Transplantation Immunology Pg. 2

Pathology Transcriber: Luke Powell 09/12/2008 (Length of lecture) Bacterial Genetics – Dr. Steyn

1. Today I’m going to give you a brief introduction to bacterial genetics. The important thing here is to understand certain concepts, and not just to memorize stuff.

2. This is the course goal: to provide you with the basic principles of microbiology and to relate the importance of these principles to the actual host/microbial interrelationships that you may encounter in your career. Emphasis will be placed on understanding the mechanisms involved in mediating oral and ocular health and disease. You need to have a broad, general concept here.

3. We’re going to talk about DNA, and I think everybody knows a little bit about what that is. Replication; why does DNA need to be replicated? Transcriptional control; we will talk a little bit about genes, and operons, and the mode of regulation. Then we’ll talk a little bit about mutations and the types of mutations that can be generated. We’ll talk about gene exchange and transfer, both very important. Lastly, we’re going to have a general discussion about technology and genetic engineering. What can be done today?

What does he do? Studies the mechanisms of pathogenesis of m. tuberculosis.

4. Let’s start. This is a diagram of a typical bacterial cell. You have the cell here and the outer membrane. Maybe an inner membrane, and periplasmic space, with peptidoglycan here which is part of the cell wall. Here you have the cytoplasm, and importantly, here you have the chromosomal DNA. Many bacteria not only have chromosomal DNA but they also have plasmid DNA, which is much smaller, circular, and is often found with many copies. These plasmids may contain antibiotic resistance markers. This (the diagram) is an example of the whole genome of m. tuberculosis. Now they have fancy technologies that allow sequencing of an entire genome within a couple of hours.

Most bacteria have one circular DNA chromosome, ranging from 1000-8000 kb. Some are small, some are large, but that’s not really an indication of anything about complexity etc. What does bacterial genome mean? It refers to the collection of all the genes present on the bacterial chromosome or extra-chromosomal elements such as the plasmids. He repeats that plasmids are extra-chromosomal.

We know that the genome contains operons. Operons are made up of genes. Bacteria have the unique characteristic that multiple adjacent genes can be co-regulated (one promoter, or switch, which controls many genes). In eukaryotes, it’s usually one gene for one promoter. But in bacteria you can have one promoter for many genes. The multiple genes controlled by one promoter are collectively called the operon.

Promoters and operators control these genes or operons. The promoter is basically the light switch. Whatever is controlling the gene is generally called the promoter. Operators are DNA elements upstream of the genes and form parts of the promoter element. So other proteins come and bind to operators in order to control the expression of operons or single genes. Transplantation Immunology pg. 2 Laura Rayne 5. Nucleic acid: where is it? Here I have an example of a number of bacteria and also viruses. So all living material contains nucleic acid, whether it’s DNA or RNA or single strand or double strand, they all contain nucleic acid. For example, textbooks would have pictures like this.

We can isolate very rapidly either the chromosomal DNA or the plasmid DNA and purify it to a very pure form. You can analyze this via agarose gel electrophoresis (think CSI). You can load the DNA on an agarose gel and separate it based on size. The white bands are the DNA. A fluorescent dye is incorporated in the gels that lights up when subjected to UV light.

Nucleic acid is present in all living organisms. We can isolate, and even manipulate, DNA.

The molecular weight ladder on the right gives the molecular weight of the unknown plasmid DNA in the picture.

6. This is a typical DNA structure; I think all of us are familiar with this. You have two helices which are held together by hydrogen bonds. The bonds come from adenine, guanine, cytosine, and thymine. A and G are purines. C and T are pyrimidines.

The important thing to keep in mind is that the GC content ratio varies between bacterial pathogens. For example, an AT rich organism will have many ATs. Other’s are rich in GC, like streptomycin or tuberculosis. This is important because the amount of GC or AT dictates the ability of the DNA to be separated.

If you heat DNA, the two strands start separating. This is called denaturing. If you have many GCs, it’s more difficult to denature because of the three hydrogen bonds. In the case of AT, it’s easier to denature because of only two hydrogen bonds.

7. So here this is illustrated. GC you have three hydrogen bonds (harder to separate), and with AT you have two hydrogen bonds (easier to separate). What this means in terms of pathogenicity, we don’t know. Can’t say that GC pathogens are more harmful than AT pathogens.

