Post on 25-Dec-2015
DNA-based information technologies
Understand use of polymorphismsDNA fingerprintingUnderstand use of proteome to determine protein functionUnderstand creation and use of DNA microarraysDescribe use of recombinant technology in plant and animal cellsUnderstand challenges in introducing DNA into animal cell
DNA technology - forensics
DNA fingerprinting taking place of fingerprints - “DNA typing”, “DNA profiling”
DNA fingerprinting based on sequence polymorphisms, single bp differences between 1 individual and another (between individuals 1bp/1000 bp) - short tandem repeats (STRs)
Sequence differences affect restriction endonuclease recognition sequences - and therefore DNA fragment sizes differ - called restriction fragment length polymorphisms - RFLPs
Use of Southern blotting
Genomic DNA sequences used in test are repetitive and are distinct (in sequence and # of repeats) from one person to another (can get rid of nonrepetitive by S1 nuclease)
Combine use of several probes and test can become so selective it can ID a single person in the entire human population
If 3 STRs match a suspect likelihood is 2000:1 that the police have the right person (nine matches makes the odds 1 billion:1)
FBI requires 13 matches (database exists)
Drawback - Southern analysis needs large amount of DNA and must be relatively fresh
To deal with this use PCR so can obtain DNA fingerprint from single hair follicle, drop of blood, and it can be years old
Once we know what every DNA sequence in the genome does we can use sample found at a crime scene to determine suspects’ build, race, eye and hair color, inherited physical defects, personality traits, etc.
From genomes to proteomes
Proteome = complement of proteins expressed by a genome
Protein function described by:
1. Phenotypic function - effect of protein on entire organism2. Cellular function - description of network of of interactions engaged in by the protein at the cellular level3. Molecular function - precise biochemical activity of protein
Comparative genomics - assign gene function by genome comparisonsConserved gene order - synteny - between human and mouse
Cellular expression patterns can reveal cellular function of a gene
DNA Microarrays - “DNA chips” - allow rapid and simultaneous screening of thousands of genes
DNA segments from known genes (up to hundreds of bp long) are amplified by PCR and placed on a solid surface using robotic devices that accurately deposit nanoliter amounts of DNA solutionThousands of such spots are deposited in a pre-designed array on a surface area of just a few square centimeters
Also can synthesize DNA directly on solid surface - photolithography
Once chip constructed can be probed with mRNAs or cDNAs from a particular cell type or cell culture to ID genes being expressed
DNA microarrayEach spot contains DNA from one of the 6200 genes in S. cerevisiae
Green dye - cells growing normally in culture
Red dye - cells 5 hrs after begin to form spores
Yellow - genes that do not change their level of expression during sporulation
Actual chip - 1.8 cm x 1.8 cm
Genome alterations and New Products of Biotechnology
PLANTS:Uses of recombinant technology in agriculture
alter nutritional profile or yield of cropsalter resistance of plants to insects, diseases, cold, salinity, drought
Bacterial plant parasite aids in cloning of plants since no naturally occurring plant cell plasmids
Use soil bacterium - Agrobacterium tumefaciensinvades plants at site of wound, transforms nearby cells, induces tumor growth (called crown gall), bacterium contain large Ti plasmid where T DNA moves from bacterium and integrates into plant cell chromosomes, 25 bp repeats and vir genes are essential
Genome alterations and New Products of Biotechnology
PLANTS:T DNA encodes enzymes that convert plant metabolites into 2 classes of compounds that benefit the bacterium
Plant growth hormones - stimulate crown gall tumor growth
Unusual amino acids - bacterial food source, only metabolized by bacterium onlyDiverts plant resources by converting them to a form that benefits only bacterium
Genome alterations and New Products of Biotechnology
PLANTS:Rare example of DNA transfer from prok to euk is a natural genetic engineering process - researchers use this to transfer recombinant DNA to plant (instead of T DNA)
Vir genes on plasmid (a) aid in transfer of foreign DNA in plasmid (b) into plant genome
Genome alterations and New Products of Biotechnology
PLANTS:
SUCCESS!!
Luciferase gene from fireflies introduced into cells of a tobacco plant
Genome alterations and New Products of Biotechnology
PLANTS:
SUCCESS!!
Production of crop plants that are resistant to herbicides, plants viruses, insects
Other benefits - increased yields & less need for use of chemicals
Expresses a gene for a protein toxin derived from Bacillus thuringiensis This toxin kills larvae of some moth species while being harmless to humans
Genome alterations and New Products of Biotechnology
PLANTS: development of soybeans that are resistant to general herbicide glyphosate (RoundUp)One treatment can last all year
Worries: evolution of glyphosate-resistant weeds, and escape of difficult-to-control recombinant plants
Genome alterations and New Products of Biotechnology
ANIMAL CELLS: want to be able to introduce foreign DNA into animal cells to study structure & function of genome, proteins, and to generate animals with new traitsUse of animal tissues (difficult to maintain & manipulate) so use cell lines/tissue culture
NO PLASMID-LIKE vector for introducing DNA into animal cells SO it is necessary to integrate DNA into host-cell chromosome
CHALLENGES: efficient delivery of DNA to cell nucleus, integration into chromosome without disrupting any critical genes
Genome alterations and New Products of Biotechnology
ANIMAL CELLS: Introduction of DNA into cells:
electroporation - very inefficientmicroinjection - inject DNA straight into nucleus using a fine needle - small number of cells treatedtakes a lot of skill
liposomes - small vesicles with a lipid bilayer, recombinant DNA inside
viral vectors - effective mechanisms for introducing foreign DNA intocells, integrate into host chromosome, retroviruses & adenovirusesmodified to serve as viral vectors into introduce foreign DNA into cells
Genome alterations and New Products of Biotechnology
ANIMAL CELLS: Introduction of DNA into cells:retroviral vectors - special regions required:• LTR for integration through homologous recombination• for packaging into viral particles
also need “helper virus” to provide genes to produce viral particle
Genome alterations and New Products of Biotechnology
ANIMAL CELLS: Introduction of DNA into cells:adenoviral vectors - lack mechanism for integrating DNA into chromosome so recombinant DNA expressed from this vector is short-lived - OK for transient gene expression
PROBLEMS WITH TRANSFORMATION OF ANY ANIMAL CELLS:Random integrationNonhomologous recombination occurs frequentlyIf disrupt essential genes - cell functions/protein alteredNew evidence that integration events can sometimes activate genes that stimulate cell division (CANCER ENSUES!)Site of integration can have an effect on expression of gene (No way to control this)
Genome alterations and New Products of Biotechnology
ANIMAL CELLS: Despite challenges, transformation of animal cells used to study chromosome structure & function, regulation & gene expression
Microinjection of DNA into nuclei of fertilized mouse eggs, those in the germline that are affected can be identified by testing their offspringCareful breeding results in a TRANSGENIC mouse line where all mice are homozygous for new gene(s)
Used to introduce gene for human growth hormone, under control of an inducible promoterFed a diet with inducer
Lots of trangenic mice have been made already - also make “knockout mice”, where a particular gene has been inactivated - can establish function of gene
Genome alterations and New Products of Biotechnology
DISCOVERY OF NEW PHARMACEUTICALSHypertension, congestive heart failure, hypercholesterolemia, & obesity are treated by a pharm drug that alters human physiology
Proteomics will help to identify future drug targets
Example: most potent vasoconstrictor - peptide hormone urotensin IIUse of proteomics, etc. helped identify its target in the cell - GPR14, an “orphan” receptor initially
Also identify new agents to treat human pathogenic diseasesID enzymatic targets in microbial pathogens