Biotechnology and Recombinant DNA Bio 205 Chandler Community College.
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Transcript of Biotechnology and Recombinant DNA Bio 205 Chandler Community College.
Biotechnology and Biotechnology and Recombinant DNARecombinant DNA
Bio 205Chandler Community College
BiotechnologyBiotechnology
• Biotechnology– The use of microorganisms, cells, or cell
components to make a product
• Foods• Vaccines• Antibiotics• Vitamins• Mining – extract minerals from ore
Recombinant DNARecombinant 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
• Insulin produced in bacterial cells
rDNA ProceduresrDNA Procedures
rDNA ProceduresrDNA 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 BiotechnologyTools of Biotechnology
• Natural Selection – normal process • Humans use Artificial Selection to
select desirable breeds of animals or strains of plants
Tools of BiotechnologyTools 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 Site Directed MutagenesisMutagenesis
Restriction EnzymesRestriction 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 EnzymesRestriction Enzymes
VectorsVectors• 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 Polymerase Chain ReactionReaction
• 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
PCRPCR
PCRPCR
Inserting Foreign DNA into Inserting Foreign DNA into CellsCells
• 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
TransformationTransformation
• 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
ElectroporationElectroporation• 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 FusionProtoplast 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
MicroinjectionMicroinjection
• Uses glass micropipette with very small diameter
• DNA injected through animal cell membrane into cell
Obtaining DNAObtaining DNA
• Biologists can get the tools for rDNA by using two main sources for genes:
• Gene Libraries• Synthetic DNA
Gene LibrariesGene 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 DNASynthetic 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 CloneSelecting 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 ProductMaking a Gene Product
• Organisms used most effectively– Eschericia coli– Saccromyces cerevisiae (Baker’sYeast)– Mammal cells– Plant cells
Applications of rDNAApplications 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 ProjectHuman 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 ApplicationsScientific Applications
• rDNA makes a large amount of copies of DNA
• Many techniques to analyze the DNA once it is made
Scientific ApplicationsScientific 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 ApplicationsScientific 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 ApplicationsScientific 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 ApplicationsAgricultural 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 PlasmidTi Plasmid
Ethics and rDNAEthics and rDNA