 You're watching FJTN, the Federal Judicial Television Network. The Federal Judicial Center presents Science in the Court Room, a series of programs for judges on science and scientific evidence. With an introduction by Stephen Breyer, Associate Justice of the Supreme Court of the United States. Welcome to the Federal Judicial Center's series of science programs for judges. This series is a response to two significant developments. One is that as time goes on, an increasing number of cases on your dockets are turning upon the application of scientific principles. Another is that recent decisions of the Supreme Court have imposed new duties on you, gatekeeping and decision-making duties that require an enhanced understanding of the scientific methodologies at issue in your cases. The series seeks to help you fulfill these duties in several ways. First, by providing you with an understanding of the basic principles governing three discrete fields of pure science, microbiology, epidemiology, and toxicology. Second, by examining methods of evaluating the reliability of evidence from those three fields of science. And third, by considering scientific evidence from these fields in the wider context of making rulings and management decisions in specific types of cases that is patent and toxic tort cases. As you know, after Markman v. Westview Instruments Inc., a patent case can turn almost entirely on your conclusions about the meaning of scientific terms in the patent claim. Because patent litigation increasingly involves questions of microbiology, the first three programs in the series will be devoted to an examination of the fundamentals of microbiology in the context of a patent case. The first two programs will be pure science lectures. In the third program, a panel of judges and attorneys will examine the Markman and related issues presented by microbiology evidence in the patent case. The series then turns to a consideration of scientific evidence in the arena of toxic tort litigation. There, as you're well aware, the parties often seek to present evidence from toxicologists and epidemiologists on the issue of causation. And Daubert and Joyner and Tkumo Tyre require you to make rulings on this evidence that may determine the outcome of the case. As such, the fourth and fifth programs of this series will address the basic principles of toxicology and epidemiology. The sixth will be a discussion by a different panel of judges and attorneys focusing on methods of evaluating toxicology and epidemiology evidence for purposes of resolving Daubert and other issues in toxic tort cases. The goal, of course, is not to turn you into scientists. Still, as you encounter terms like recombinant DNA, antisense, or nucleotides, and phrases like no observable effect level and cohort study, you may find yourself thinking you are on a longer and more arduous journey into the realm of scientific inquiry than you'd like to take. I can only respond that I think it is a journey you must take. Indeed, that we all must take to be effective judges in this day and age. Why? Because it will increase our knowledge about the science we can expect to encounter in our cases. And as such, increase our chances of achieving justice in them. And so, in the spirit of scientific inquiry and in the pursuit of justice, let us begin. Science in the Court Room, Program 1, Core Concepts of Microbiology. This lecture is presented by Edward S. Makarski, Jr., Professor of Microbiology and Immunology at Stanford University School of Medicine. Professor Makarski received his doctorate in microbiology from the University of Iowa and conducted postdoctoral studies in microbiology at the University of Chicago. So among the key scientific concepts to have actually come into the courtroom over the last 10 or 15 years are from the field of biology. Today, I'm going to try and cover several key concepts in basic microbiology, the biology of microorganisms, as well as molecular biology, molecular biology having grown out of an understanding of the basic molecules of life. The key concepts we're going to try and cover are shown on this first slide. The genes that are common to all organisms carry the blueprint for each organism. So genes are the essence of the information that encodes life. All genes are made up of a molecule called DNA, deoxyribonucleic acid. DNA is a template, and DNA is used in the course of gene expression. That is to say, the expression of the genes into proteins that carry out many functions in life. The gene, the intermediate in expression of those genes is RNA. So RNA is sort of a messenger service. Copies DNA and carries it out to encode proteins. Proteins carry out most of the work of life. They're the enzymes. They're the things you can see, skin, eyes, most of the structure of organisms. So the molecular dogma we're going to cover today is that information flow and something about the organisms in which that information flow operates. DNA to RNA to protein. That's called the molecular dogma. It's been coined about 40 years ago now. And DNA is a universal code. So all life is DNA-based. All life has the same basic code and carries out the same basic process. And that's going to be important as we get through