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