Biotechnology
The first thing we need is a language lesson. All citizens of the 21st century should know the meaning of the following terms. If you don't, you can look them up in a science dictionary or an online reference source. Wikipedia is a good example.
http://en.wikipedia.org/wiki/Main_Page
You might also try the genetics glossary at:
http://helios.bto.ed.ac.uk/bto/glossary/
Words and phrases you must know:
Clone*
DNA probe
cDNA
transformation
DNA hybridization
plasmid
GMO
virus
vector
insert*
gene insertion
restriction enzyme
protein sequencing expression
central dogma
restriction map
DNA sequencing
agarose gel
acrylamide gel
electrophoresis
nucleotides
A, G, C and T
bases, base pairs
bands
(on a gel)
E.coli
fluorescent dyes, fluorescence
Gene
shotgun cloning & shotgun sequencing
Ti plasmid, T DNA
locus
opines
allele
DNA
Agrobacterium tumefaciens
ligase, ligation
PCR (polymerase chain reaction)
RNA
DNA polymerase
TAQ polymerase (used in PCR)
screening
antibiotic resistance gene
* can have several meanings and may be used as a verb or a noun.
Some basic techniques:
http://www.bioinformatics.nl/webportal/background/techniques.html
Extraction:
DNA can be extracted from organisms by mechanical disruption of cells followed by chemical extraction and purification of the DNA. DNA dissolves in some liquids but not in others - this allows the DNA to be separated from other substances by rounds of solution and centrifugation. For example, DNA becomes a white solid in cold ethanol. It can then be centrifuged down into a pellet. The liquid containing impurities is discarded and the pellet containing DNA can be re-suspended in a special salt solution.
Restriction digests:
DNA can be cut up into fragments using restriction enzymes. Restriction enzymes come from bacteria. There are hundreds of different restriction enzymes. Each one looks for a specific short (4 - 6 bases) sequence of DNA and cuts the DNA wherever it finds this sequence. DNA pieces called restriction fragments are the result. If we use more restriction enzymes then the fragments get smaller as they are cut at a greater number of places. Each restriction enzyme or combination of enzymes yields a characteristic set of DNA fragments when used on a particular organism or individual.
When a mixture of these fragments is run on an agarose or acrylamide gel, they separate by size to give a banding pattern that is characteristic for that particular combination of DNA and restriction enzyme.
Using this technique it is possible to deduce the relative positions of the restriction sites (where the enzymes cut the DNA) on the original DNA molecule. This is called a restriction map.
Restriction enzymes can be used to cut open a vector such as a plasmid. This allows scientists to glue or "ligate" a DNA fragment into the vector.
Using probes
DNA
probes are short sections of DNA (usually 6 - 20 bases) that can be used to
look for other (usually larger) pieces of DNA containing a region with complementary
sequence. They work because double and single stranded DNA and RNA can stick
to (or "hybridize" with) DNA molecules with the same or a similar
base sequence. Probes can be chemically bonded to radioactive or fluorescent
markers that can then help scientists to identify a particular fragment of
DNA from among thousands of other fragments that they may be working with.
Probes can be made synthetically using whatever small piece of information
is already known about the particular piece of DNA that you want to find.
For example, by sequencing a short piece of an interesting protein you can
deduce first the amino acid sequence of part of the protein, then part of
the DNA sequence of the gene that made it. You can now make a probe that will
stick to the gene that made the protein or to other similar genes that you
want to study.
Inserting DNA fragments into a vector:
The
enzyme "DNA ligase" is used to glue fragments into a vector. Success
can be observed by running the results on a gel. The new vector plus insert
should be bigger (thus move more slowly) than the original vector.
Cloning the fragment:
Thousands
of copies of a given fragment can be made by placing the vector containing
the fragment into a bacteria and allowing the bacteria to reproduce.
A set of identical DNA molecules made this way is sometimes called a clone,
although this word is used in other ways too. The fragment can also be copied
by using polymerase chain reaction.
Polymerase chain reaction:
A technique which uses a heat-resistant DNA polymerase to make many copies of a given DNA molecule, or to locate and make many copies of a small lenth of DNA from within a much larger DNA molecule. The technique involves a cycle of heating and cooling which causes DNA strands to separate, then copy themselves. Each cycle of heating and cooling lasts a few minutes and doubles the amount of DNA present. This technique is often used in forensic science to "amplify" (increase) a tiny trace of DNA evidence found at a crime scene. The special enzyme used in this procedure was originally isolated from bacteria that live around hot volcanic springs at the bottom of the ocean.
Making cDNA
cDNA is made using an enzyme called "reverse transcriptase". This enzyme comes from a virus and can be used to turn RNA back into DNA (essentially reversing the process of transcription). mRNA in the cell cytoplasm only comes from genes that are currently active. By turning mRNA back into DNA , scientists can determine which genes are active at any given time. This makes it easier to understand how the cell uses and regulates its genetic material. Its a bit like intercepting all the outgoing messages from the town hall that tell a city what to do.
Microarray analysis
Also called DNA chips. Miccroarrays are small trays about the size of a microscope slide with a large number of DNA fragments stuck to spots that are arranged on the slide in a specific grid-like arrangement. Typically, the entire collection of fragments correspond to the entire genome of a well-known organism, broken up into thousands of pieces. The identity and arrangement of genes within the organism is usually well known, and their location on the grid is recorded in an acompanying data file. When the DNA on a single spot binds to test DNA that is applied to the chip, the spot lights up, and can be easily seen when the chip is examined under a microscope. The identity of that gene can now be read from a reference list that describes the identity of all the individual spots that make up a microarray. The location and identitifcation of any glowing spots is accomplished and recorded automatically using digital imaging. This form of analysis is extremely fast and efficient.