this lecture because it's what scientists have used to begin to understand genomics, to understand how to manipulate and do gene therapy, genetic engineering cloning. If I go now to the next slide, this is a cartoon taken from one of my daughter's books. It's actually a book from the Colchping Harbor Lab, a famous lab on Long Island. It's done a lot of molecular biology research, but it basically shows us a cartoon of some plants and some insects and basically says, if I think it's the beetle saying, I suppose you realize that if my DNA had been in a different order, I could have been a brain surgeon. And in fact, that's true. DNA is a very simple molecule. We'll get into how it's composed. And in fact, the order of the building blocks of DNA really determine everything about an organism. So for a human, it's the genes that dictate and determine what is human. Genomes from different organisms are very, very greatly with regard to size and with regard to the sequence of the building blocks. And so here we've got on the first line, the genome of bacteria. Bacteria are microorganisms. They're single cell microorganisms. A genome of a bacteria is a single DNA molecule, which we'd call a chromosome. It's a collection of genes or genomes, chromosome. And it's 4 million building blocks. The building blocks, we're going to name bases in a little while, but the building blocks are 4 million of them arranged in a particular order. And they encode some 4,000 genes. So you need 4,000 different genes or coding sequences to make a bacterium, a single cell organism. Bacteria are prokaths, one type of life. The kingdom of life is broken into prokaths and eukaryotes pretty much. So another example would be a eukaryote. Humans, a higher order organism. Human genome is 23 different molecules, pairs of chromosomes. We have a lot of other characteristics, sexual reproduction. But 23 pairs of chromosomes totaling some 3 billion. So nearly 1,000 times more complex than bacteria. And the estimate right now for the human genome is that there are some 120,000 genes. So humans or eukaryotes, they're more complex. They have larger genomes. They have more genes. Bacteria are microorganisms. They're simple. They have a smaller number of genes. All of those genomes, either organism that I've just used an example or any other living thing on Earth, has a genome made up of DNA. So now if we go on, we just talk about not just the genome, but the cells that make up different organisms. Because that's also an order of complexity here that we should all understand. On the next slide, it shows that bacteria, as I've already said, are microscopic. They're microorganisms, single cells. And down there at the bottom in the panel, an arrow is pointing at a little speck. That is a bacteria, not magnified very much there, because it's shown next to a human cell. Human cells are larger than bacterial cells. But indeed, humans are made up of lots of different cells. Some 75 trillion cells. So we're each made up of lots of cells. And each of our cells is larger than a bacterial cell. But indeed, the basic makeup of each is a common set of molecules and codes. So the movie now, which is down there in that panel, shows the bacterium being engulfed by this human cell. That human cell is actually a blood cell called a leukocyte, a white blood cell. It's a polymorphonuclear cell, polymorph. And its job in life is actually to engulf bacteria. So when you have a pussie wound or when you have a situation where you've got an infection, your immune cells, the white blood cells come in and take care of those bacteria. It's shown here just for size comparison. So now, if we go back to DNA and go to the next slide, we're going to look at the makeup, the structure of DNA over the next series of slides. I said that DNA is made up of relatively simple building blocks. And those simple building blocks, we're going to call G, A, T, and C. They're abbreviations for the bases that make up DNA. But they serve the purpose here. So the order of G, A, T, and C really determines what a particular DNA molecule is. It's what a particular organism's DNA sequence is. And it plays a central role in determining what organism is what. So the order of these four nucleotides in a bacterium genome, bacterial genome, is different than the order of these bases in a human. Even though there's 4 million of them in a bacterium and 3 billion of them in humans, the sequences are very different as well. So the numbers and the sequences. And that's really a very important part of how life is defined. So DNA uses these four building blocks. And as I show them, they're paired. G colon C, A colon T, G colon G. DNA is a double helix. And that's what's shown in the cartoon there on the right. The double helix is two ribbons. And the cartoon shows the different bases as little arms shooting out and sort of opposing one another all the way down the ribbon. So DNA is a series of base pairs or nucleotide base pairs, as they're called. And so DNA is a double helix made up of these four bases in a variety of different orders. Next slide. So these four bases, if we really expand it, and this cartoon expands the double helix into the actual chemical components, which aren't