Overview

-       Terminology

-       Tools

-       Restriction enzymes

-       Vectors

-       PCR – Polymerase Chain Reaction

-       Techniques

-       DNA transfer methods

-       Obtaining DNA

-       Selecting clones

-       Making products

-       Applications

 

Terminology

-       Biotechnology: The use of microorganisms, cells, or cell components to make a product.

-       Foods, antibiotics, vitamins, enzymes

-       Recombinant DNA (rDNA) technology: Insertion or modification of genes to produce desired proteins

-       Vector: Self-replicating DNA used to carry the desired gene to a new cell

-       Clone: Population of cells arising from one cell, each carries the new gene

-       Selection: Culture a naturally occurring microbe that produces desired product

-       Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait

-       Site-directed mutagenesis: Change a specific DNA code to change a protein

-       Select and culture microbe with the desired mutation

 

Tools - Restriction Enzymes

-       Cut specific sequences of DNA

-       Destroy bacteriophage DNA in bacterial cells

-       Cannot digest (host) DNA with methylated cytosines

 

Tools - Vectors

-       Carry new DNA to desired cell

-       Shuttle vectors can exist in several different species

-       Plasmids and viruses can be used as vectors

 

Tools – PCR

-       Polymerase Chain Reaction

-       Enzymatic reproduction of DNA

-       Used to

-       Clone DNA for recombination

-       Amplify DNA to detectable levels

-       Sequence DNA

-       Diagnose genetic disease

-       Detect pathogens

 

Techniques – DNA Transfer

-       DNA can be inserted into a cell by

-       Electroporation

-       Transformation

-       Protoplast fusion

-       Gene gun

-       Microinjection

 

Techniques – Obtaining DNA

-       Genomic libraries are made of pieces of an entire genome stored in plasmids or phages

-       Complementary DNA (cDNA) is made from mRNA by reverse transcriptase

-       Synthetic DNA is made by a DNA synthesis machine

 

Techniques – Making Products

-       E. coli

-       Used because it is easily grown and its genomics are known

-       Need to eliminate endotoxin from products

-       Cells must be lysed to get product

-       Saccharomyces cerevisiae

-       Used because it is easily grown and its genomics are known

-       May express eukaryotic genes easily

-       Mammalian cells

-       May express eukaryotic genes easily

-       Harder to grow

 

Therapeutic Applications

-       Human enzymes and other proteins

-       Subunit vaccines

-       Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines

-       Gene therapy to replace defective or missing genes

 

The Human Genome Project

-       Nucleotides have been sequenced

-       Human Proteome Project may provide diagnostics and treatments

-       Reverse genetics: Block a gene to determine its function

 

Scientific Applications

-       Understanding DNA

-       Sequencing organisms' genomes

-       DNA fingerprinting for identification

 

Forensic Microbiology

-       PCR

-       Primer for a specific organism will cause application if that organism is present

-       Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle

-       Reverse-transcription (RT-PCR): Reverse transcriptase makes DNA from viral RNA or mRNA

 

Nanotechnology

-       Bacteria can make molecule-sized particles

 

Using Agrobacterium

-       Bt toxin

-       Herbicide resistance

-       Suppression of genes

-       Antisense DNA

-       Nutrition

-       Human proteins

 

Safety Issues and Ethics of Using rDNA

-       Avoid accidental release

-       Genetically modified crops must be safe for consumption and for the environment

-       Who will have access to an individual's genetic information?