Post Tagged with: "history"

Polymerase chain reactions, so good they invented it twice.

I’ve recently been preparing some new courses which have given me the opportunity to browse through the literature from the dawn of molecular biology. And in the process I came across a 43 year old paper entitled  “Studies of Polynucleotides XCVI. Repair replication of short synthetic DNA’s as catalyzed by DNA polymerase.” by Kleppe  and Khorana in the Journal of Molecular Biology. Its an elegant manuscript that describes how DNA polymerase can replicate a DNA strand but only if there is a section of duplex DNA, known a as primer, from which it can start.

So Klepper et.al. started off with a bit of DNA that looked like this:

and after incubating with DNA polymerase ended up with a DNA sequence with the gaps filled in, like so.

 

Well isn’t that nice?

But the really intriguing bit is the last paragraph of the discussion.

.. the DNA duplex would be denatured to form single strands. This denaturation step would be carried out in the presence of a sufficiently large excess of the two appropriate primers. Upon cooling, one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme. … After every cycle of repair replication, the process of strand separation would have to be repeated. Experiments based on these lines of thought are in progress.

Wow, what a cliff hanger. Kleppe has just described polymerase chain reactions (PCR), the now ubiquitous method for amplifying DNA. But this was 14 years before Kary Mullis (with Saikia as first authur)  published the first application of  PCR in Science.

But despite the tantalising ending to Kleppe’s paper, nothing else emerges from Khorana’s group to that effect. He never published those experiments. And indeed no one else picked up on the idea until Kary Mullis ran with it.

Its an interesting story that’s been brought up plenty of times before, but having stumbled across the original paper describing the ‘invention’ of PCR and given the big DNA anniversary next week, I thought I’d put it out there again.

By April 15, 2013 2 comments chemical biology, Uncategorized

Shades of Gray, The Curious History of LCDs

Prof. George Gray

Today is the 40th anniversary of an innovation in chemistry that has had, arguably, a greater impact on our society than any of the Chemistry Nobel Prize winning achievements in the past 40 year. But the man responsible, George Gray, is only known in select chemistry circles (apart from maybe a few travellers boarding a train traveling between London and Hull that bears his name). Yet you are almost certainly reading this blog on a device that owes its existence to Gray. For he and his small team, of just two post-docs, developed the first liquid crystals that were viable in liquid-crystal displays (LCDs). Forty years ago today his work was published, triggering a multi-billion dollar industry and making today’s abundance of flat screen devices possible.

The breakthrough that emerged from Gray’s small group was the synthesis of 4-Cyano-4′-pentylbiphenyl (5CB). It had a nematic liquid crystalline phase between 22C and 35C which made it the first material that could form the bases of viable LCDs.*

4-Cyano-4′-pentylbiphenyl

Just like so many great innovations getting to this point had been far from easy, largely because there was little appetite for funding research on molecules that, at the time, had no clear applications. Turning liquid-crystals from curiosities into the ubiquitous technologies that they are today required both a burning need for new displays and the foresight of one of the more colourful government ministers.

Enter John Stonehouse, Minister of State for Technology under the UK Prime Minister Harold Wilson. Stonehouse wanted a technology capable of producing flat screen colour displays (a good 30 years before LCD TVs became the norm) with the aim of replacing cathode ray tubes that were costing the Ministry of Defence colossal sums (more than the development costs of Concorde) in royalties. So in 1968 he set up a working group consisting of military brass, civil servants and scientists to find a suitable replacement technology. The way the contracts were distributed is a far cry from how things are done today. The story goes that at one of the group’s meetings liquid-crystals were proposed as a candidate. But the key speaker was unable to answer a question about why light from the projector generated such curious patterns as it reflected off the vials of liquid-crystals. There followed an embarrassingly long silence before a voice piped up from the back of the room exclaiming “I wonder if I can help”. That voice was George Gray’s and come the end of the meeting he and his team of chemists at the University of Hull were awarded the contract to deliver room temperature liquid-crystals.  That they did and the results were patented and published by 1973 with the first LCDs in commercial devices the following year. (Cyril Hilsum was chairing the session and he was recently filmed recounting his memory of the  meeting and the development of LCDs. You can watch it here  http://youtu.be/AaB902_ds-g?t=34m3s )  

