1. Biotechnology and Recombinant DNA
Chapter 9

2. Biotechnology and Recombinant DNA
The use of microbiological and biochemical techniques to solve problems and produce product
Recombinant DNA techniques
Methods used to manipulate DNA to intentionally genetically alter organisms through genetic engineering
Often to give organisms more useful traits

3. Fundamental Tools of Biotechnology
Basic components of molecular biologist’s “toolkit”
Restriction enzymes
Gel electrophoresis
DNA probes
Primers

4. Fundamental Tools of Biotechnology
Restriction enzymes (Extracted from Bacteria)
Naturally occurring enzymes that cut DNA into fragments
Cut in predictable and controllable manner
Generates pieces of DNA called restriction fragments
These fragments can be joined to new fragments
Enzymes produce jagged cuts called sticky ends
Ends anneal together to form new strand
DNA ligase covalently joins fragments
file:///N:/Biology/Power%20Points/20_Lectures_PPT/media/20_03RestrictionEnzymes_A.swf

5. Fundamental Tools of Biotechnology
Gel electrophoresis
Used to separate DNA fragments according to size
DNA is put into wells in gel
Gel subjected to current
DNA moves through the gel
Fragments are separated according to size
Large fragments remain high in the gel
Small fragments migrate lower
Gel must be stained to view DNA
Stained with ethidium bromide solution

6. Fundamental Tools of Biotechnology
DNA probes
Used to locate nucleotide sequences in DNA or RNA
Probe is single-stranded piece of DNA tagged with detectable marker
Location can be easily determined
Probe will hybridize to complementary fragment of interest

7. Using a DNA probe to find the colony with the gene of interest
Alignment Marks

8. Fundamental Tools of Biotechnology
Primers
Single stranded DNA fragments that bind sequences of DNA
Used in in vitro DNA synthesis
Primer serves fragment for addition of DNA nucleotides

9. Applications of Genetic Engineering
Genetically engineered bacteria
Genetic engineering relies on DNA cloning
Process of producing copies of DNA
Cloned DNA generally combined with carrier molecule called cloning vector
Insures replication of target DNA

10. Applications of Genetic Engineering
Genetically engineered organisms have variety of uses
Protein production
DNA production
Researching gene function and regulation
file:///N:/Biology/Power%20Points/20_Lectures_PPT/media/20_04CloningAGene_A.swf

11. Applications of Genetic Engineering
Protein production
Produce commercially important proteins
Pharmaceutical proteins
Human insulin
Vaccines
Hepatitis B vaccine
Commercially valuable proteins
Chymosin An enzyme that catalyzes the coagulation of milk used in the production of cheese

12. Applications of Genetic Engineering
DNA production
Researches interested in acquiring available sources of specific DNA fragments
Fragments used for
DNA study
Looking genomic characteristics
DNA vaccines
Looking at injecting DNA of pathogen to produce immune response

13. Applications of Genetic Engineering
Researching gene function and regulation
Function and regulation can be more easily study in certain bacteria
E. coli used often due to established protocols
Gene expression can be studied by gene fusion
Joining gene being studied to reporter gene
Reporter gene encodes observable trait
Trait makes it possible to determine changes in gene

14. Applications of Genetic Engineering
Genetically engineered eukaryotes
Yeast serve as important eukaryotic model for gene function and regulation
Plant or animal that receive engineered gene termed transgenic organism
Examples of genetically altered plants include
Pest resistant plants
Corn, cotton and potatoes
Herbicide resistant plants
Soybeans, cotton and corn
Plants with improved nutrient value
Rice
Plants as edible vaccines
Bananas and potatoes

15. Applications of Probe Technologies
Variety of technology employ DNA probes
Colony blotting
Southern blotting (check for specific DNA in electrophoresis samples)
Fluorescence in situ hybridization (FISH) (check for specific DNA sequences in whole chromosomes)
DNA microarray

16. Applications of Probe Technologies
Colony blotting
Used to detect specific DNA sequences in colonies grown in agar plates
Colonies are transferred in place on nylon membrane
Colony blots are used to determine which cells contain genes of interest

17. Applications of Probe Technologies
Southern blotting
Uses probes to detect DNA sequences in restriction fragments separated using gel electrophoresis
Application of Southern blotting is locating DNA sequences similar to ones being studied

18. Applications of Probe Technologies
Fluorescence in situ hybridization (FISH)
Uses fluorescently labeled probes to detect certain nucleotide sequences
Detects sequences inside intact cells
Specimens are viewed using fluorescence microscopes
FISH can be used to identify specific properties of bacteria
Mycobacterium tuberculosis in sputum sample

19. Applications of Probe Technologies
DNA microarray technologies
DNA arrays are solid supports with fixed patterns of different single stranded DNA fragments attached
Enables researches to screen sample for numerous sequences simultaneously

20. Applications for DNA Sequencing
Knowing DNA sequence of particular cell helps identify genetic alterations
Alterations that may result in disease
Sickle cell anemia
Due to single base-pair change in a gene
Cystic fibrosis
Caused by three base-pair deletion

21. Applications of Polymerase Chain Reaction
Creates millions of copies of given region of DNA in matter of hours
Technique exploits specificity of primers
Allows for selective replication of chosen regions
Termed target DNA
Large amounts of DNA can be produced from very small sample
Care must be taken to prevent contamination with external source of target DNA
Basis for false-positive test results
PCR Presentation

22. Techniques Used in Genetic Engineering
Obtaining DNA to be cloned
Generally through cell lysis
Generating a recombinant molecule
Restriction enzymes and ligases are used to create a recombinant molecule
Introducing recombinant molecule into new host
Host acts as an “incubator” for DNA replication
DNA-mediated transformation often used to get DNA into host

23. Techniques used in Probe Technologies
Probe technologies include
Colony blotting
Southern blotting
FISH
Microarray technology

24. Techniques used in Probe Technologies
Techniques in colony and Southern blotting
Blotting steps transfer sample to nylon membrane
Probe is added
Probe hybridizes with complementary sequence
Process is used to locate positions of hybridized probe

25. Techniques used in Probe Technologies
Techniques used in FISH
Sample preparation is critical
Methods used depend on type of organism
Specimen is applied to glass slide
Fluorescent label is applied and incubated
Incubation allows for hybridization
Specimen is view with fluorescence microscope

26. Techniques Used in DNA Sequencing
Dideoxychain termination
Elements for termination reaction include
Single-stranded DNA template
Primer that anneal to template
DNA polymerase
Each of the nucleotide bases
One of these bases is labeled with marker for detection
Dideoxynucleotides
Like deoxynucleotide counterparts but lack 3’ OH
Incorporation causes chain termination
Special gel electrophoresis used to separate DNA fragments by size

27. Techniques Used in DNA Sequencing
Automated DNA sequencing
Most automated systems use fluorescent dyes to detect newly synthesized DNA
Gel electrophoresis used to separate fragments into colored bands
Laser used to detect color differences
Order of color reflects nucleotide sequence

28. Techniques Used in Polymerase Chain Reaction
Starting with double stranded DNA molecule, process involves number of amplification cycles
PCR requires three step amplification cycle
Step 1: double stranded DNA denatured by heat
Step 2: primers anneal to complementary sequence of target DNA and DNA synthesis occurs with heat stable DNA polymerase
Step 3: duplication of target DNA
DNA is amplified exponentially