8. DNA Replication. If the bacteria has to divide, the chromosomal DNA also has to divide. How does that take place? It can be complicated, but for today we will simplify it quite a bit. Here you have a typical bacterial cell; the cell elongates and the DNA must be duplicated.

The next step is that the cell wall and plasma membrane begins to invaginate and a septal crosswall forms, separating the cells. A large complex of proteins is involved here.

The proteins involved in replication are typically good drug targets. A lot of drug companies focus on these proteins to design drugs to inhibit them. If you can inhibit bacterial DNA replication, you stop all growth, and so control virulence.

What environmental signals are needed to tell the bacteria to replicate? Many bacteria enter a persistent, or dormant state. Bacillus forms spores. Tuberculosis enters a dormant state. When Transplantation Immunology pg. 3 Laura Rayne bacteria start forming spores, they are resistant against drugs because their metabolism is shut down. That’s why the replication step is important for drug design.

Replication of chromosomal DNA is initiated at a very specific site. Some organisms can start at different sites, but bacteria only start at one site, called the oriC. It requires many enzymes. Large amounts of protein complexes are required to divide. These proteins must recognize the oriC, separate the strands, and start replicating the DNA.

Two things to remember: new DNA is synthesized semi-conservatively, which means that, in the daughter cells, one strand will be original and the other strand will be newly synthesized. Another important point is that DNA synthesis proceeds bi-directionally. When you have an origin of replication (oriC), the DNA helices are melted away, allowing replication to occur in both directions.

9. When the DNA separate, you get single strand DNA. These single strands act as templates for the second strand to be made. The double helices open, and then small DNA single strand elements attach themselves to complementary strands. These small fragments are called Okasaki fragments, and they function as primers for synthesis.

Again, remember bi-directional and semi-conservative.

At the end, you get two new chromosomes. In each of these chromosomes, one strand is from the mother cell and you also have a newly synthesized strand.

10. This is one way of duplicating DNA by the bacterium itself. I’m going to take a step back and say that the cell is very efficient in replicating DNA, but we can do it even faster. We can amplify DNA within fifteen minutes. You can take a small amount of DNA and amplify it a million million fold in a very short period of time. Most labs do this today.

This is a technique called the polymerase chain reaction (PCR). Here we have double stranded DNA which we’ve extracted (from hair, bones bacteria, etc.). We denature the DNA in a small tube by simply heating it up to 95 degrees. At this temp the two strands separate and become single strand DNA.

Now, beforehand, you’ve already decided to amplify a specific gene. You know the identity of the gene. What you’ve done is synthesized small oligonucleotides (small pieces of DNA) that are in the specific region of your gene. You send the info to a company and two days later you get DNA back in a little tube. You add these single strand primers to the reaction. These primers are complementary to the gene that you would like to amplify.

Once your DNA has been denatured, you cool it down, and very quickly the single strand oligonucleotides anneal to the complementary single strand DNA.

Also in the reaction is an enzyme called Taq polymerase. It recognizes the annealing of the oligonucleotide to the DNA, and it starts polymerizing the complementary strand at that point. It does this at 72 degrees. Transplantation Immunology pg. 4 Laura Rayne You end up with the original DNA strand with a complementary synthesized strand attached to it. So you’ve doubled your DNA. Do this for thirty cycles, and you have a lot of DNA.

It’s a cycle of denature and high temp, anneal and polymerize at low temp, back up to high temp, then low temp, etc. Gives you 1 -> 2 -> 4 -> 8 -> 16 -> … DNA in a very short period of time.

11. Let’s talk about the transcription control. How are genes regulated? We’ll talk about basic prokaryotic gene organization, the lac operon, induction of the lac operon, control of the lac operon, and two-component systems in bacteria.

12. The central dogma here is that you had DNA which is being transcribed to mRNA. The mRNA is translated to protein via ribosomes.

13. Let’s look at the basic prokaryotic gene organization. This is your DNA. Here you see four genes; A, B, C, and D. This is an example were ABCD is in an operon. These genes can be spaced a couple of bases apart, or they could overlap. But they constitute an operon. Here you have a promoter region, which is like a light switch. And you also have a terminator, which is a vaguely defined sequence which is basically a stop sequence. When transcription occurs here, mRNA knows when to stop based on the terminator.

