594648,2450,Spring2010,DNA_Paper

Notes:
DNA chains coiled & floated lurid blue and pink images of electric molecules injected
themselves somewhere between in my mind. Tonight I’m cooking, the enzymes and the
chemicals I have at Cetus are my ingredients. (p.1)

There are pressing reasons to want to read DNA; children are born with genetic defects,
sometimes with tragic consequences. Such things could be predicted and averted if we could
only read the DNA blueprints. Then there is the looming, but not so pressing, reasons for
knowing DNA –the ones that extend out to horizons mankind has not yet reached. Understanding
the intricate mechanisms of our own genes will have more than just medical impact. Having a
detailed understanding of why children resemble their parents will lead to genetic manipulating
by those who may prefer alterations over strict duplication. Genetic engineering will not be a
new endeavor; evolution is always and has always been a genetic engineer. The DNA molecules
in our cells are our history, and they are the stuff of which our future will be crafted. (p.2)

Oligonucleotides that are easily made, like a FIND sequence in a computer search, a short string
of nucleotides in a synthetic molecule might be able to define a position along a much longer
natural DNA strand. Natural DNA is tractless coil, like an unwound and tangled audiotape on the
floor. A chemical program “FIND” a specific sequence on DNA with 3 billion nucleotides and
then display that sequence to a human. A reiterative mathematical procedure where you apply
some process to a starting number to obtain a new number, and then you apply the same process
to the new number, and etcetera. If the process is multiplication by 2, then the result of many
cycles is an exponential increase in the value of the original number. 2 -> 4 -> 8 -> 16 -> 32 and
so on. (p.3)

A short synthetic piece of DNA to find a particular sequence and then start a process whereby
that sequence would reproduce itself over and over. A concept not out of the question given one
of the natural functions of DNA is to reproduce themselves –every time a cell divides into two
daughter cells. Synthetic DNA would stick to a longer strand of DNA in a specific way if the
sequences matched up some-where on the long piece. A thousand out of 3 billion in the human
genome would be no trivial feat, but it wouldn’t be enough, I just needed to find one place. I
could use another short piece to narrow the search, which would bind to a sequence just down
the chain from the first sequence and use the natural properties of DNA to replicate itself. That
sequence between the two short search strings would replicate the hell out of it. (p.6)

I could make a zillion copies but they’d always be the same size. I had just solved two major
problems in DNA chemistry: abundance and distinction. (p.7)

To amplify DNA by the repeated reciprocal extension of two primers hybridized to the separate
strands of a particular DNA sequence. Extending the primer on a single-stranded DNA template
would make a double-stranded DNA molecule that you could heat-up and turn into two more
single-stranded DNA templates, the process repeatable. (p.9)

It was not all that easy to imagine during the postcloning, pre-PCR year, to accept the fact that
you could have all the DNA you wanted. I originally wanted to amplify a 400-nucleotide
594648,2450,Spring2010,DNA_Paper

fragment of Human Nerve Growth Factor, which Genetech had cloned and just published an
article on in Nature. What had taken Genetech months to obtain, I would reproduce in hours.
(p.10)

I put the human DNA and HNGF primer in a little screw-cap tube with an O-ring and a purple
top, boiled for a few minutes, cooled, added DNA polymerase, closed the tube and left it at 37
degrees. I poured cold Becks into a 400-mililiter beaker and left. I figured the reaction would
take place by itself. The next day I went to the lab and there was no sign by ethidium bromide of
any 400-nucleotide fragment. The unpleasant prospect of cycling the rxn by hand, which meant
adding the thermally unstable polymerase after every cycle & a hell of a lot more work for me,
was daunting. Finally I retreated from the idea of starting with human DNA and settled on
something simpler, called a plasmid. (p.12)

The first successful experiment happened on December 16th, 1983. I took the autoradiogram out
of the freezer and developed it. There was a little black band which meant I was going to be
famous. I was aided in this process by mathematician, Fred Faloona. We had just changed the
rules in molecular biology. (p.13)

PCR was a tool I created because I needed it to do an experiment. If I had had more knowledge
about what I was doing, PCR would have never been invented.