so important to us here, but it gives you a feeling for what kinds of chemical bonds are involved, the four bases are made up of a organic molecule that is partly sugar and partly what is called nucleotide base. Linked by various chemical bonds, both covalent and non-covalent, that make up this double helix. So there's a chemical basis of DNA. And there's now a higher role for DNA as a coding sequence, as a sequence that carries the code for what is each organism that has a DNA genome. If we go on now to the next slide, this double helix goes back from a very highly magnified double helix into what looks like a split open egg, which is a depiction of a cell. So as I said, all organisms are made up of cells. All organisms have DNA. This would be a typical human cell, blood cell that's in a cartoon version. The DNA is contained inside the cell, inside a nucleus in that cell. And every cell in the body would have a nucleus, and that nucleus would have the same genome, the same DNA molecules as every other cell. If we now look on the next slide at a very simple organism, a bacterium, bacteria are really very simple cells. They don't have a nucleus, for example, as human cells have a nucleus that contains the DNA. They just have a little separate compartment in their cell called a nucleoid that has their DNA genome. And again, their genome is just one molecule of DNA. Very long, four million bases, but one molecule. Here on the left is a cartoon depiction of a bacterium, which allows me to actually go through the parts of a bacteria. And on the right is an actual photomicograph of a magnified bacterium, which you really can't tell any of the parts. You might be able to tell that it's bounded by sort of a wall on the right. But on the cartoon side, we can look at the bacterium. It's cut open. We can see it's got a kind of fuzzy surface, because it's got these components called pili, which are important in the life of the bacterium. It's got long flagella sticking out at the bottom, which are used to swim around and find nutrient sources. And inside the bacterium, besides the nucleoid, there's ribosomes. And we'll talk about ribosomes. They're involved in the synthesis of proteins, which are the molecules that carry out most of the functions in life. And you can see a cell wall and a cell membrane kind of depicted there. If we go now to the next slide, this is going to be a little movie, which is showing how bacteria move around. And here it comes kind of like the Goodyear blimp. But this is a very small organism. The flagella are off to the right. We're going to zoom in and look inside the cell. And you can see sort of the cell wall and lots of components in there. It's just a cartoon version. This shows how the bacterial genome is dividing. And as the bacterial genome replicates and divides, then the cell divides. And so bacteria go through life with a simple mission, and that's to make more of themselves. They do it by this binary fission, where they duplicate their DNA by a mechanism of DNA replication. And then the cell splits by binary fission. I think we'll go through this one more time, just to highlight the aspects of what's going on. So again, a bacterium will be growing, moving around. It has inside the cell many components to carry out the functions of life. And one of the most important things is for that bacterial genome to replicate and make a second copy of itself. And for the cell to divide and make more copies of bacteria, because that's the only thing that bacteria exist for is to make more of themselves. That's their desire in life. So now if we go on to the next slide, this is a cartoon depiction of DNA replication. That last movie didn't really show as clearly as this cartoon shows. Bacterial genome is really just one very, very large circle of nucleic acid. It replicates by copying itself into two circles, and then the cell divides. This is a really simple cartoon version of what we saw in the movie just previously. And the two daughter cells then go off merrily and have their own lives. So each bacterial genome is a circle of some 4 million base pairs of DNA. DNA replication is one of the key aspects of life. You have to be able to make more DNA from existing DNA for life to propagate itself and have more of anything. Bacteria just want to make more of themselves. Many people want to have kids and things like that. So it's a very popular sport here. DNA replication is the core to this process. DNA replication is an inherent quality of DNA. As I've already said, it's a double helix. It's a complementary copy of itself in those two helices that are formed from these base pairs. And so as the DNA helix is copied into additional strands, and you can see here a replication fork where there's two progeny coming off from the original strands up above, you end up with two exact copies of what was the parental molecule. So every time a cell divides, the entire genome is copied, true of all organisms. Base pairing, the A to T, G to C relationship is retained. And it's the key to making exact copies of the DNA each time it's made. The product is the same as the starting material, so an organism like a bacterium remains the same bacterium every time it divides. And the