Ever wondered how living things are able to function and exist in such diversity of features?

Scientists had been pondering about this for centuries, until 1953 when an American man ran into a pub in Cambridge shouting to his British collaborator that they've discovered the secret of life. (No he was not inebriated as I am sure that’s what the customers of ‘The Eagle’ that afternoon probably thought.) The answer lies in an intricate set of instructions stored within every living being. This set of instructions, used by living organisms in growth, development, functioning and reproduction, is stored in a molecule called Deoxyribonucleic acid or DNA.  The two gentlemen in the pub James Watson and Francis Crick understood the structure of the DNA based on cutting edge X-Ray crystallography work done by Rosalind Franklin and Maurice Wilkins.

So how does this store such a code?

DNA, as the scientists in 1953 understood, is a long polymer consisting of repeating units called nucleotides coiled up in a double helix which is held together by hydrogen bonds and made up of 4 nucleic-acid bases : adenine (A), cytosine (C), guanine (G) and thymine (T). The genetic code in turn made from three-letter 'words' called codons formed from a sequence of three bases (e.g. ACT, CAG, TTT). These codons form a gene that influences certain observable traits of a living organism. Sequencing is the process of determining the precise order of bases in a DNA and over the years this has become an integral part of fundamental biological research as well as in fields such as medical diagnosis, biotechnology, forensic biology, virology and gene therapy.

Sequencing over the years…..

The first DNA sequencing experiments used a laborious chromatography technique. It became much faster and easier after the development and commercialization of fluorescence based approaches. This has resulted in scientists making huge strides in understanding DNA and developed several ground breaking applications. The greatest achievement enabled by this technique is however, the 15 year project that deciphered the human genome (the set of instructions that defines the human organism). This resulted in huge investments in developing techniques that increase the speed and decrease cost of sequencing. Over the past decade or so this cost dropped from $100 million per human genome to only $1,000 which is faster than that affected by Moore’s law and this is popularly called as Flateley’s law  after its then Chief Executive Jay Flateley of Illumina. Inc. which was the driving force behind this revolution. 

Broadly speaking most sequencing techniques use the process of creating multiple copies of DNA artificially or sequencing by synthesis. This takes advantage of a unique property of DNA bases to bind specifically to each other i.e., A to T and G-C only. When creating the artificial copies they are tagged fluorescently and read out using a highly sensitive camera. This was traditionally done in a long process involving several steps and Illumina were able to refine this process using flow cells made on glass and further developments used miniaturized flowcells using components called microfluidics made using plasma etching technologies.  Over the years there have been several competitors like Thermo fisher, Pacific Biosciences and many other who have used various architectures of microfluidics from channels and wells on glass, silicon and polymers to read out this sequence using fluorescent tags. This still remains one of the most accurate ways to sequence DNA and in combination with semiconductor fabrication technologies the cost are falling further.

However, over the last 5 years or so there has been an evolution of a sequencing philosophy called single molecule sequencing. This set of techniques is aimed at literally reading each base as you would if you had a molecular picture of the DNA. Progress in this field has already been rapid and promises to drive down the costs down to $100/genome and eventually to $10/genome. Several players like the Roche backed Genia, Quantum Biosystems have joined this game but the clear frontrunner is the Oxford based Oxford Nanopore who already offer several sequencers based on single molecule sequencing. In particular they ‘read’ the DNA code by passing the DNA (which has a small negative charge) through a very small pore using electric fields across an electrolytic solution. The magnitude of the current observed is specific to the base that passes through and the changes in current can be translated into the sequence. There are several variations of this technique but the idea of sequencing by passing DNA through a nanopore is already touted as the next disrupter in DNA sequencing.

Nanopore sequencing currently uses protein membranes and pores which work well but have limited scaling up potential and suffer from stability issues. This is where MEMS fabrication technology using plasma processing techniques come into play and enable creation of mechanically stable ultra-thin membranes which is a subject of intense R&D today as the race towards the $10/genome heats up.

Would you like to fabricate novel DNA sequencing devices using plasma etch and deposition techniques and solve $10/genome challenge? Come talk to us at Plasma-experts@oxinst.com.

Author: Dr Ravi Sundaram

#Bio #DNA #plasma #MEMS #microfluidics