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DNA-based information technologies
Human Genome Project strategies
Understand use of polymorphisms
DNA fingerprinting
Understand use of proteome to determine protein function
Understand creation and use of DNA microarrays
Describe use of recombinant technology in plant and animal cells
Understand challenges in introducing DNA into animal cell
From genes to genomes
From genes to genomes
LINES - long interspersed elements (6 to 8 kbp)
SINES - short interspersed elements (100 to 300 bp)
Retroviruslike transposons - “trapped” in genome
SSR - simple sequence repeats
SD - large segmental duplications
? - genes encoding RNAs?remnants of transposons
Major landmarks in DNA sequencing
* 1975 The first complete DNA genome to be sequenced is that of bacteriophage φX174
* 1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation”; Frederick Sanger,
independently, publishes "DNA sequencing by enzymatic synthesis".
* 1980 Frederick Sanger and Walter Gilbert receive the Nobel Prize in Chemistry
* 1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb.
* 1986 Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the first semi-automated
DNA sequencing machine.
* 1987 Applied Biosystems markets first automated sequencing machine, the model ABI 370.
* 1990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum,
Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (at 75 cents (US)/base).
* 1995 Craig Venter, Hamilton Smith, and colleagues at The Institute for Genomic Research (TIGR) publish the first
complete genome of a free-living organism, the bacterium Haemophilus influenzae. The circular chromosome contains
1,830,137 bases and it marked the first use of whole-genome shotgun sequencing, eliminating the need for initial mapping
efforts.
* 1995 Richard Mathies et al.. publish fluorescence energy transfer dye-based sequencing.
* 1996 Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm publish their method of
pyrosequencing.
Competition to sequence DNA genome
(NIH)
Overlapping clones in a DNA library
(Celera)
“whole genome shotgun approach” - no physical map
Random sequencing of DNA genome
Human Genome project strategy
Necessary to have computer-power
to match random segments and
overlaps
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 organism
2. Cellular function - description of network of of interactions engaged in by the protein
at the cellular level
3. Molecular function - precise biochemical activity of protein
Comparative genomics - assign gene function by genome comparisons
Conserved 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 solution
Thousands 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 microarray
Each 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 crops
alter 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 tumefaciens
invades 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 -
Unusual amino acids -
stimulate crown gall tumor growth
bacterial food source, only metabolized by
bacterium only
Diverts 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 traits
Use 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 inefficient
microinjection - inject DNA straight into nucleus
using a fine needle - small number of cells treated
takes a lot of skill
liposomes - small vesicles with a lipid bilayer, recombinant DNA inside
viral vectors - effective mechanisms for introducing foreign DNA into
cells, integrate into host chromosome, retroviruses & adenoviruses
modified 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 integration
Nonhomologous recombination occurs frequently
If disrupt essential genes - cell functions/protein altered
New 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 offspring
Careful 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 promoter
Fed 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 PHARMACEUTICALS
Hypertension, 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 II
Use of proteomics, etc. helped identify its target in the cell - GPR14, an “orphan” receptor initially
Also identify new agents to treat human pathogenic diseases
ID enzymatic targets in microbial pathogens