Topic 12 Genetic Engineering Selective Breeding vs. Genetic Engineering Polymerase Chain Reaction, Gel Electrophoresis & DNA Sequencing HGP & DNA Profiling in Forensics Stem Cells & Cloning DNA Probes, PGD & Gene Therapy

Selective Breeding vs. Genetic Engineering Topic 12: Lesson 1 Resources: Read Chapter 11.1-2A pp. 218-221 Kahn Academy – Restriction Enzymes Lab – Restriction Enzymes Selective Breeding vs. Genetic Engineering

Selective Breeding = Artificial Selection Humans have been using artificial selection for thousands of years

Artificial Selection in Pets & Livestock Selective breeding takes many generations to achieve desired results, but can be achieved without much technology

Genetic Engineering Directly inserting, removing, or changing an organism’s DNA using technology Genetically Modified Organisms (GMOs) contain DNA from other organisms (transgenic organisms)

NOT Examples of GMOs Grafting Hybrid animals

Example of GMO: Bt Corn European corn borer lays eggs in corn plants and the larvae feed on the corn leaves Scientists inserted a gene from a soil bacteria, Bacillus thuringiensis, that is deadly to the corn borer in the corn

How to Make a Transgenic Organism Obtain source DNA (gene) Obtain a plasmid (circular DNA) from bacteria Use restriction enzymes to cut out the gene of interest and to cut the plasmid to receive the gene Mix source DNA gene with plasmid to form recombinant DNA DNA ligase will seal the phosphodiester bonds Insert recombinant DNA into host cell

Restriction Enzymes

Transgenic Plant with DNA from Bacterium

Polymerase Chain Reaction, Gel Electrophoresis & DNA Sequencing Topic 12: Lesson 2 Resources Read Chapter 11.2B-C pp. 223-4 Virtual Lab – PCR DNA Learning Center – PCR MIT Open Courseware – PCR Kahn Academy – PCR Virtual Lab – Gel Electrophoresis Kahn Academy – Gel Electrophoresis Kahn Academy – DNA Sequencing AK Lectures – Sanger DNA Sequencing Polymerase Chain Reaction, Gel Electrophoresis & DNA Sequencing

PCR Amplifies Genes 95°C 54°C 72°C

PCR = Copying DNA by Alternating Temperatures One gene can be copied millions of times within 1-2 hours A three step process is repeated for 30-40 times A thermal cycler machine controls temperature changes STEPS OF PCR Denaturation (95°C; 201.2°F) – the double stranded DNA opens into two pieces of single stranded DNA Annealing (54°C; 129.2°F) – primers pair up with the single stranded DNA template; DNA (Taq) polymerase begins to copy the template Extension (72°C; 161.6°F) – DNA (Taq) polymerase is at its optimum temperature and completes copying the double stranded DNA

Uses of PCR First step in each of the following practices Establish paternity DNA fingerprinting Study human evolution Diagnose genetic diseases Clone DNA of dead organisms

Gel Electrophoresis

Sorting Molecules by Size

DNA Sequencing Finding the Order of Nucleotides in DNA

Using Gel Electro for DNA Sequencing

Different People Produce Different Bands on Gels SNP/RFLP STR

Paternity Testing

Human Genome Project & DNA Profiling in Forensics Topic 12: Lesson 3 Resources Read Chapter 11.2B pp. 222-3 Bozeman Science – DNA Fingerprinting Naked Scientists – How Does DNA Fingerprinting Work? Activity – Who Killed Sharon? Human Genome Project & DNA Profiling in Forensics

Human Genome Project Aim to sequence the 3.2 billion base pairs Completed in 2003 Found 25,000 genes 400,000 proteins 1.5% of DNA codes for protein

Finding Variation in DNA Each human’s DNA is 99.9% identical Large variation is found in between the genes in non-coding regions

FBI Using DNA Fingerprinting The FBI uses 13 different STRs to determine the matching identity of a person

Stem Cells & Cloning Topic 12: Lesson 4 Resources Read Chapter 11.3A-B pp. 226-228 Worksheet – Stem Cells & Cloning NOVA – Stem Cell Video NOVA - Stem Cell Poll Module 6 – Contrasting Cases of Animal Modifications Stem Cells & Cloning

From Zygote to Morula to Blastula to Gastrula

3 Germ Layers

What is a Stem Cell?

Stem Cells Embryonic Stem Cells Adult Stem Cells Totipotent – give rise to all cell types in the body Derived from zygote cell or morula (first 8 cells) Pluripotent – give rise to many but not all types of cells in the body Derived from Inner Cell Mass of blastocyst

Cloning As a Means of Asexual Reproduction Many organisms using cloning to create identical copies of themselves Plants, fungi, bacteria, and some animals use asexual reproduction Human can use cuttings to clone fruit trees and flowers

Gene Cloning DNA/Molecular/Recombinant/Gene – DNA from one organism is combined with a vector and then put into a host cell mRNA from eukaryotic cell is copied into cDNA cDNA will be spliced into a vector plasmid with a restriction enzyme The recombinant vector (cDNA + vector) is inserted into a host cell Once in the host cell, the recombinant vector will replicate before cell division The cell will make the proteins encoded in the cDNA

Reproductive Cloning (SCNT)

Therapeutic Cloning Stem cells are extracted from a 5 day old embryo; the embryo is destroyed but the stem cells can transform into any of the 220 human cell types

DNA Probes, PGD & Gene Therapy Topic 12: Lesson 5 Read Chapter 11.4 pp. 229-31 PGD Video – Genetics & IVF Institute Do you want to know your chances of getting Alzheimer's disease? Module 4 – Max’s Case DNA Probes, PGD & Gene Therapy

Probes Look for Genes DNA probes detect specific nucleotide sequences in DNA DNA probes are ssDNA strands that are complementary to a certain gene Labeled with a fluorescent tag of radioactive isotope Target sequence DNA is heated so it separates DNA probe binds to region that it is complementary to Scientists detect radioactivity or use a fluorescent light to determine if the gene is present

FISH

Preimplantation Genetic Diagnosis If two carriers of a particular disease want to be sure that they have a baby that is disease free, PGD can reduce the odds in vitro fertilization occurs (IVF) Morula is formed and one of the cells is selected for diagnosis PCR amplifies the region of DNA where the suspected allele may be DNA probe determines if the alleles are present of the recessive condition Embryo can be discarded or implanted back into the woman’s body if it is considered a healthy cell

Genotyping Using Gel Electro

Testing 1 of 8 What bioethical issues present themselves when PGD is applied? Who should do PGD? Mothers over 35 Repeated miscarriages Previous pregnancy with chromosomal abnormality X-linked carrier mothers Unexplained infertility IVF failures

Fixing Bad Genes Most genetic diseases do not have a cure, but gene therapy could provide new treatment options Gene therapy corrects defective genes Swaps for bad copy, reverses mutation, turns off the gene A vector (vehicle) that can deliver a gene to all of the cells that are “infected” Viruses and plasmids are the two most commonly used vectors

Treating Cystic Fibrosis with the Cold Virus Viruses can act as a vector for CF patients The virus is introduced to the lung cells with a normally functioning gene The CF gene is replaced by the normal gene

RNA Interference RNAi can prevent translation of mRNA silencing the gene RNAi bonds to a complementary mRNA strand to make a double stranded and nonfunctional mRNA