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DNA chains coiled & floated lurid blue and pink images of electric molecules injected

themselves somewhere between in my mind. Tonight Im cooking, the enzymes and thechemicals 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 couldonly read the DNA blueprints. Then there is the looming, but not so pressing, reasons forknowing DNA the ones that extend out to horizons mankind has not yet reached. Understandingthe intricate mechanisms of our own genes will have more than just medical impact. Having adetailed understanding of why children resemble their parents will lead to genetic manipulatingby those who may prefer alterations over strict duplication. Genetic engineering will not be anew endeavor; evolution is always and has always been a genetic engineer. The DNA moleculesin 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 stringof nucleotides in a synthetic molecule might be able to define a position along a much longernatural DNA strand. Natural DNA is tractless coil, like an unwound and tangled audiotape on thefloor. A chemical program FIND a specific sequence on DNA with 3 billion nucleotides andthen display that sequence to a human. A reiterative mathematical procedure where you applysome process to a starting number to obtain a new number, and then you apply the same processto the new number, and etcetera. If the process is multiplication by 2, then the result of manycycles is an exponential increase in the value of the original number. 2 -> 4 -> 8 -> 16 -> 32 andso on. (p.3)

A short synthetic piece of DNA to find a particular sequence and then start a process wherebythat sequence would reproduce itself over and over. A concept not out of the question given oneof the natural functions of DNA is to reproduce themselves every time a cell divides into twodaughter cells. Synthetic DNA would stick to a longer strand of DNA in a specific way if thesequences matched up some-where on the long piece. A thousand out of 3 billion in the humangenome would be no trivial feat, but it wouldnt be enough, I just needed to find one place. Icould use another short piece to narrow the search, which would bind to a sequence just downthe chain from the first sequence and use the natural properties of DNA to replicate itself. Thatsequence between the two short search strings would replicate the hell out of it. (p.6)

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

To amplify DNA by the repeated reciprocal extension of two primers hybridized to the separatestrands of a particular DNA sequence. Extending the primer on a single-stranded DNA templatewould make a double-stranded DNA molecule that you could heat-up and turn into two moresingle-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 thatyou could have all the DNA you wanted. I originally wanted to amplify a 400-nucleotide

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fragment of Human Nerve Growth Factor, which Genetech had cloned and just published anarticle 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 purpletop, boiled for a few minutes, cooled, added DNA polymerase, closed the tube and left it at 37degrees. I poured cold Becks into a 400-mililiter beaker and left. I figured the reaction wouldtake place by itself. The next day I went to the lab and there was no sign by ethidium bromide ofany 400-nucleotide fragment. The unpleasant prospect of cycling the rxn by hand, which meantadding 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 onsomething simpler, called a plasmid. (p.12)

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

PCR was a tool I created because I needed it to do an experiment. If I had had more knowledgeabout 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 turningout better than others. Often by the 10 th cycle, it would not completely double. Something wasrunning 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 manycases there simply werent enough enzymes to react with the DNA that had to be processed. Thelimiting factor in doing PCR is simply the # of molecules of the enzyme. Enzyme amounts areexpressed 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 thesolution. Keep the # of things the enzyme is going to interact with smaller than the number ofmolecules of the enzyme. Simplicity is embarrassing when you have to work for months toachieve it. (p.99-100)

PCR is a chemical procedure that would make the structures of the molecules of our genes aseasy to see as billboards in the desert and as easy to manipulate as tinker-toys. It would findfragments of DNA and multiply them billions of times. And it would do it quickly. Theprocedure would be valuable in diagnosing genetic diseases. It would find infectious diseases bydetecting the genes of pathogens that were difficult or impossible to culture. PCR would solvemurders 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 fromthe DNA in ancient specimens. The branching and migrations of early man would be revealedfrom fossil DNA and its descendent DNA in modern humans. And when DNA is finally foundon other planets, it will be PCR that would tell us whether we had been there before or whetherlife on other planets was unrelated to us and had its own separate roots. (p.105)

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

PCR has many applications in modern genetics but it has become particularly important in theclinical diagnosis of genetic disease and in the field of forensic science. It is now commonpractice to screen the genetic recipes of prospective parents and fetuses for disease-causinggenes, when there is a history of an inherited disorder. By using primers specific only tosequences found in the disease causing gene, a PCR test can reveal whether an individual carriesthe 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 nowhere to bind and yield a blank result.

PCR is used in a similar way as a diagnostic test for the presence of disease-causingorganisms such as the HIV-virus & Mycobacterium, which causes tuberculosis. With HIVtesting, DNA is extracted from the patients blood and a PCR rxn is carried out.

One of PCRs 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 ofpotential 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 beextracted 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-knownand serious genetic diseases. The results of such a test can assist in the decision making processof childbearing by providing a positive or negative confirmation of genetic risks involved.

Cystic fibrosis is caused by a defective protein which, in unaffectedindividuals, maintains a chemical equilibrium across the membranes of cells. Affectedindividuals have abnormal mucus in their respiratory tract and digestive system causingbreathing and digestion complications. In addition, the mucus is a prime auger forbacterial quorums that can lead to infections such as pneumonia. Two apparently healthyparents, both with a history, can consult a genetic counselor, trained and educated aboutall aspects of genetic testing, as to the statistics of their progenys likelihood ofsusceptibility. DNA from a buccal salivary swab may indicate whether one or both