1. Biotechnology and Recombinant DNA
Chandler Community College

2. Biotechnology Biotechnology Foods Vaccines Antibiotics Vitamins
The use of microorganisms, cells, or cell components to make a product
Foods
Vaccines
Antibiotics
Vitamins
Mining – extract minerals from ore

3. Recombinant DNA Usually commercial or medicinal value Insulin Vaccines
Amplify DNA
Enzymes
Hormones
Insect resistant plants
Recombinant DNA (rDNA)
Also called genetic engineering
Inserting genes into cells to make chemicals that the organisms do not naturally make

4. rDNA Procedures
Insulin produced in bacterial cells

5. 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

6. Tools of Biotechnology
Natural Selection – normal process
Humans use Artificial Selection to select desirable breeds of animals or strains of plants

7. 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

8. Site Directed Mutagenesis

9. 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

10. Restriction Enzymes

12. 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

14. 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

15. PCR

16. PCR

17. 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

18. 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

20. 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

22. 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

24. Microinjection Uses glass micropipette with very small diameter
DNA injected through animal cell membrane into cell

25. Obtaining DNA
Biologists can get the tools for rDNA by using two main sources for genes:
Gene Libraries
Synthetic DNA

26. 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

28. 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

30. 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

31. Making a Gene Product Organisms used most effectively Eschericia coli
Saccromyces cerevisiae (Baker’sYeast)
Mammal cells
Plant cells

32. 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

33. 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

34. Scientific Applications
rDNA makes a large amount of copies of DNA
Many techniques to analyze the DNA once it is made

35. 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

36. 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

38. 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

39. 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

40. Ti Plasmid

41. Ethics and rDNA