Chapter 13: Gene Technology

Genetic Engineering The process of manipulating genes for practical purposes Involves building recombinant DNA = DNA made from two or more different organisms Example: using bacteria DNA to make human insulin by transferring human insulin gene to bacteria

Recombinant DNA

Steps in a Genetic Engineering Experiment

Step One DNA with gene of interest from organism (insulin gene) and DNA from vector (agent used to carry gene of interest into another cell) are cut using restriction enzymes = bacterial enzymes that recognize and bind to specific short sequences of DNA, and then cut the DNA between specific nucleotides.

Step Two Recombinant DNA is produced by using DNA ligase to help combine the two DNA fragments to each other.

Step Three The gene is cloned when bacteria are allowed to reproduce. Gene cloning =the process of making many copies of the gene of interest by allowing the host cell to reproduce (binary fission in bacteria)

Step Four Cells are screened to isolate the cells that produced the gene of interest from those that did not.

Additional Info on Making Recombinant DNA Enzyme EcoRI –recognizes a palindrome (same forwards and backwards) on both DNA strands and makes the cut there strands are complimentary to each other and are called sticky ends –now both DNA segments can be joined together at this point

EcoRI

Confirming a Cloned Gene is Present = Southern Blot Step One = DNA from each bacterial clone colony is isolated and cut into fragments by restriction enzymes.

Southern Blot cont’d Step Two: DNA fragments are separated by gel electrophoresis –a technique that uses an electrical field within a gel to separate molecules by their size and charge. Smallest fragments move fastest and further down the gel once electricity is applied.

Southern Blot cont’d Step Three: DNA bands are transferred (blotted) directly onto a probe-moistened piece of filter paper. Probes = radioactive- or fluorescent-labeled RNA or single-stranded DNA pieces that are complementary to the gene of interest

Southern Blot cont’d Step Four: Only DNA fragments that contain the gene of interest bind to the probes.

Other Types of Blots Northern blot –used to identify RNA, not DNA, fragments Western blot –used to identify proteins

Genetic Engineering in Medicine and Society

Medicines Proteins needed in large quantities to treat illnesses Diabetes Hemophilia anticoagulants (treat heart attack patients)

Vaccines Used to make vaccines –Vaccine = a solution containing all or part of a harmless version of a pathogen (disease-causing microorganism)

Vaccines cont’d immune system recognizes pathogen’s surface proteins antibodies are made to protect against future invasions Traditionally, vaccines made by immobilizing or killing a harmful pathogen. However, there is a high risk of not completely killing pathogen and innocently infecting patients.

Genetic Engineering in Medicine and Society Genetic engineering eliminates this risk by modifying harmless bacteria or viruses with the pathogen’s surface proteins. The harmless microorganisms now appears harmful but is not.

Polymerase Chain Reaction (PCR) Method that allows for increase in available amount of DNA material w/in a few hours so a DNA fingerprint can be made Important for diagnosing genetic disorders and solving crimes

Goal of Genetic Engineering Cure genetic disorders Gene therapy: a technique that involves putting a healthy copy of a gene into the cells of a person whose copy of the gene is defective

Diseases Being Treated with Gene Therapy Cancer SCID (Severe Combined Immunodeficiency) Cystic Fibrosis Familial hypercholesterolenemia Hemophilia Fanconi’s anemia Rheumatoid arthritis

Techniques Used to Identify Organisms DNA fingerprint: a pattern of dark bands on photographic film that is made when an individual’s DNA fragments (RFLPs) are separated by gel electrophoresis, probed, and then exposed to an X-ray film. Each individual has a unique banding pattern (except identical twins)

DNA Fingerprinting

Human Genome Project GoalGoal: determine the nucleotide sequence of the entire human genome and map the location of every gene to each chromosome by 2003 or sooner (FYI: mission accomplished) Work underway to map out genomes of other organisms

Genetic Engineering in Agriculture Transporting genes into plants can improve crops Help make insect, drought, and disease resistant Can potentially help alleviate hunger problems and food shortages

Gene Technology in Animal Farming Efforts underway to improve animals’ abilities to resist disease Use animals to produce human proteins for medicines through use of transgenic animals: animals that have foreign DNA in cells Think of these animals as vectors like the bacteria were in the earlier slides

Gene Technology in Animal Farming

The Making of Dolly Mammary cells were extracted and their cell cycles were stopped Egg cells were extracted and the nucleus from each removed and discarded A mammary cell was placed next to the “empty” egg cell

The Making of Dolly An electric shock opened up the cell membranes so that the cells fused. Cell division was triggered. The embryo developed in vitro and was later implanted into a surrogate mother. After a 5-month pregnancy, a lamb was born that was genetically identical to the sheep from which the mammary cell was extracted.