DNA Technology Breeding strategies Mutations Artificial - PowerPoint PPT Presentation

DNA Technology

Breeding strategies  Mutations Artificial selection Biotechnology alteration of organisms or their components with specific applications in mind Genetic engineering - Direct manipulation of genes Recombinant DNA - technology

https://sites.google.com/site/asoftsb/home/methods-of-selective-breeding/examples

http://cal.vet.upenn.edu/projects/genetic/inbreed/images/diagram2.gif

Basic DNA Techniques Gene cloning • amplification of genes • production of protein products Purification and Incorporation of Identification and fragmentation DNA into isolation of GENE using CLONING OF INTEREST RESTRICTION VECTORS (i.e. ENZYMES plasmids) Incorporation of SELECTION of GROWING of plasmids into hosts carrying selected host cells bacterial host desired in culture medium through recombinant gene TRANSFORMATION

Restriction Enzymes • aka restriction endonucleases • produced by Bacteria/Archaea • cut at (restriction sites) that are 4-8 base pairs long • long DNA fragments can be cut to produce restriction fragments • e.g. EcoRI (G*AATTC)

Constructing recombinant DNA with the aid of restriction enzymes A comprehensive list is available at http://www.sciencegateway.org/RES /index.html

DNA Insertion • conditions that allow host cells to take up the vector – high salt concentration – high temperature (~42 o C) – electric pulse – microinjection

Selection of transgenic host cells • recombinant molecules must be separated from molecules consisting of just donor or just plasmid DNA • plasmid used contains genes for resistance against certain antibiotics • large number of cells grown from single cell carrying recombinant plasmid • w/in hrs, the a whole culture of cells containing the recombinant DNA

http://filebox.vt.edu/users/chagedor/biol_4684/Methods/vector.gif

Genomic Library • Complete set of plasmid- containing cell clones

Using cDNA • difference between prokaryotes & eukaryotic DNA • DNA copied from mature mRNA transcript by reverse transcription • cDNA library

Basic DNA Techniques • DNA Amplification – method of creating multiple copies of a particular segment of DNA – e.g. growing recombinant cells polymerase chain reaction (PCR)

Polymerase Chain Reaction  Makes use of Taq polymerase  Steps: 1. Denaturation 2. Annealing 3. Extension  Each PCR cycle of heating and cooling the DNA mixture doubles the number of DNA molecules.

Basic DNA Techniques  Gel Electrophoresis  technique used to distinguish DNA molecules  applied electric field forces DNA to migrate through a medium and distance themselves from one another by length <16.2-2>

Basic DNA Techniques • DNA Sequencing – Sanger method - for determining the nucleotide sequence of a piece of DNA – many copies are needed (by cloning or PCR)

QUIZ 2 (by pair) 1. Describe 1 natural way to manipulate DNA. 2. Differentiate between biotechnology and recombinant DNA technology. 3. What are restrictzion enzymes? 4. What is the role of ligase in DNA recombination? 5. How can we ensure that only the host cell with the gene of interest will be cloned? 6. What enzyme is needed in creating cDNA? 7. What are the three steps in PCR? 8. How does gel electrophoresis separate DNA of different lengths? 9. What is the use of fluorescently-labeled bases in DNA sequencing?