A Brief History of DNA Sequencing

Technical revolutions that got us to modern DNA sequencing.

DNA is an incredibly elegant molecule- that much was apparent as soon as its structure came out. It is structurally tough, and its principles of information storage and replication are easy to understand. It is one instance where evolution came with, not a messy hack, but brilliant simplicity, which remains universal over all the life that we know. While its modeled structure was immediately informative, it didn't help to figure out its most important property- its sequence. Methods to sequence DNA have gone through an interesting evolution of their own. First were rather brutal chemical methods which preferentially cut DNA at certain nucleotides. Combined with the hot new methods of labeling the DNA with radioactive P32, and of separating DNA fragments by size by electically pushing them (electrophoresing) through a jello-like gel, this could give a few base pairs of information.

A set of Maxam-Gilbert reactions, with the DNA labeled with 32P and exposed to X-ray film after being separated by size by electrophoresis through a gel. Smallest are on the bottom, biggest fragments on on the top. Each of the four reactions cleaves at certain bases, as noted at the top. The intepretation of the sequence is on the right. PvuII is a bacterial enzyme that cleaves DNA, and this (palindromic) sequence noted at the bottom is the site where it does so.

Next came the revolution led by Fred Sanger, who harnessed a natural enzyme that polymerizes DNA in order to sequence it. By providing it with a mixture of natural nucleotides and defective ones that terminate the extension process, he could easily develop far bigger assortments of DNAs of various lengths (that is, reads) as well as much higher accuracy of base calling. The chemistry of the Maxam-Gilbert chemical process was quite poor in base discrimination. This polymerase method also eventually used a different isotope to trace the synthesized DNAs, S35, which is less powerful than P32 and gave sharper signals on film, which was how the DNA fragments were visualized after laid out and ordered by size, by electrophoresis.

The Sanger sequencing method. Note the much longer read length, and cleaner reactions, with fully distinct base specificity. dITP was used in place of dGTP to help clarify G/C-rich regions of sequence, which are hard to read due to polymerase pausing and odd behavior in gel electrophoresis. 

There have been many technological improvements and other revolutions since then, though none have won Nobel prizes. One was the use of fluorescent terminating nucleotides in place of radioactive ones. In addition to improving safety in the lab, this obviated the need to generate four different reactions and run them in separate lanes on the electrophoretic gel. Now, everything could be mixed into one reaction, with four different terminating fluorescent nucleotides in different colors. Plus, the mix of synthesized DNA products could now be run through a short bit of gel held in a machine, and a light meter could see them come off the end, in marcing order, all in an automated process. This was a very significant advance in capacity, automatability, and cost savings.

Fluorescent terminating nucleotides facilitate combined reactions and automation.

After that came the silicon chip revolution- the marriage between Silicon Valley and Biotech. Someone discovered that silicon chips made a good substrate to attach DNA, making possible large-scale matrix experiments. For instance, DNA corresponding to each gene from an organism could be placed at individual positions across such a chip, and then experiments run to hybridize those to bulk mRNA expressed from some organ or cell type. The readout would then be fluorescent signals indicating the level of expression of each gene- a huge technical advance in the field. For sequencing, something similar was attempted, laying down all possible 8 or 9-mers across such a chip, hybridizing the sample, thereby trying to figure out all the component sequences of the sample. The sequences were so short, however, that this never worked well. Assembling a complete sequence from such short snippets is nearly impossible.

What worked better was a variation of this method, where the magic of DNA synthesis was once again harnessed, together with the matrix layout. Millions of positions on a chip or other substrate have short DNA primers attached. The target DNA of interest, such as someone's genome, is chopped up and attached to matching primers, then hybridized to this substrate. Now a few amplification steps are done to copy this DNA a bunch of times, all still attached in place to the substrate. Finally, complementary strands are all melted off and the single DNA strands are put through a laborious step-by-step chemical synthesis process, similar to how artifical DNA is made to order, across the whole apparatus, with chemicals successively washed through. No polymerase is used. Each step ends with a fluorescent signal that says what the base that just got added was at that position, and a giant camera or scanner reads the plate after each pass, adding +1 to the sequence of each position. The best chemical systems of this kind can go to 150 or even 300 rounds (i.e. base pairs), which, over millions of different DNA fragments from the same source, is enough to then later re-assemble most DNA sequences, using a lot of computer power. This is currently the leading method of bulk DNA sequencing.

A single DNA molecule being sequenced by detecting its progressive transit through a tiny (i.e. nano) pore, with corresponding electrical readout of which base is being wedged through.

