BiotechnologyPacket #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 includeRetrovirusesAdenovirusesHerpes simplex virusRhinovirusHuman 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

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 ProbesSouthern 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

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!

Review