1. DNA Technology
Packet #27

2. 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.

3. 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.

4. 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.

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

6. 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.

7. 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.

8. Homework Assignment
What are some of the advantages of having a cDNA library?

9. 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.

10. 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.

11. Homework Assignment
Define Northern Blot.
Define Western Blot.

12. 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

13. Polymerase Chain Reaction II
Used to help amplify DNA from crime scenes and archaeological remains

14. 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

15. 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!