I'm a biologist. I'm in the biology department.
You're the one! You're the one's going to make all that trouble ... making
everyone the same, rearranging the chromozones, or whatever it is. Isn't
NICK (with that small smile)
Not exactly: chromosomes.
I read somewhere that science fiction is really not fiction at all ... that
you people are rearranging my genes, so that everyone will be like everyone
else. Now I won't have that! It would be a shame. I mean ... look at me!
Is it really such a good idea ... if everyone was forty something and looked
-Edward Albee, Who's Afraid of Virginia Woolfe?1962
Biotech Campbell and Reece, Chapter 20
It's not possible for a professor to lecture from old notes in biolog, especially
for topics like this
TRANSPARENCY (Fig. 20.8)
When voltage is applied, proteins in detergent migrate through a gel based
on their size.
"PAGE" (polyacrylamide gel electrophoresis) and western blotting
Here is a photo
of the rig used to pour the gel with wells depicted in the transparency.
Here is a picture
of the power supply and the electric hookup to make the proteins migrate.
Here is a photo
of the gel with proteins spread out after the proteins have been stained.
This shows the
apparatus to transfer proteins from the gel.
The blot, with
radioactively labeled antibody to the protein of interest, are put together
in a cassette.
Film (unexposed) and blot are sandwiched in the casette until the film is
exposed, then it is developed (figure)
to make an autoradiogram.
Here is some work
from my laboratory to determine if a protein called PLC (phospholipase C)
is decreased by vitamin A deprivation; protein molecular weights are shown.
The word "clone" has several meanings. One of the best known to
the lay public started when a sheep named Dolly was made from a nucleus
from another sheep. But for now:
TRANSPARENCY (Fig. 20.1) In lay terms, cloning is chopping a gene at both
ends, putting it in and growing it up in bacteria. Plasmids are important.
This allows manufacture of proteins of interest, and examples are given:
growth hormone and a clot disolving enzyme. Also, bacteria can be altered
with inserted genes so that they do useful things like clean up oil spils.
Crops can also be altered like inserting resistance to pests.
TRANSPARENCY (Fig. 20.2)
restriction nucleases often cut at "palindromes"
"restriction" restrict phage infections in bacteria
"Able was I ere I saw Elba" Napoleon "Madam I'm Adam"
Eco R1 E. coli
staggered cuts with single stranded cohesive ends
The1978 Nobel prize
in Physiology and Medicine was awarded jointly to: WERNER ARBER , DANIEL
NATHANS and HAMILTON O. SMITH for the discovery of restriction enzymes and
their application to problems of molecular genetics
TRANSPARENCY (Fig. 20.3) If the same restriction enzyme is used to gut a
piece of DNA with the gene of interest and the plasmid, they will have complementary
sticky ends and will stick together. Though putting antibiotic resistance
into bacteria may seem dangerous (and early on, precautions were regulated)
this allows you to screen for bacteria that have the new plasmids you have
made (since only those bacteria will survive if ther is antibiotic in the
DNA has a tendency to anneal, hybridization, and this is real useful.
TRANSPARANCY (Fig. 20.4) How do you test for the gene you want?
Spread thinly on petri dish so that each clump is one clone.
Pick up some on paper denature and hybridize to radioactive probe. Do autoradiography.
Then go back to dish to recover clone.
Here's a photograph
I took of a petri dish with colonies.
TRANSPARENCY (Fig. 20.6) chop up whole genome (genes which are and are not
This works pretty well except chopping chops some genes in half.
Chop plasmids (with same enzyme) mix, put back in (bacteria),
Select on antibiotic and keep growing, then you have a genomic library.
A genomic DNA library is, good for telling you introns, promotors, also,
it's a big mess.
TRANSPARENCY (Fig. 20.5) it is possible to make DNA out of mRNA - cDNA (complementary).
You can make a cDNA library.
Note that the cDNA library would be specific for different cell types, deprnding
on which cells are expressed.
It's a lot of trouble to clone a gene.
TRANSPARANCY (Fig. 20.7) DNA polymerase which works on single stranded DNA.
prize in chemistry was shared and awarded in part to KARY B. MULLIS
for his invention of the polymerase chain reaction (PCR) method.
One strand is sense strand, the other is the antisense strand, and they
If you know sequence at beginning and end of the gene, you make one primer
for the sense strand at the beginning of the gene and another for the antisense
strand at the end of the gene.
These will copy gene and then continue beyond in the first round, which
is a small problem.
But on next round, going the other way stops at the end of the gene.
If 98oC to denature, cool to 60oC for polymerase to add to prime, that would
require new polymerase at each cooling, but Thermus aquaticus from Yellowstone
hot spring polymerase.
It all becomes very automated, and here's a picture
I shot of one of the Biology Department's PCR machines.
TRANSPARANCY (Fig. 20.9) restriction fragment length polymorphisms
In general, a marker is something like fruit fly eye color (something that
has a phenotype (trait)), that can be used for genetic mapping but RFLPs
can be markers too.
TRANSPARENCY (Fig, 20.10) Southern blotting (for DNA) [northern - mRNA,
western - protein]
TRANSPARENCY (Fig. 20.15) Sometimes a RFLP marker can be closely linked
to a specific disease-causing genetic allele.
TRANSPARENCY (Fig. 20.17) This is the idea behind DNA fingerprinting
Get different cuts by cutting with different nucleases, use different cuts
to walk to gene of interest
TRANSPARANCY (Fig. 20.11)
TRANSPARENCY (Fig. 20.16) - if a gene is in a retrovirus, it can be put
into a particular somatic tissue and get incorporated into the genome. Note,
there are orders of magnitude different ethical issues for the germ line!
TRANSPARENCY (Fig. 20. 14a) Microarray - test for expression of all the
genes (supplies are expensive)
This page was last updated 8/2/02
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