Here we have a +1. The +1 is the transcriptional start site (TSS). In other words, at this +1 position, the synthesis of mRNA starts, not the synthesis of a protein, but of mRNA. If you look at the start of gene A, this is where translation starts. So translation starts at one position, and transcription at another. Translation can only start once you have mRNA. I want to make it clear that the transcript might be longer than all these genes, and based on the profile, you would have translation that might start within the transcript somewhere.

The promoter is where the action is. It determines when genes will be switched on or off.

In our mouth, there’s 600 species of bacteria (not all can be cultured). There the bacteria are exposed to a wide range of environmental stresses such as saliva, food drinks, etc. This causes stress on the bacteria. The bacteria adapt to that stress in a very precise way, whether you increase the salt, change the temperature, or whatever, the bacteria will adapt. The conditions must be sensed by the bacteria and then relayed back to promoter regions on the DNA. The cell is told whether or not to turn certain promoters on and off based on environmental factors.

So the promoter regions of important genes are always the primary focus of research.

Here you would have an operator sequence and a -10 and -35 region (all in the promoter). This promoter region is the area to which RNA polymerase binds. RNA polymerase is a big protein complex that consists of multiple subunits, and it also contains a wide range of regulatory proteins (transcription factors). Those transcription factors are usually being produced in response to changing environmental signals. Transplantation Immunology pg. 5 Laura Rayne In a particular environment, the bacteria will sense this, and relay the signal to a regulatory protein which affects the RNA polymerase, telling it which genes to switch on. RNA polymerase is just a massive collection of proteins.

14. Let’s zoom in a little bit more on the promoter properties. Again, you have the transcriptional start site here where RNA polymerase binds. Here the sequence of the DNA is recognized by the RNA polymerase. One mRNA is being synthesized, proteins must be translated from the transcript. There you have the translational start site (ATG). Again, you have a -10 and -35 region that are the contact sites of RNA polymerase. -10 and -35 generally have consensus sequences (TATAAT for -10, TTGACA for -35). You have a spacer of about 17 bases, another one 5-9 spaces, and then you have an A or G sequence ( see picture).

In some cases, regulatory proteins could prevent the RNA polymerase from binding to the -10 or -35 region, or you could get mutations in those regions that might prevent binding of RNA polymerase.

15. This is just a very basic overview of transcription. The four steps of transcription. Again you have double stranded DNA and you have the RNA polymerase complex. It recognizes the promoter regions upstream. And you have a gene, and then a terminator sequence.

So the first step is initiation, in which RNA polymerase binds the promoter and begins unwinding the DNA helix.

The second step is elongation, in which RNA polymerase moves along the DNA in a 5’ to 3’ direction.

Third step is continued elongation. The unwound DNA moves along with RNA polymerase. As it moves along, the DNA unwinds, and there are proteins responsible for this unwinding.

The fourth step is termination and release of the mRNA and RNA polymerase at the terminator sequence. For many bacteria, there is a more or less conserved sequence for termination, but it’s not really known.

So this is how you transcribe DNA to for a transcript. Now the mRNA is recognized by translational machinery. Actually, translation occurs before transcription is complete. That is an error in the slide.

16. Let’s look at a very classic example: the lac operon, which has been found to have negative regulation. Here you have DNA with three genes, Z, Y and A which are three different genes that code for three proteins, or enzymes (they are B-galactosidase, Permease, and Transacetylase). Here is the promoter sequence that is recognized by RNA polymerase. But upstream of the operon, you have a regulatory gene, gene I, which is a repressor.

This is an example of a repressor, gene I. When it is transcribed, it gives an active repressor that recognizes the operator sequence. It binds to the operator and then sterically inhibits, or prevents, or even competes with RNA polymerase at that particular site. In other words, the repressor blocks Transplantation Immunology pg. 6 Laura Rayne RNA polymerase from performing its function. You get no lac mRNA, and no protein. This is negative regulation.

Again the repressor protein is produced and it binds to the operator sequence, preventing RNA polymerase from interacting with the promoter. Transcription is blocked.

17. However, you can induce the lac operon as well. This is a classic example developed in the 60s. Now this technique is the most widely used genetic selection system in the field of genetic engineering. For example, if you want to induce the lac operon, you can add lactose, which is an artificial inducer. Lactose binds to the active repressor, preventing it from binding to the operator sequence. So there is no inhibition of the RNA polymerase binding to the operator sequence. The result is production of protein.