Unfortunately, our DNA has lots of repetitive and junky areas which read sizes of even 300 bases can not do justice to. We have thousands of derelict transposons and retroviruses, for instance, presenting impossible conundrums to programs trying to assemble a complete genome, say, out of ~200 bp pieces. This limitation of mass-sequencing technologies has led to a niche market for long-read DNA sequencing methods, the most interesting of which is nanopore sequencing. It is almost incredible that this works, but it is capable of reading the sequence of a single molecule of single stranded DNA at a rate of 500 bases per second, for reads going to millions of bases. This is done by threading the single strand through a biological (or artifical) pore just big enough to accommodate it, situated in an artifical membrane. With an electrical field set across the membrane, there are subtle fluctuations detectable as each base slips through, which are different for each of the four bases. Such is the sensitivity of modern electronics that this can be picked up reliably enough to read the single thread of DNA going through the pore, making possible hand-held devices that can perform such sequencing at reasonable cost.

All this is predicated on DNA being an extremely tough molecule, able to carry our inheritance over the decades, withstand rough chemical handling, and get stuffed through narrow passages, while keeping its composure. We thought we were done when we sequenced the human genome, but the uses of DNA sequencing keep ramifying, from forensics to diagnostics of every tumor and tissue biopsy, to wastewater surveillance of the pandemic, and on to liquid biopsies that promise to read our health and our future from a drop of blood.

• Is originalism progressive?
• Crypto is insane- a climate disaster, ponzi-ridden, crime-facilitating, sanctions-busting.
• Who lies, and who believes them? It tends to be a consistent demographic.
• The insurrection is not over.
• "The Capitalist system was never designed to satisfy societal needs."
• How do you pronounce "mayonnaise"?
• Big box and big parking lot solar? Obviously a good thing.
• Jane Fonda is onboard.

(No) Sympathy for the Devil

Blaming ourselves for Russia's attack on Ukraine.

Here we are, in a time warp back almost a century. A European country has elected an authoritarian leader, on the support of a doddering president. That leader went on to resolve the economic and politicial crisis of the country, mostly by taking complete control himself and forming an increasingly repressive fascist state. Nationalist propaganda and lies were ceaselessly conveyed through the state media, paving the way for attacks on other countries, generally portrayed as critical to protect fellow countrymen being oppressed there. The aggression and the lying escalated until here we are, in a full scale international war, with distinct chances of becoming a world war. 

In the US, there are strange convergences of support for the Russian side of this conflict. Those on the fringe left can not tear themselves away from respect for the Russia that was the Soviet union and vanguard of world communism. Nor can they resist bashing the US. The far right is infatuated with the new Russia, with its super-Trump leadership, free-wheeling criminality, and clever propaganda, as many Americans were of Hitler back in the day. But a third stream comes from the foreign policy establishment- the realists, who think spheres of influence are the most normal, god-given organizing principles of international affairs. Thus China should be given its suzerainty over South East Asia, including Taiwan, and Russia over its near abroad, whatever the people actually living there may think. We are to blame for pushing NATO to Russia's borders, we are to blame for injuring Russia's sensitivities and pride, and we have caused their invasion of Ukraine, by luring Ukraine to the West with our sweet blandishments.

Well, each of those views is out of touch in its own way, but the last is especially curious. For what was the post-World War 2 order about, if not about civilized behavior among nations, letting each seek prosperity and freedom, in peace? The realist view would plunge us back into medieval power relations, or perhaps the three-sphere world of George Orwell's 1984. It consigns small countries to the depredations of bullies like Russia, who can not make friends in a civilized manner, but, in Ukraine, has strained every nerve to corrupt its political system, destroy its internet, and obliterate its sovereignty and economy.

It is obvious to all, including Russia, that NATO was and remains a defensive alliance, of countries intent above all else to rebuild after World War 2 without further aggressive encroachment by Russia. And once the Soviet Union fell apart, the Eastern Bloc countries fled as fast as they could to the West, not because they wanted to attack Russia in a new World War 3, but quite the opposite- they wanted to pursue the promise of freedom and prosperity in peace, without bullying from Russia. Russia's much vaunted "sensitivities" are nothing more than toxic, domineering nostalgia for their former oppressive empires, of both Czarist and Soviet times. As the largest country in the world, one would think they have enough room, but no, their sense of greatness, unmatched by commensurate cultural, economic, or moral accomplishment, demands bullying of its neighbors. More to the point, their current system of government- autocracy / fascism by ceaseless lying and propaganda, would be impaired by having their close neighbors have more open, civilized systems. 

