1. Biotechnology
Chapter 17

2. DNA Manipulation
Restriction endonucleases revolutionized molecular biology
Enzymes that cleave DNA at specific sites
Used by bacteria against viruses
Restriction enzymes significant
Allow a form of physical mapping that was previously impossible
Allow the creation of recombinant DNA molecules (from two different sources)

3. 3 types of restriction enzymes
Type I and III cleave with less precision and are not used in manipulating DNA
Type II
Recognize specific DNA sequences
Cleave at specific site within sequence
Can lead to “sticky ends” that can be joined
Blunt ends can also be joined

5. DNA ligase Joins the two fragments forming a stable DNA molecule
Catalyzes formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides
Same enzyme joins Okazaki fragments on lagging strand in replication

6. Gel Electrophoresis Separate DNA fragments by size
Gel made of agarose or polyacrylamide
Submersed in buffer that can carry current
Subjected to an electrical field
Negatively-charged DNA migrates towards the positive pole
Larger fragments move slower, smaller move faster
DNA is visualized using fluorescent dyes

9. Transformation Introduction of DNA from an outside source into a cell
Natural process in many species
E. coli does not
Temperature shifts can induce artificial transformation in E. coli
Transgenic organisms are all or part transformed cells

10. Molecular Cloning Clone – genetically identical copy
Molecular cloning – isolation of a specific DNA sequence (usually protein-encoding)
Sometimes called gene cloning
The most flexible and common host for cloning is E. coli
Vector – carries DNA in host and can replicate in the host
Each host–vector system has particular uses

11. Vectors Plasmids Small, circular chromosomes
Used for cloning small pieces of DNA
3 components
Origin of replication – allows independent replication
Selectable marker – allows presence of plasmid to be easily identified
Multiple cloning site (MCS)

13. This process involves three steps:
Molecular hybridization is the most common way of identifying a clone in a DNA library
This process involves three steps:
Plating the library
Physically the library is a collection of bacteria or viruses in bacteria
Replicating the library
Screening the library
Probe is specific sequence of interest

15. DNA Analysis Restriction maps
Molecular biologists need maps to analyze and compare cloned DNAs
Initially, created by enzyme digestion, separation by electrophoresis, and analysis of resulting patterns
Many are now generated by computer searches for cleavage sites

16. Southern blotting
Sample DNA is digested by restriction enzymes and separated by gel electrophoresis
Double-stranded DNA denatured into single-strands
Gel “blotted” with filter paper to transfer DNA
Filter is incubated with a labeled probe consisting of purified, single-stranded DNA corresponding to a specific gene

20. Northern blotting Western blotting
mRNA is separated by electrophoresis and then blotted onto the filter
Western blotting
Proteins are separated by electrophoresis and then blotted onto the filter
Detection requires an antibody that can bind to one protein

21. RFLP analysis Restriction fragment length polymorphisms
Generated by point mutations or sequence duplications
Restriction enzyme fragments are often not identical in different individuals
Can be detected by Southern blotting

23. DNA fingerprinting
Identification technique used to detect differences in the DNA of individuals
Population is polymorphic for these markers
Using several probes, probability of identity can be calculated or identity can be ruled out
First used in a U.S. criminal trial in 1987
Tommie Lee Andrews was found guilty of rape
Also used to identify remains

24. DNA Analysis

25. Polymerase chain reaction (PCR)
Developed by Kary Mullis
Awarded Nobel Prize
Allows the amplification of a small DNA fragment using primers that flank the region
Each PCR cycle involves three steps:
Denaturation (high temperature)
Annealing of primers (low temperature)
DNA synthesis (intermediate temperature)
Taq polymerase

26. After 20 cycles, a single fragment produces over one million (220) copies!

28. Applications of PCR Allows the investigation of minute samples of DNA
Forensics – drop of blood, cells at base of a hair
Detection of genetic defects in embryos by analyzing a single cell
Analysis of mitochondrial DNA from early human species

29. Genetic Engineering Has generated excitement and controversy
Expression vectors contain the sequences necessary to express inserted DNA in a specific cell type
Transgenic animals contain genes that have been inserted without the use of conventional breeding

30. In vitro mutagenesis
Ability to create mutations at any site in a cloned gene
Has been used to produce knockout mice
A known gene is inactivated
The effect of loss of this function is then assessed on the entire organism
An example of reverse genetics

31. Medical Applications
Medically important proteins can be produced in bacteria
Human insulin
Interferon
Atrial peptides
Tissue plasminogen activator
Human growth hormone
Problem has been purification of desired proteins from other bacterial proteins