When lactose is present, the lac genes can be produced because there is no inhibition.

Again, he takes it back to the varying conditions bacterial pathogens encounter in the body. Bacteria can experience things such as low salt, high salt, temperature, oxygen, nitric oxide, free radicals, osmotic pressure, pH. These are all important signals that either allow or prevent bacteria from causing disease. The dangerous pathogens are capable of responding to these environmental signals, circumventing the host immune response.

A fundamentally important question in bacterial pathogenesis is to figure out what are the environmental signals, and how are they being sensed by the bacteria, and how does the bacteria respond to those signals. Again, if you know the mechanisms of action of those events, and you know the genes involved, you can design effective drugs and vaccines.

For example, RNA polymerase is a good target for drugs. Unfortunately, pathogens sometimes have human homologues. If you have a drug against a bacterial protein, but the protein has a strong human homologue, you might kill the bacteria and the human as well. That’s not a good drug.

So, all these proteins (the ones he’s been talking about) are potential targets for drugs.

18. I’m not going to go into much depth here. All I’m going to say is that in terms of positive control of the lac operon, you have another protein, CAP (catabolite gene activator protein). CAP is another transcription factor that associates with RNA polymerase to provide another level of regulation. This protein responds to the glucose level inside the cell. When the cell experiences of low or high glucose, it is capable of modulating the lac operon.

So besides the lac I repressor, you have CAP, and really a whole bunch of other factors to.

19. In sum here, you guys can read through this, what is negative control, and what is positive control.

20. Now, probably one of the most important classes of proteins in bacteria is the so called two- component protein class. The word makes it easy: two-component. Usually, it’s two important proteins that are in an operon next to each other. Transplantation Immunology pg. 7 Laura Rayne One of the proteins functions as a sensor, which senses a specific environmental signal. That information is then relayed to the second protein, which is a regulatory protein that associates with RNA polymerase to specifically induce a subset of genes in response to the environmental signal.

For example, a two-component system is a regulatory system that ties the expression of specific genes to chemical signals in the environment. Here you have the KdpD and KdpE system, which very specifically respond to changes in osmotic stress in bacteria. These proteins are universally conserved. Most bacteria have this two-component system.

Here you have one protein KdpD, which is in the membrane. The sensor protein in two-component systems is always membrane associated. The sensor contains a specific domain which senses the environmental signal. We only know a few signals that specifically are being sensed by these proteins. So it has a sensing domain that’s responsible for sensing the environmental signal.

One the signal is sensed, the information causes a structural change in the protein, allowing it to become phosphorylated. One the membrane protein is phosphorylated, it transfers the phosphate to a second protein. In this case, it’s KdpE, the regulatory protein.

Once KdpE is phosphorylated, it can bind RNA polyermase as a regulatory protein.

So it’s very simple. The membrane sensor protein is a histadine kinase which phosphorylates itself at the histadine residue in response to a signal. The moment it does this, it transfers the phosphate to the second protein (KdpE, the “response regulator”), which goes on to act as a regulatory protein as it binds RNA polymerase.

This is classic system to respond to pH, osmotic pressure, and temperature, and varying magnesium levels.

21. We’re going to talk about mutation, repair and recombination. We can quickly talk about single point mutations, DNA repair mechanisms, general excision repair, and silent mutations. This is elementary.

22. You guys can read through this slide. What is a mutation, what is a single point mutation, etc.

23. Let’s move over to single point mutations. Transition is an example where a purine is replaced with a purine (A with G or G with A), or where a pyrimidine is replaced with a pyrimidine (T with C or C with T)

Tranversion mean purine replaced with pyrimidine.

24. Exposure to UV light is always very important. It’s not good for you. If you take bacteria and expose them to UV light, you can very quickly kill them. This is because the UV light attacks the DNA and forms thymine dimers. Some bacteria is very resistant to UV light because they have protective proteins that coat the DNA protecting it from UV light.

The tymine dimers cannot act as templates for DNA replication. You have a problem. Transplantation Immunology pg. 8 Laura Rayne 25. DNA repair mechanisms: this is very easy. You have direct DNA repair with enzymatic removal. You have DNA bases which have been modified, and the DNA enzyme comes and repairs it. You have excision repair, post-replication repair, SOS repair, and error prone repair.

