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

Photolithography

Use DNA microarray to answer:“which genes are expressed at a given stage in development?”

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