machinery for DNA replication, which isn't shown here, this is just a replication fork, there'd be machinery in there actually doing the work, would be made up of proteins such as DNA polymerase, which is an enzyme that polymerizes or copies and makes this DNA molecule. So we go to this slide. This is some practical utility of DNA replication. So genome replication provides more copies of chromosomes. But in using a DNA template and bases and DNA polymerase, that DNA template can be copied into additional exact copies. And one of the practical uses has been the polymerase chain reaction, something we refer to as PCR. You'll see that in the newspaper as well as in scientific literature. PCR is a way to make many copies of a small amount of DNA. And it's been very important. And for example, forensics or in paternity, where if you can get a little bit of a blood sample from an individual, you can collect the DNA, a small amount PCR up regions that vary from individual to individual. So each organism is defined by a genome. But each individual has slightly different individual features, and those are also based in slightly different individual genes. And those differences can be used to actually identify the sequence of those DNA molecules can be used to identify individuals. And PCR is a way to make a lot of DNA available to, for example, forensics to figure out what individual that DNA came from. So that's become a very, very important part of, I think, molecular biology in the courtroom. It's considered a very important way to identify an individual unambiguously. Go to the next slide. Well, genome replication is only part of the process of life. Gene expression is the second. So the DNA is a template to make more of itself. We call that replication, DNA replication. The DNA is also a template to make individual gene products. Those gene products are proteins, by and large. We'll call them proteins. And proteins like DNA polymerase carry out a lot of the machinery that carry out a lot of the functions of life. And we're going to talk about this information flow now of DNA making RNA intermediate making proteins, each of them acting as a template. So RNA transcription produces an intermediate messenger RNA from a DNA template. That's the first step in gene regulation. And then the second is protein translation. That is, say, the reading of that RNA intermediate into protein produces a final product. And that final product is used to constitute most of the structures and functions that we call life, the metabolic activity of a cell. Both RNA and DNA are made up of bases. And proteins are made up of amino acids. We're going to come back to talk about that in some detail. So here's the dogma in a single slide. The dogma of DNA makes more DNA. That's replication. DNA also is a template for RNA transcription, the first step in gene expression, and translation the production of proteins off of that RNA intermediate. DNA, again, on the top here is shown as a double helix, a very small double helix. RNA is shown as a red kind of hairy looking molecule coming off of a DNA template in the middle of that panel. That RNA is an exact copy of the DNA from which it was made. And it's used, then, it is red, I should say, by a ribosome, which translates that red messenger RNA into proteins. That's the central dogma. That's what all life does. That's an important concept, I think, to understand much of the molecular biology and genomics and gene cloning that's gone on for the past couple of decades. If we go on now to the next slide. So transcription makes an exact copy. I show here on the top a DNA double helix, here laid on its side instead of standing straight up, but basically the same double helix. It's got these base pairs, G paired to C, A paired to T, and you can make various combinations on either strand. So it's made up of base pairs. It's a double helix. RNA is a single strand. It is an exact copy of one of the two DNA strands. And usually a gene is one of the two DNA strands being copied into RNA. But it's an exact copy. And it's made up of four bases. Just like DNA is made up of four bases, they're chemically slightly different. And we give them the letters G, A, C, and U, whereas it's G, A, C, and T in the case of DNA. So very much similar. The next slide, then, is a movie depiction of this first step in gene expression, which is transcription. You see the RNA polymerase moves in in binds. And if we can stop this slide for a moment, the movie, you can see the RNA polymerase has recognized a place on this double helix, which is drawn in a cartoon version underneath that big blob that moved in, the RNA polymerase. RNA polymerase is a big protein. It's composed of many different proteins, but it's a big thing that can synthesize RNA off a DNA template. It comes down and recognizes a site on the DNA, which we call promoter, because it's the place where the RNA polymerase starts to make RNA. It's got to have a starting point. And it's important that it pick the right starting point because each gene is in a different location on the genome. So if humans have 120,000 genes in three billion base pairs, the RNA polymerase has to know where to find those genes and start transcribing not just