SOS repair is very interesting. For example, when you expose bacteria to UV light, there is a massive induction of a large set of genes in response to UV light, as if the bacterial cell knows that there are big DNA problems that need to be fixed. Lots of proteins are induced which run along the DNA to try and fix errors caused by the UV light.

You need to be familiar with this terminology, so that you can read papers and textbooks and know what they are talking about.

26. General excision repair. Here you have a mutated base. The damaged base is being recognized by an endonuclease, which cleaves DNA in the middle of DNA, not on the sides. Here it’s being nicked, and then the exonuclease can carry out excision of the fragment with the damage base.

So you get nicked by endonuclease, and then the exonuclease chews away the strand. DNA polymerase then fills in the gap. A ligase can ligate the nicks to give double stranded DNA again.

27. Silent mutations. You are always going to get mutations. Every colony has at least one mutation in the bacterial cell. Not all mutations are necessarily bad. If you look at the codon, we know that the AUG or ATG eventually results in a methionine. The next triplet CAG results in a glycine. Depending on the codon bias of the organism, you might be able to swap out the third position in the codon without changing the amino acid. So for example here, you can swap out the G with an A, and this doesn’t change the ability of the organism to generate the same amino acid. That’s why it’s a silent mutation; it doesn’t change anything.

28. Gene exchange in prokaryotic cells. There are three ways in which genes can be exchanged. Nowadays the focus is on studying communities of bacteria, not just single species. We are figuring out that the community of organisms is capable of doing things that individual organisms cannot do. They are dependent on each other.

This means that if you have a lot of bugs mixed up, some bacteria can spontaneously release DNA, and other bacteria can take it up. That DNA can be recombined and incorporated into the other bacterial species. This is an example of transformation.

Transduction is where you have bacteriophages (viruses) which insert into a bacteria and recombine into the bacterial genome. When phage particles are produced, it cleaves the phage DNA back out, but takes bacterial DNA with it. When this new phage infects another bacteria, it can transfer some of the original bacterial DNA to the new bacterial cell.

Third, you have conjugation, where conjugation tubes (pilli) can transfer chromosomal or plasmid DNA from one species to another. Transplantation Immunology pg. 9 Laura Rayne If you think about all these mechanisms, it’s very plausible that all sorts of genes, including antibiotic resistance markers can all be transferred. That’s what is important.

29. For example, transformation. You have a donor cell. The cell is being lysed. The DNA is released, and the DNA then enters the recipient cell to integrate with its DNA. These can be two different bacterial species. If you have antibiotic resistant markers here (on the donor cell), they can be transferred to the recipient cell.

The same is true with transduction. We have a cell filled with phages. The phages contain bits and pieces of the bacterial DNA. When the phages are released, they can transduce that bacterial DNA into a different species.

30. Conjugation is another example in which you can get the transfer of not only chromosomal DNA but also plasmid DNA from one cell to another.

31. When you go back to plasmids, these are extra-chromosomal DNA elements. Usually, they contain antibiotic resistant markers (genes). This is an example of pGEM which we use in the lab to clone genes in.

32. If you think about bacteriophages, this is how they look. The phage recognizes specific cell surface receptors on the bacterial cell. Once it recognizes the cell, it attaches and injects DNA into the bacterial cell. This DNA integrates into the chromosomal DNA. In a nutshell, bacteriophages infect bacteria. They are not cellular, and cannot grow outside the bacteria. Some contain capsids, and tails, and can contain RNA or DNA.

At some point, because the viral DNA integrated into the bacterial DNA, virus particles can be synthesized. Remember that bits and pieces of bacterial DNA get incorporated into the new phages. Once the bacterial cell lyses, and all the phages are released, the phages can infect other cells and transfer some of the original bacterial DNA.

33. In the lytic cycle, you have bacteriophages injecting DNA and it get integrated into the chromosome. The bacteria start replicating. These individual cells, at some point in time, start producing phage particles. At some point, the bacterial cell is so stuffed with phages that the cell lyses, releasing bacteriophages to go start another cycle. They can end up forming a plaque of the lysed bacteria.

In contrast, in lysogenic life cycle, the phage DNA simply keeps on replicating with the host chromosome (no viral particle formation).

