Biotechnology Chapter 17

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 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

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

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

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

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

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)

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

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

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

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

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

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

DNA Analysis

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

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

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

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

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

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