anywhere. So it finds a promoter. Now if we go on with the movie, we'll see that the RNA polymerase is now where we focused in, magnified, opens up the DNA double helix and starts making an RNA, which is a red line here, off of one template, one strand as a template. And you see that RNA polymerase moving along and leaving a red single-stranded molecule beyond. Now we've focused in even closer, and you're going to see the individual bases come in and those little energetic connections are chemical bonds that are formed to put the bases into a polymer form, which is the RNA. And each of them is dictated by the sequence on the DNA. They're each a base pair, and they're each specifically encoded. So the sequence of bases on a bacterial genome is going to encode a gene at the bacterial gene based on the RNA, based on the DNA making a particular RNA, and then bacterial proteins, whereas human genes have a different order of those four bases, make a different set of, and make human proteins. Now if we go, well, we can summarize that by going through this diagram here. Here the RNA polymerase is, again, shown as a blob, and it recognizes the DNA template as a first step. That's the promoter. It comes in and recognizes a particular site on the DNA. It then opens up those strands of DNA and starts a process of copying the DNA into an RNA molecule, just by incorporating those individual bases, as we just saw in the movie. The RNA is made as a copy, and it goes anywhere from a few hundred bases to thousands of bases. Genes vary in size from a very small few hundred bases to thousands of bases. In any case, the RNA has a defined size, and it's basically the gene intermediate. For the intermediate for gene expression, it carries a particular set of information. So we now go on and ask, how is that information used? We're going to go to the next slide, which is a cartoon, again from this Cold Spring Harbor series, called DNA Is Here to Stay. And it's a cartoon that, I think, captures the next step, which is really the most complicated step of all the life processes, the trying to decode this four base intermediate, this RNA, that's made up of four different bases, into a very complex set of proteins that are made up of a lot of different amino acid building blocks. So let's go through the cartoon first. The copy strand, as it says in the upper left panel, is now complete and leaves the chromosome. So the RNA says to it, the chromosome, I'm off. I'm out of here. Now to look for a ribosome. The ribosome is the machinery for translation for making protein. Here, it's down on the bottom left panel. The copy strand finds a ribosome, which isn't too hard, because there's thousands in each cell. Each cell in any organism has a lot of protein translation machinery, something that cells do all the time and do lots of. And so the messenger RNA says aha, and the ribosome says hi. Then the ribosome binds to this RNA and starts to read the bases. And so the copy strand sticks to the ribosome, which reads the A's, C's, G's, and U's. And now the ribosome knows the order in which to join up amino acids. It's sort of like reading a sentence and knowing what the sentence means. The ribosome can read those four bases and make a complex protein out of it. We're going to talk about how that happens. But in any case, the bottom right panel says, and all this happens millions of times a minute every day inside cells of your body and every organism on Earth. And the amino acids are going off. We're seeing in chorus. I'm sure we're amino acids and we make protein. So now if we go on to the maybe less colorful but factual process here, this movie will cover the translation step. But first up at the top, you see the RNA polymerase that's making mRNA, messenger RNA. And you see the ribosomes coming in and grasping that messenger RNA. And off the bottom there comes a protein. We're going to zoom in on this. And let's stop right here for a minute if we can. Because we just zoomed in and we've got the messenger RNA on the left. And you can see the series of bases. It's up vertical at this point. And you've got these squiggly things coming in and recognizing essentially three bases at a time. Those squiggly things are transfer RNAs. They're a particular kind of RNA, different than messenger RNA, but also RNA, that at one end read the RNA by having these three base recognition sites. And at the other side have individual amino acids that make up protein. And so the circles on the right side of this frame in the movie show different amino acids being brought in. So indeed, the RNA is read kind of like a sentence in three-letter words that tell the ribosome what amino acid to put into that protein. And there's some 20 different amino acids. So there's a very large variety of order to those amino acids to make up protein. So we can get the enormous diversity of life out of a very simple four base template on messenger RNA. If we continue the movie, and we'll go through this movie twice, but let's just finish it, you see the transfer RNA comes in. You get a new amino acid added, S-E-R just got it added. And then one more comes in, ALA gets added, just reading along the messenger RNA. So if we go through again, again, we see