34. These are two forms of transduction (he’s not going into this). He reads something to illustrate the importance of genetic exchange: there’s an estimated 10^29 of bacteria in the world’s ocean, and 10^30 viruses in the ocean. It’s been calculated that if only one in 100,000,000 infections brings a fragment of bacterial DNA to a recipient cell, there’s still ten million billion gene transfers occurring per second in the ocean. That is about 10^21 infections per day, which is an incredibly large amount of DNA exchange that’s occurring. It’s about a million cells per mL of seawater. If you swallow sea water, you have a million phages in your body. Transplantation Immunology pg. 10 Laura Rayne 35. Transposons. They are mobile genetic elements that can transfer DNA within a cell from one position to another in a genome, or between different molecules of DNA. For example, you have donor DNA, and it contains a so-called transposon. This transposon is a chunk of DNA that contains three or four genes, including antibiotic resistant markers. So three or four genes are flanked by two small little DNA sequences called inverted repeats.

This chunk of DNA has the ability to jump around randomly through different stages. For example, when it experiences stress, it starts jumping around. You have target DNA, it might recognized a small GC sequence, and boom! The transposon jumps into the target DNA.

The implications are obvious. The transposon could contain antibiotic markers, which could be transferred when DNA are being released and taken up by other species.

36. On the other hand, it all depends on where it’s jumping to. It could jump right in the middle of a gene and inactivate that gene. You can then cause either drug susceptibility or drug resistance. For example, here you have a transposon with two inverted repeats and the donor site. Boom! It inserts itself into gene B, which becomes inactivated. Gene B might be important for drug resistance or susceptibility, but it is now knocked out.

37. OK, this is just the components; you have three genes. B-lactamase is an example of an antibiotic marker that is being transferred by transposons. You have a resolvase which recognizes these two inverted repeats. And then you have the transposase that is participating in the overall excision of the DNA and the insertion of the DNA into a new site. This whole region (Tn3) is a classic example of a transposon.

38. To just come back to chromosomal DNA transfer, or exchange of chromosomal DNA. Here you have a genome of a bacterial species that contains a capsule. This bacterial species can be lysed, releasing naked DNA. The DNA can be taken up by non-encapsulated cells. Now those cells have the DNA that codes for a capsule, and they can produce a capsule.

39. This is a very important slide. This is a good example where DNA transformation and conjugation and transposition give real life examples of antibiotic resistance. So you have this VRE strain here with chromosomal and plasmid DNA. This DNA can be released, and another bacterium can take this DNA up via either transduction or transformation and incorporate it into its own DNA, generated methicillin resistant staph aureus (MRSA).

On the other hand, DNA can also be transferred via conjugation of the plasmid. The plasmid might also contain a transposon. Once the plasmid is being transferred, the transposon can hop into the endogenous plasmid here, which leads to the exchange of DNA via conjugation and transposition to eventually end up not only with a methicillin resistant strain, but a methicillin vancomycin resistant strain (MVRSA).

(Last two paragraphs were a little confusing, but his point is that all of these elements of genetic recombination can work together to make a bacterial cell extremely antibiotic resistant) Transplantation Immunology pg. 11 Laura Rayne You usually get these outbreaks in hospitals.

40. I’m going to quickly tell you about genetic engineering. We can take DNA from bacteria and clone it into certain vectors. We can overexpress genes. You can purify proteins. These can be of therapeutic value when you produce certain cytokines or very important proteins. You can genetically engineer plants in this way, and we have the tools to do this in the lab.

41. Continuation from above.

42. For example, restriction enzymes. These are enzymes that recognize DNA at very specific sites. For example, you have enzymes called EcoR1 or Sma1. With EcoR1, you get sort of “sticky ends.” On the other hand, Sma1 gives sort of “blocked ends.” Using these, we can amplify and clone DNA into whatever vector we would like to.

43. This is an example; you guys can go through this on your own.

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45. Recent advances. There’s over 600 microbial species in the human oral cavity, but only half of them have been successfully colonized. But nowadays, instead of taking two years or five years to sequence a whole genome, they can do it in a couple of hours. You can sequence bacterial species in six hours.

He tells the story of a guy who went out into the ocean and extracted DNA and just sequenced it all. He didn’t even distinguish between different species.

So now you don’t need to colonize individual species, just sequence it all because it’s possible.

454 Life Sciences company can sequence 25 million bases at 99% accuracy in one four hour run.

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