DNA Technology Packet #27

Introduction Since the 1970’s, humans have been attempted to manipulate and modify genes in a way that was somewhat predictable. 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 Introduce the new DNA into a living cell for replication Recombinant DNA technology isolates and amplifies specific sequences of DNA by incorporating them into vector DNA molecules.

Recombinant DNA Made by splicing a DNA fragment of interest into a small quickly dividing replicating molecule (plasmid). An organism containing an artificially inserted, foreign piece of DNA, is considered as being transgenic. Transgenic organisms allow gene targeting and mutagenesis screening that help identify the function of a gene and its protein product.

The Making of a Transgenic Organism Organism, from which the DNA of interest is extracted, is called the donor. The DNA of interest is excised using “scissors” known as a restriction enzyme. The DNA, into which the DNA of interest (from the donor), is a vector. Normally a bacterial plasmid The DNA, from the donor, is inserted into the vector via multiple methods DNA ligase is used to join the DNA fragments together. Receiving organism is replicated.

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

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.

Genomic Library & cDNA 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 of having a cDNA library?

Genetic Probes 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 must have been separated into two strands via artificial denaturation—heat.

Genetic Probes Southern Blot Technique DNA fragments, produced using restriction enzymes, are separated via gel electrophoresis. Fragments are then denatured Fragments are blotted onto a nitrocellulose or nylon membrane.

Homework Assignment Define Northern Blot. Define Western Blot.

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

Polymerase Chain Reaction II 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 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!