In the early years of PCR, no one could figure out why certain methods of doing it were turning
out better than others. Often by the 10th cycle, it would not completely double. Something was
running out ot something was being made that interfered with the process. (p.96)

When I compared the # of molecules in each of my ingredients, it became obvious that in many
cases there simply weren’t enough enzymes to react with the DNA that had to be processed. The
limiting factor in doing PCR is simply the # of molecules of the enzyme. Enzyme amounts are
expressed in units of activity, how many molecules of something it will turn into something else,
under a standard set of conditions per unit of time. Simply counting the molecules was the
solution. Keep the # of things the enzyme is going to interact with smaller than the number of
molecules of the enzyme. Simplicity is embarrassing when you have to work for months to
achieve it. (p.99-100)

PCR is a chemical procedure that would make the structures of the molecules of our genes as
easy to see as billboards in the desert and as easy to manipulate as tinker-toys. It would find
fragments of DNA and multiply them billions of times. And it would do it quickly. The
procedure would be valuable in diagnosing genetic diseases. It would find infectious diseases by
detecting the genes of pathogens that were difficult or impossible to culture. PCR would solve
murders from DNA samples in trace materials –semen, blood hair. The field of paleobiology
would blossom b/c of PCR. Its practitioners would inquire into the specifics of evolution from
the DNA in ancient specimens. The branching and migrations of early man would be revealed
from fossil DNA and it’s descendent DNA in modern humans. And when DNA is finally found
on other planets, it will be PCR that would tell us whether we had been there before or whether
life on other planets was unrelated to us and had its own separate roots. (p.105)

NTS # 2
594648,2450,Spring2010,DNA_Paper

PCR was invented by American Geneticist Kary Mullis, who was awarded the Nobel Prize for
his work. He claimed the idea came to him after a drug induced hallucinatory trip into a DNA
molecule.

PCR has many applications in modern genetics but it has become particularly important in the
clinical diagnosis of genetic disease and in the field of forensic science. It is now common
practice to screen the genetic recipes of prospective parents and fetuses for disease-causing
genes, when there is a history of an inherited disorder. By using primers specific only to
sequences found in the disease causing gene, a PCR test can reveal whether an individual carries
the gene. If it is present, the produced copies will be detected by electrophoresis and ethidium-
bromide staining; if the individual does not carry the defective gene, the primers will have no
where to bind and yield a blank result.
PCR is used in a similar way as a diagnostic test for the presence of disease-causing
organisms such as the HIV-virus & Mycobacterium, which causes tuberculosis. With HIV
testing, DNA is extracted from the patients blood and a PCR rxn is carried out.
One of PCR’s great strengths is that it works with even the smallest amount of DNA. For
this reason, it has become an invaluable tool in forensic science. Crime scenes are full of
potential genetic clues criminals often leave unwittingly behind. Blood, skin, hair, saliva &
sometimes sexual fluids, are all bodily tissues that contain the suspects’ dog tags. DNA can be
extracted from a single hair follicle was so minute as to be undetectable, until PCR.
Is used to test whether prospective parents are carriers for some of the more well-known
and serious genetic diseases. The results of such a test can assist in the decision making process
of childbearing by providing a positive or negative confirmation of genetic risks involved.
Cystic fibrosis is caused by a defective protein which, in unaffected
individuals, maintains a chemical equilibrium across the membranes of cells. Affected
individuals have abnormal mucus in their respiratory tract and digestive system causing
breathing and digestion complications. In addition, the mucus is a prime auger for
bacterial quorums that can lead to infections such as pneumonia. Two apparently healthy
parents, both with a history, can consult a genetic counselor, trained and educated about
all aspects of genetic testing, as to the statistics of their progeny’s likelihood of
susceptibility. DNA from a buccal salivary swab may indicate whether one or both