All this has a religious aspect that is interesting to note as well. Ukraine recently extricated itself from domination by the Moscow orthodox church, becoming autocephalic, in the term of art. The process shows that even in this supposedly supernatural sphere of pure timeless principle, tribalism and politics are the order of the day. Not to mention propaganda, and fanciful philosophy and history. The narratives that Russia as spun about Ukraine and its invasion are particularly virulent, unhinged, and insulting, insuring that Ukraine would never, in any sane world, want to have anything to do with their neighbor. It is one more aspect of the Russian aggression that spares us from needing to sympathize overly with its "sensitivities".

So, what to do? It is not clear that Ukraine can withstand Russian attacks forever. They have stopped Russia in its tracks, thanks to a lot of Western assistance. They have millions of men under arms, compared to a much smaller invasion force. They have motivation and they have the land. But they need heavier weapons and they need to preserve their air power. With those two things, they could turn the tide and drive Russia out. Without them, they will probably only manage a stalemate. Western sanctions have imposed highly justifiable pain on Russia itself, but historically, such sanctions tend to have as much countervailing effect, consolidating pro-government attitudes, as the opposite. So barring a dramatic turn of events at the top of the Russian system, which is highly unpredictable and rather unlikely, we are facing a very drawn out and destructive war in Ukraine.

In a larger sense, we are facing something far more momentous- the rise and assertion of autocracy (not to say fascism 2.0) as a competing world order. Russia's pattern has been clear enough (and historically eerie)- escalate their aggression and ambition as far as they can get away with. And China is watching carefully. The ability of the West to punish Russia for its completely immoral and cruel attack on Ukraine, and deter future repetitions, will shape the next century. Russia has decisively broken the borders and tranquility of the post-World War 2 order, and that has caused many, especially in Europe, to wake up and realize that coasting along on US coat tails is not enough- they have to actively participate in sanctioning Russia, in resolving their dependence on Russian fossil fuels (as if that had not been patently obvious before), and strengthening the collective defence, as expressed in NATO. Western leaders should make it clear that Putin and his key lieutenants will never be allowed to personally enter the West without being shipped off the Hague for trial. And we should give Ukraine what it needs to defend itself.

Finally, what of our own culpability? Not so much in mistreating Russia, which we have done only to a slight degree, but in committing war crimes of our own, in attacks of our own, based on lies of our own, on innocent countries far distant. I am speaking of Iraq (which ranks first among several other cases). While our justification for that war was far better than Russia's in Ukraine, it was still poor, still caused hundreds of thousands of deaths, was grievously misconceived and mismanaged, and has left a political ruin, not to mention a geopolitical mess. This alone should make George W Bush rank among the worst of US presidents- significantly lower than Trump, who for all his destructiveness, did not destroy whole countries. We should be willing to put Bush and others who made those decisions to an historical and international account for their actions, in a spirit of historical rectitude.

• In praise of Washington's teaming minions.
• New thoughts on an old book.
• A song for Ukraine.

DNA Damage Domain Declines to Bind DNA

How one protein domain changed through time.

The BRCA1 and BRCA2 genes are notorious for harboring mutations that increase susceptibility to breast cancer (thus their name, breast cancer type 1 (or 2) susceptibility protein). They have therefore been intensively studied for what they do in the normal course of our cellular lives. Their common naming does not mean they are similar- their structures are completely different. They play related, but distinct, roles in DNA repair, which is naturally influential in our susceptibility to cancer caused by DNA mutations.

An article some time back delved into the history of one domain of the BRCA1 protein, tracing how its functions have changed significantly over evolutionary time. BRCA1 is a large gene encoding a large protein, (1863 amino acids long), composed of several domains. Proteins frequently possess several domains in order to integrate several functions in an orderly way, such as binding a few different partners that together form a complex and carry out some function. Modular protein domains facilitate evolution by being easily duplicated, transferred, and generally being able to be passed around, thanks to rearrangement mutations. BRCA1 has domains that bind to at least 11 other proteins,  most of which play some role in DNA damage responses. So it is a key protein, and damage to it has correspondingly bad effects. 

The domains of BRCA1. Each one has some role in the protein's function, which integrates responses to DNA damage. The BRCT domains are on the very end, right side. NLS is nuclear localization (import) sequence, and NES is the nuclear export signal. These would be typically regulated by other interacting proteins or phosphorylation, to control the access of BRCA1 to the nucleus.

The domain of interest here is the BRCT, or BRCA1 C-terminal domain. It is ~90 amino acids long and BRCA1 has two of them, side by side. Other work has shown that it binds to other proteins, but only after they have been modified by phosphate addition. The DNA damage sensor ATM is one such kinase that adds phosphates to BRCA1 targets such as Abraxis. Thus the BRCT domain plays the key role of bringing this DNA damage repair integrating protein to the right sites, where there is DNA damage to repair. 

