Download Biotechnology and Recombinant DNA

Biotechnology and
Recombinant DNA
Bio 205
Chandler Community College
Biotechnology
• Biotechnology
– The use of microorganisms, cells, or cell
components to make a product
• Foods
• Vaccines
• Antibiotics
• Vitamins
• Mining – extract minerals from ore
Recombinant DNA
• Recombinant DNA
(rDNA)
– Also called genetic
engineering
– Inserting genes into
cells to make chemicals
that the organisms do
not naturally make
• Usually commercial or
medicinal value
– Insulin
– Vaccines
– Amplify DNA
– Enzymes
– Hormones
– Insect resistant
plants
rDNA Procedures
• Insulin
produced in
bacterial cells
rDNA Procedures
• Human Growth
Hormone (hGH)
• Some humans lack
hGH
• Use to get hGH
from the dead
• Now bacteria are
used to make the
hormone
Tools of Biotechnology
• Natural Selection – normal process
• Humans use Artificial Selection to select
desirable breeds of animals or strains of
plants
Tools of Biotechnology
• Mutation
– Responsible for diversity of life
– New bacteria strains can be created by exposing
them to mutagens
• Chemicals or UV radiation
– Produced over 1000 new strains of penicillin
producing organisms
• Site Directed Mutagenesis
– Used to make a specific change in a gene
Site Directed Mutagenesis
Restriction Enzymes
• Restriction Enzymes
– Special class of DNA-cutting enzymes that
exist in many bacteria
– Used naturally by some bacteria to kill invading
viruses
• Cuts, or digests, only one particular
sequence of nucleotide bases in DNA
• Cuts the same way each time
Restriction Enzymes
Vectors
• A Plasmid or virus used in genetic
engineering to insert genes into a cell
– A mosquito is a vector also – inserting parasites
into a human body
• Vehicle for the replication of a desired
DNA sequence
• Shuttle Vector can be used to move cloned
DNA sequences among organisms
• Viral DNA can be used as a vector
– Retroviruses, adenoviruses, herpesviruses
Polymerase Chain Reaction
• Polymerase Chain Reaction (PCR)
• Process by which small pieces of DNA can
be quickly amplified for analysis
– Only good for small pieces of DNA
– Within 30 minutes the sample can be amplified 1
billion times
• Used for diagnostic tests to detect the
presence of infectious particles
PCR
PCR
Inserting Foreign DNA into Cells
• rDNA must first be manipulated outside the
cell before it can be inserted into a new cell
• Several ways to introduce DNA into new
cells
– Transformation
– Electroporation
– Protoplast fusion
– Microinjection
Transformation
• Procedure where cells can take up plasmids
(DNA) from the surrounding environment
• The cell receiving the new DNA must be
Competent
• Made able to take up DNA
• Chemicals used to prepare the cell
– Calcium chloride
– Heat also used
Electroporation
• Uses an electrical current to form
microscopic pores in the membrane of the
cells receiving the DNA
• Procedure works on most cells
• Some must be made into protoplasts first
– Removing the cell wall
– The plasma membrane is more susceptible for
receiving DNA
Protoplast Fusion
• Protoplast in solution will fuse at slow but
significant rate
• Adding polyethylene glycol will increase the
frequency of fusion
• New “hybrid” cell is formed by the fusion of
the two cells
• Used more in plant and algae cells
Microinjection
• Uses glass micropipette with very small
diameter
• DNA injected through animal cell membrane
into cell
Obtaining DNA
• Biologists can get the tools for rDNA by
using two main sources for genes:
• Gene Libraries
• Synthetic DNA
Gene Libraries
• DNA is extracted from a cell
• Broken into smaller pieces of the cell’s entire
genome
• Pieces are then spliced into a plasmid or a
virus to make a collection of clones
• The collection of clones (one clone for each
fragment) containing different fragments of
DNA from a single organism
• Each organism and it’s DNA fragments has
a “book” of genetic information
Synthetic DNA
• Some genes can be made in the cell (in-vitro)
• Synthesis machines
• Used to enter a desired sequence of
nucleotides
• Like a word processor
• Order is determined by a the use of stored
nucleotides and reagents
Selecting a Clone
• Must select the particular cell that contains
the specific gene of interest
– Can be difficult (number of cells)
• Blue-White Screening procedure
• Colony Hybridization
– DNA probes
– Radioactive marker or
– Fluorescent marker
– Identifies the marker
– Photography
Making a Gene Product
• Organisms used most effectively
– Eschericia coli
– Saccromyces cerevisiae (Baker’sYeast)
– Mammal cells
– Plant cells
Applications of rDNA
• Therapeutic (pharmaceuticals)
– Insulin production
– Somatostatin production
– Subunit vaccines
• Contain protein portion of a pathogen
– DNA vaccines
– Gene therapy
• Replace a defective gene with a better one
– Gene silencing
• Defense in microbes against viruses
Human Genome Project
• 13-year effort to sequence
the entire human genome
• Used rDNA technology
• 3 billion nucleotide pairs
• 20,000 to 25,000 genes
• Only 2% of genome
produces functional
products
• 98% is junk DNA
Scientific Applications
• rDNA makes a large amount of copies of
DNA
• Many techniques to analyze the DNA once
it is made
Scientific Applications
• DNA sequencing
– Determination of the exact sequence of
nucleotides
• Random Shotgun sequencing
– Small pieces of a genome are sequenced and
then assembled using a computer
• Bioinformatics
– The science of understanding the function of
genes through computer assisted analysis
Scientific Applications
• Proteomatics
– Science of determining all of the proteins
expressed in a cell
• Southern Blotting
– Hybridization technique that determines the
sequence of a gene
– Electrophoresis
– Used for genetic screening for certain diseases
Scientific Applications
• Forensic Microbiology
– Identification of a particular DNA from many
others
– DNA Fingerprinting
• Determines paternity
• Blood analysis
• Etc.
• Nanotechnology
– Design and manufacture of extremely small
electronic circuits using molecular level of matter
– Robots and computers
Agricultural Applications
• Developing new strains of plants
• Resistance
• Crossing plant species
• Cloning of plants
• Plant breeding
• Ti plasmid used to introduce genes into
plants
• Produces stronger plants
Ti Plasmid
Ethics and rDNA