the RNA polymerase reading that DNA into RNA coming off the bottom of the RNA polymerase. Ribosomes come in and recognize that as messenger RNA, bind to it, and start to synthesize proteins. If we go close up and look at what's happening there, the RNA is being, the ribosome is orchestrating the reading of the messenger RNA by transfer RNAs that bring particular amino acids in in an order that dictates what is the final gene product that's made, the protein that's made. And so you get the enormous diversity of 4,000 different proteins in E. coli or in any bacterium, and the 120,000 different proteins that can be made by the human genome from this very simple process. If we go to the next slide, we'll talk a little more detail about translation. So just to summarize, RNA polymerase produces this messenger RNA intermediate. Ribosomes decode words of three bases into, which are called codons, into each individual amino acid. There are 20 different amino acids that make up all the proteins in nature. Each has their own set of codons so that each amino acid is matched up with a three base word. And proteins are made up of strings of amino acids. Proteins are basically linear arrays of amino acids that fold up into different forms and take on different functions. But they're determined essentially by the sequence of that messenger RNA intermediate. So if we go now and look without a movie, but in static form, what happened when a ribosome moved on the messenger RNA? Here the messenger RNA is a blue line. And the ribosome is a glob sitting on there with two tRNAs in position. And the ribosome is moving from left to right on this messenger RNA. And you can see that there are four codons that have bars over them that have already incorporated, served as template to incorporate amino acids. And the arrows point to the amino acids on that growing protein chain that was encoded as that ribosome moved along. So that's the entire nature of the ribosome reading messenger RNA and being able to make a very different molecule of protein made up of amino acids from that template. So now if we go to the fact that replication, transcription, and translation are all universal processes, the same 20 amino acids are used throughout nature, whether it's a bacteria, a plant, or an animal. The proteins that are made, and the size is quite variable from a few dozen amino acids, very important neuropeptides or growth hormones or very small molecules, sometimes only 12 or 20 amino acids, but proteins range up in size to thousands of amino acids. The RNA polymerase, for example, that we saw making messenger RNA, that's a very, very large protein made up of thousands and thousands of amino acids. The possible arrangement of the 20 amino acids is one of the secrets of life. Not every possible arrangement is used in nature, but there are sufficient variation, there are very sufficient possibilities that the existence of all the different proteins that make up all the different organisms is assured. Bacteria again, those 4,000 genes encode 4,000 proteins. And in human beings, those 120,000 genes encode 120,000 different proteins. And that's the same as the gene number that we started off with in this series of slides for those two respective types of organism. So finally, if we go to the conclusions from this section of the lecture, we've talked about the fact that DNA is the basis of genes and that every organism on Earth has a genome made up of DNA. That's the universal code of life. RNA is transcribed from DNA to allow an intermediate messenger RNA that is used to produce proteins or gene products. DNA and RNA are made up of four bases, essentially. And they're strands of those bases, polymers of those bases. Proteins are translated from RNA and are made up of amino acids. And they're strings of amino acids. The three base codons on RNA encode each amino acid that tells the protein, tells this machinery what protein to make. And proteins end up carrying out most of the work of life and also are the structure that you see. You don't see DNA when you look at hair as a protein. Skin as a protein, by and large. Molecular dogma, then, is DNA flows to RNA flows to protein. And the universal code, all life is DNA-based. We've talked about three processes of molecular biology. Replication of DNA, transcription of RNA, and translation of proteins. Replication of DNA is the copying of a DNA template using bases, four bases, to make more DNA. It's a process that's necessary for all life forms to produce more of themselves. Transcription is a process that uses four bases as well to make a messenger RNA intermediate. That messenger RNA is used itself as a template to encode proteins. That process is called translation. Translation is done by ribosomes on messenger RNA and uses amino acids as the building blocks. All told, this process is the molecular dogma or the molecular biology of life. So those are the core concepts that we need to understand to understand biology. Microbiology and molecular biology forms a core of understanding that's been implied in science to both recombinant DNA, gene therapy, and much of genomics. Next time we'll talk about the concepts of recombinant DNA. I want to thank you for your attention.