DNA Technology Breeding strategies Mutations Artificial - - PowerPoint PPT Presentation

DNA Technology

Biotechnology

alteration of organisms or their components with specific applications in mind Breeding strategies 

Artificial selection

Mutations

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

Identification and isolation of GENE OF INTEREST Purification and fragmentation using RESTRICTION ENZYMES Incorporation of DNA into CLONING VECTORS (i.e. plasmids) Incorporation of plasmids into bacterial host through

TRANSFORMATION

SELECTION of hosts carrying desired recombinant gene GROWING of selected host cells in culture medium

Gene cloning

• amplification of genes
• production of protein products

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 (~42oC) – 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

• f 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

• f 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

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Basic DNA Techniques

• DNA Sequencing

– Sanger method - for determining the nucleotide sequence of a piece of DNA – many copies are needed (by cloning

• r 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?