Structure of the BRCT double domains in BRCA1 (E). The pocket that binds a phosphorylated serine residue on a partner protein such as abraxis is shown in teal, and in (C), close up. (B) shows a single BRCT domain.

This paper did a sensitive computer search for all possible versions of this domain in all available species and proteins, finding it in 23 human proteins, and in species all the way back to bacteria, so is quite ancient. And the phylogeny they reconstruct indicates that the original versions of these domains had a different function, which was to bind DNA directly, at sites of DNA damage! Such frayed ends also have phosphate groups, so it isn't a huge leap from one function to another. Additionally, other examples of BRCT domains have dispensed with phosphate-dependent binding altogether, but simply bind other proteins regardless. This transition may have happened after phosphorylation became the central way to alert the cell, and key proteins, to the existence of DNA damage, instead of dealing with it solely through enzymes that find & fix such damage directly. This transition allowed a much more robust response by cells, which now includes halting the cell division cycle and activating other stress responses to help the cell recover.

Some of the BRCT domains (along with many others) found in various species and their proteins.

The BRCT domain is mostly used among proteins involved in DNA repair, and even in humans some versions bind DNA directly (PARP1, RFC1). So through the long path of evolution, this single domain has stuck generally to its original role, while it also- along with the organisms and proteins it acts within- diversified and ramified in its functions. From an initial role in direct DNA damage and end recognition, it has become a card-carrying member of the bureaucracy of the cell, playing regulatory and organizing roles within numerous actors important to DNA handling and repair. It is a classic story of how eukaryotes used their surfeit of energy and material resources to develop whole orders of novel molecular, and concomitant outward, complexity.

• There are a lot of places we shouldn't get our energy from.
• But we are hopelessly dependent and immature.
• Partisan hack on the Supreme Court.
• What the Russians think of negotiation.
• Is it more than a job? Should it be?
• Ruminations on war.

Toxic Nostalgia

Making Russia great again.

What is it about the past? Even though we are condemned to live in the future, we can't stop fantasizing and fetishizing the past, and wanting to go back. On the gentle side, Proust wrote nothing but loving remembrances of his (sometimes mortifying) past, trying to evoke its moods, textures, smells, and feelings. But why does nostalgia so often curdle into bloodlust and terror? For that is where the Russian autocrat is going with his nostalgia for the Soviet era when Stalin ruled even more autocratically over a well-cowed populace extending from Hungary to the Pacific. Ah, those were the days!

It isn't just our current crisis- far from. The Trumpists want to make America great... again. The Muslim jihadists are bent on reproducing the pre-eminent dominance of Islam of 1300 years ago. The Serbs hearken back to their own grand empire of 700 years ago. Shia muslims fetishize their losses and in a theology of repair and redemption. Jews have both bemoaned their losses of their great kingdoms two millennia ago, and militantly sought their promised land back. And fundamentalists of all stripes yearn to get back to the basic tenets of their faith- the pure origins of incendiary belief and miracles.

It all seems a little over-determined, as though the operative emotion isn't nostalgia exactly, but powerlust, seizing on whatever materials come to hand to say that we as some tribe or culture are better and deserve better than we've got. While the future remains ever shrouded, the past is at least accessible, if also rather protean in the hands of dedicated propagandists. In Russia's case, not only did Stalin help start World War 2 by co-invading Poland, but the prior holocaust/famine in Ukraine, followed by the transplacement of millions of Russians into Ukraine.. well, that all makes this current bout of nostalgia far from sympathetic, however well-twisted it has been for internal consumption. Of course the propaganda and the emotion is mostly instrumental, in a desperate bid to fend off the appearance of happy, secure, and prosperous democracies on Russia's borders, which is the real danger at hand, to Putin and his system.

In remembrance of Russia's great patriotic war, which it helped start.

Yet, such nostalgia is strongly culturally binding, for better or worse. Rising states may have short histories and short memories, resented as the nouveau-rich on the world stage. They are not "as good" in some essential way as those whose greatness has passed into the realm of nostalgia. Worth is thus not in the doing but in some ineffible essentialist (read nationalist/tribal) way that is incredibly resistant to both reason and empathy. It is analogous to "nobility" in the class structure within most societies. In the US, we seem on the cusp (or past it) of our time atop the world stage. Do we then face hundreds of years of regret, comforting ourselves with tales of greatness and seething resentment?

With echos of a deeper past.

• Could the West have been smarter; more generous?
• Apparently, we are all going to die.
• Tires are bad.