At one time the molecules that Gray invented accounted for over 90% of all the liquid-crystals in the world’s calculators, digital watches and LCD clocks. So what became of the money that flowed in via the patents? Well the Ministry of Defence owned most of the intellectual property and made a tidy sum which offset the money they were still paying for cathode ray tubes.  Meanwhile the University of Hull, like most UK academic institutions at the time, didn’t think it was its place to own intellectual property, so the remainder of the royalties went to Gray and his team. But Hull wasn’t left completely out of pocket, the MOD continued to invest in LCD research in Hull until the patents ran out in 1992.

As for Stonehouse he may well have been blessed with the foresight to back LCDs, but he wasn’t so hot with his own businesses. Shortly after the first LCD devices were being manufactured his clothes were found piled on a beach in Florida with no sign of his body. He had apparently committed suicide after a series of disastrous business ventures. In reality he had faked his own death and was winging his way to Australia to start a new life with his mistress. The law caught up with him, briefly mistook him for Lord Lucan before sentencing him to several years in gaol. As if that wasn’t enough intrigue for one man he also turned out to be a Czech spy!

References:

1)  Gray, K.J. Harrison, J.A. Nash. New Family of Nematic Liquid Crystals for Displays, Electronic letters. 9:6. pp 130-131, 1973

2)   Hirohisa Kawamoto, The History of Liquid-Crystal Displays. PROCEEDINGS OF THE IEEE, 90: 4. pp 460-500.  2002

Footnote:

* A working range of 22 to 35C was not, of course, anywhere near sufficient for saleable LCD display.  That came about via a series of  mixtures of 5CB with new cyanobiphenyls which eventually settled on a quaternary mixture known as E7.

Composition of E7. From ‘The History of Liquid Crystal Displays’

 

 

Originally posted (as a slightly different version) in the Guardian.

By March 22, 2013 1 comment synthetic chemistry

The most beautiful wrong ideas in science

Over the summer I’ve been preparing some new biochemistry courses. This has given me the excuse to browse through the literature from the dawn of molecular biology, starting with Watson and Crick’s DNA structure paper from 1953 1. From there, I quickly got distracted (as is the way when you are supposed to be doing something else) by the amazing ideas and theories that followed this seminal paper.  It must have been a fabulous time, there were so many questions and little data to restrict  thought and so some of the most beautiful wrong ideas in science were hatched.

One of the most pressing questions of the time was how does DNA code for proteins? The problem was simple. There are just 4 bases in DNA, adenine (A), thymine (T), guanine (G) and cytosine (C). But DNA codes for 20 different amino acids found in proteins. So how can a 4 letter alphabet be translated into a 20 letter alphabet?  Most of the subsequent ideas seems to stem for a desire to get 20 from 4.

Gamow's Diamond Code.

The first serious stab at answering this conundrum came from a surprising direction. George Gamow was a theoretical physicist and cosmologist, who is more famous for the Big Bang Theory than his contributions to molecular biology. Nevertheless his background didn’t stop him publishing, in Nature, an intriguing theory for the genetic code 2.

Gamow proposed an overlapping triplet code. It had to be a triplet of bases because a doublet only produces 16 (4 x 4) combinations, whilst a triplet produces 64 possible combinations, more than enough to code for all the amino acids. He suggested the triplet codes overlapped because it allows the double stranded helix of DNA to act as a direct template on which the amino acids can be assembled into proteins. So, for example the sequence ATGCTA would contain the triplets ATG, TGC, GCT,CTA each of which would code for a different amino acid.

Gamow also proposed a mechanism (which became known as Gamow’s Diamond Hypothesis) to back up his theory. Each amino acid would fit directly into distinct diamond shaped pockets formed within the  grooves of DNA where the 4 sides of each pocket would be defined by the 4 bases. And when he did the math, hey presto, it turns out there are 20 possible uniquely shaped pockets!

