Chapter 20/21 Biotechnology

Focus of Chapter An introduction to the methods and developments in: Recombinant DNA Genetic Engineering Biotechnology

Recombinant DNA DNA in which genes from different sources are linked. Ex: the “green” mice

Genetic Engineering The direct manipulation of genes for practical purposes. Ex: Using E. coli to produce human insulin.

Biotechnology The use of living organisms or their components to perform practical tasks. Ex: the use of bacteria to digest oil spills.

Restrictive Enzymes Cut DNA at specific nucleotide sequences called “restriction sites”. Used to "cut and splice" DNA. Obtained from bacteria. Ex. EcoRI and Hind III

Plasmids Used extensively in Biotechnology and Recombinant DNA. Serve as a “vehicle” for transporting genes. Comment – other “vehicles” are used in other methods

Steps for Plasmid Use 1. Get the DNA for the trait. 2. Insert DNA into the plasmid. 3. Bacterial Transformation. 4. Identification of the new trait.

Insertion Placing foreign DNA into a plasmid. Open plasmid with enzymes to create “sticky ends”. Splice the new DNA and plasmid together.

Transformation Placing the plasmid into a bacterial cell. Reminder - our lab – need to know for AP testing purposes.

Methods Temperature shock & salt treatment Electric current Injection

Identification Screening the altered cells for the desired gene. Ex: Antibiotic sensitivity or the expression of a “new” trait (color, glowing etc.).

Example Applications 1. Insulin 2. Human Growth Hormone 3. Other Proteins

Comment Gene can’t be above a certain size (12 kb) or a plasmid won’t work. mRNA must not need splicing to remove introns.

DNA Sources 1. Organism - use a section of their chromosome. 2. cDNA - Complementary DNA - created copy of DNA from the mRNA transcript to avoid introns. Uses reverse transcriptase.

Other Vehicles BACs – Bacteria Artificial Chromosome – handle inserts of 100-300 kb YACs – Yeast Artificial Chromosome – have a centromere and telomomere, handle inserts >300 kb HACs – Human Artificial Chromosome

Nucleic Acid Probes Used to find a specific DNA sequence in a mixture of DNA pieces

How Used DNA is denatured and cut to produced ss pieces. Piece of compliment DNA is added as a “probe”. The probe has been “labeled”. Look for where the probe goes in the DNA sample.

Probe Diagram

DNA Microarray

PCR Polymerase Chain Reaction Method for making many copies of a specific segment of DNA. Also called “DNA Amplification”.

PCR - Method 1. Separate strands by heating (denature the DNA). 2. Cool slightly. 3. Build new strand from primers and nucleotides. 4. Repeat.

Importance - PCR Can amplify any DNA with as little as one original copy. Very useful in a variety of techniques and tests.

Gel Electrophoresis Technique will be covered in lab. Used to separate mixtures of DNA or proteins.

RFLP Analysis Restriction Fragment Length Polymorphisms. Method for detecting minor differences in DNA structure between individuals. Common in DNA fingerprinting

Method 1. Digest DNA with restrictive enzymes. 2. Separate pieces by Gel Electrophoresis 3. Identify sequences with probes.

RFLP - Results Patterns of DNA markers or DNA fingerprint Markers are inherited in a Mendelian pattern and can show relationships (Pedigree studies).

DNA Sequencing Uses dideoxynucleotides which stop DNA Polymerase at a known point in DNA replication. Builds new DNA from single strand DNA. Produces fragments of different lengths.

DNA Sequencers

Application DNA sequence is read base by base. By sequencing overlapping pieces of chromosomes, the entire genome of an organism can be read. (chromosome walking)

Cloning of Organisms Reproducing an organisms by asexual means. Commonly done in plants. Shows the concept of “Totipotency” – that a single cell can develop into a new organism.

Cloning in Plants

Cloning in Animals Has been done by nuclear transplantation. Examples – Dolly Many other vertebrates have now been cloned.

Dolly Picture

Stem Cells Stem Cell – an unspecialized cell that reproduce itself or differentiate into other cells.

Types of Stem Cells Embryonic – from an embryo. Adult – found in various tissues of the adult body.

Research Attempting to use stem cells to replace damaged cells or body parts. If can use own stem cells, avoids tissue rejection problems. Ex – grow a new bladder.

DNA Technology: Applications 1. Basic Research 2. Medical 3. Forensics 4. Agricultural

Basic Research 1. DNA and protein studies 2. Evolution 3. Gene structure and control mechanisms.

Medical Uses 1. Diagnosis of Diseases 2. Gene Therapy 3. Vaccines 4. Pharmaceutical Products

Gene Therapy

Forensic Uses DNA fingerprints for crime solving – used in every TV crime show DNA identification records – standard for the military

Comments Links suspect bodily to the crime scene, but doesn’t prove they committed the crime. Results take MUCH longer than on TV shows. Analysis of old evidence is reversing some sentences.

Agricultural Uses 1. Animals Increased milk production Increased feed utilization Increased meat production

PharmAnimals

Agricultural Uses 2. Plants Herbicide resistance Slow spoilage of fruits Insect resistance – BT corn Nitrogen-Fixation ability

Genetically Modified Organism or GMO Produced by direct genetic manipulation, not traditional breeding practices. FDA just approved sale of GMO animal products for human consumption. Bioethics concerns

Future Of DNA Technology Cloning of higher animals. Stem Cells - growth of replacement tissues and organs. Gene therapy to correct DNA defects. ?

Summary Know the basics of some of the DNA technology techniques. Bacterial transformation lab How Gel electrophoresis works Restriction enzymes

Summary Watch the news for DNA technology discoveries. Be able to discuss one recent event.