By extracting various types of DNA from an organism and applying it to the chip, it is possible to determine information such as which genes are active at any given time, which genes are inactive and which genes are totally absent. It is also possible to determine other information such as the number of copies of a particular DNA sequence that exist in an organism and the degree of similarity of DNA from different organisms.
After a DNA
chip has been used to test a DNA sample, the DNA that was examined can be
washed off and the chip can be used again. This system will probably be developed
into a variety of genetic tests that may performed in doctor's offices.
http://www.sciencemag.org/feature/e-market/benchtop/micro.shl
DNA sequencing:
See below:
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
http://www.llnl.gov/str/Balch.html
Transforming dicots using Agrobacterim tumefaciens:
http://commtechlab.msu.edu/sites/dlc-me/zoo/microbes/agrobacterium.html
http://helios.bto.ed.ac.uk/bto/microbes/crown.htm
Agrobacterium
contains a special plasmid called the Ti (tumor inducing plasmid) which can
inject a portion of itself (called T DNA) into some plants. T DNA integrates
into the plant nucleus and causes a "crown gall tumor" to form,
as well as instructing the plant how to make Agrobacterium's favorite food;
chemicals called opines. Genes added to the T DNA of the Ti plasmid (by humans)
stand a good chance of being expressed by the plant. This is the basis of
many genetically modified plants.
Transforming plants using a DNA gun
http://www.nal.usda.gov/pgdic/Probe/v2n2/particle.html
http://opbs.okstate.edu/~melcher/MG/MGW4/MG434.html
If you want to buy one look here:
http://fisio.dipbsf.uninsubria.it/scuola/Bulletin_9075.pdf
Transforming
plants using electroporation
Take a plant cell and use enzymes to strip away the cell wall.
The
resulting bag of cytoplasm is called a protoplast.
Use a series of electric shocks to punch holes in the bag, then immerse the
cell into a fluid containing the DNA you want to introduce.
Under the right conditions the cell will now regrow its wall and start to
divide.
Eventually it will develop into an adult plant.
Other ways of getting DNA into organisms (not necessarily plants):
Start here and follow links:
http://opbs.okstate.edu/~melcher/MG/MGW4/MG43.html
More about transgenic crops:
http://wwwfac.mcdaniel.edu/Biology/botf99/biotech/techniq.htm
http://www.businessweek.com/1999/99_51/b3660136.htm
Examples:
Here is a nice page of examples and descriptions of the role of genetics and genetic engineering in many aspects of everyday life. This site also includes links to educational online radio programs.
1) Golden rice
http://www.biotech-info.net/golden.html#research
http://www.biotech-info.net/rice_boost.html
http://www.uky.edu/Agriculture/Entomology/entfacts/fldcrops/ef130.htm
http://www.ars.usda.gov/is/AR/archive/feb02/corn0202.htm
3) Roundup ready corn - coming soon roundup ready canola
(Glyphosphate resistence)
http://www.poptel.org.uk/panap/archives/carmen.htm
http://www.kehoe.org/owen/soybean/
4) Flavr Savr tomato
Calgene's Flavr Savr tomato
was enhanced with bacteria to toughen it up for shipping and to increase its
shelf life. Approved for commercial distribution in 1994 and later marketed
under the homey name "MacGregor's," this was the first genetically
engineered whole food to hit grocery stores. Calgene removed it from the market
after it flopped commercially. It tasted awful.
5)
Green fluorescent protein (GFP)
Usually used in conjunction with special confocal microscope. Can produce especially nice 3d images and movies of cellular activity:
http://www.plantsci.cam.ac.uk/Haseloff/CONFOCAL/subcelldyn.html
more examples of wierd glowing stuff:
http://www.bbc.co.uk/science/genes/gene_safari/wild_west/glowing_gallery2.shtml
6) Bioplastics
http://www.biology.iupui.edu/biocourses/N100/goodfor10.html
http://atmizzou.missouri.edu/apr04/PlasticPlants.htm
7) Various phytoremediation and bioremediation projects.
EPA's guide to phytoremediation and bioremediation.
Links related to phytoremediation:
8)
Edible vaccines
http://www.bbc.co.uk/science/genes/gene_safari/pharm/edible.shtml
9 ) Monsanto's terminator gene
http://www.hort.purdue.edu/newcrop/proceedings1999/v4-127.html
http://news.bbc.co.uk/1/hi/sci/tech/465222.stm
10 Double muscle animals
http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtml
11) "hardworking" gene
http://news.bbc.co.uk/2/hi/science/nature/3557310.stm
Potential problems with GM products:
Unpredictable
effects and often dissapointing results.
Loss of business in countries that won't accept GM products.
Contamination and risk to ecosystem.
Possible health issues.
http://www.mercola.com/2003/jul/2/gm_crops.htm
"Poison Plants?" - Article pertaining to the controversy over genetic engineering.
Other Problems With Biotechnology:
Martha Crouch is a famous plant biologist who at the height of her successful research career, stunned the science community by announcing that she would no longer perform any scientific research.
She lives in a house made of all-natural materials, has no motor vehicle and uses virtually no electricity.