The numerology is compelling but there is a significant limitation to this theory; it does not allow all possible combinations of amino acids. This problem is best illustrated with a dipeptide. In the overlapping triplet hypothesis a dipeptide would be coded by 4 bases, which results in 256 (4 x 4 x 4 x 4) possible combinations. However, given the 20 amino acids, there are 400 (20 x 20) possible dipeptide sequences. So 144 dipeptide combinations are not possible. Of course this limitation should be easily testable by simply looking at protein sequences and seeing if there are more than 256 dipeptide combinations. But let’s remember that at the time (1954) the data on protein sequences was pretty limited (Sanger only published the first protein  sequence in 1951 3). So the test had to wait until 1957, by which time there were just enough sequence data for Brenner to publish the clearly titled paper “On the Impossibility of all Overlapping Triplet Codes in Information Transfer from Nucleic Acid to Proteins” 4.

So back to square one. And Francis Crick steps back into the picture.

Crick saw the problem like so: The genetic code had to consist of non-overlapping triplets of bases. But if that is the case how can one triplet be distinguished from the next. After all there is no punctuation in the DNA. Its like trying to find the three letter words in SATEATEATS without any commas. They could be  SAT EAT EAT  or ATE ATE ATS or TEA TEA depending on where you start. So Crick decided there must be  “codes with out commas”. And that’s what he called his paper 5.

It was a brilliantly elegant theory. He took the 64 triplet codes and put them together in groups according to whether they had the same circular permutations i.e. ACG, CGA, and GAC are in one group, CCG, GCC and CGC form a second group and so on. He then hypothesised that only one sequence from each group would be used to code for an amino acid. These he called ‘sense’ codons. The remainder were termed ‘nonsense’. So if ACG and CCG are sense then the sequence ACGCCGACG can only be read ACG CCG ACG, because CGC CGA both give nonsense codons.

Also into the nonsense pile went AAA, TTT, GGG and CCC because when they appeared they would cause ambiguity about where a codon starts (e.g. is CCCCGGG read CCC CGG or CCC, GGG).  So thats 64 possible codons, subtract CCC, GGG, AAA and TTT leaves use with 60. Of those remaining only every third codon is ‘sense’. EUREKA! theres the 20 codons needed to code for the 20 amino acids. Brilliant, everything fits perfectly.

The comma free code was so elegant and the numbers fitted so well that everyone believed it for  the best part of 5 years. Until, that is, pesky experimental data got in the way. In 1961 Marshal Nirenberg and Johann Matthaei produced a stretch of RNA composed of uracil (RNAs equivalent of thymine) 6. When they added it to a mix of ribosomes, tRNAs and amino acids the result was a polypeptide of pure phenylalanine. And so a theory that was too elegant for nature was shot down in flames.

The rest of the story is in the text books.

1. Watson, J.D. & Crick F.H.C. A Structure of Deoxyribose Nucleic Acid. Nature, 1953. 171:737-738 

2. Gamow, G.  Possible relation between deoxyribonucleic acid and protein structures.  Nature, 1954. 173:318.

3. Sanger, F & Tuppy, H. The amino acid sequence in the phenylalanyl chain of Insulin. I. The indentification of lower peptides from partial hydrolysates. Biochem J. 1951 49:463-81.

4. Brenner, S.  On the impossibility of all overlapping triplet codes in information transfer from nucleic acid to proteins. Proceedings of the National Academy of Sciences of the U.S.A. 1957. 43:687–694.

5. Crick, F. H. C., J. S. Griffith and L. E. Orgel. Codes without commas. Proceedings of the National Academy of Sciences of the U.S.A. 1957 43:416–421.

6.  Marshall N.W., and  Matthaei, J..The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proceedings of the National Academy of Sciences of the U.S.A. 1961 47:1588–1602

 

 

By August 16, 2012 9 comments Uncategorized