membrane is bathed in a labeled probe for a specific DNA fragment. The selected DNA fragments are cut out of the gel

Homework Assignment Define Northern Blot. Define Western Blot.

Making Copies of DNA in a Lab Setting

Introduction Once a sequence of DNA (DNA fragment) has been isolated, it is sometimes necessary to make large amounts of that sequence for study.

Polymerase Chain Reaction Allows rapid, efficient amplification of DNA sequences of interest. In vitro technique Researchers target a particular DNA sequence, by specific primers, and then clone the DNA sequence by heat resistant DNA polymerase. Used to help amplify DNA from crime scenes and archaeological remains

Gene Therapy

Simple idea—hard to practice The use of sequencing, cloning and vector insertion techniques to deliver working versions of genes to individuals who are born with deleterious mutant versions of the gene. Germ Line Therapy Somatic Gene Therapy

Genetic Engineering & Food

Genetic Engineering of Agricultural Species Foreign genes, under study, for insertion into commercial plant species. Helps provide Selective herbicide resistance Increased yield Plant-grown vaccines and pharmaceuticals Improved nutrient balance Problems? Human allergic reactions to foreign proteins Increased use of herbicides “jumping” of plasmids from commercial crops to weed species. Eco-mayhem!

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