Forbes, Sept 8, 2014 (II)

choi

Matthew Herper, Genetics’ Engineer. DNA sequencing is revolutionizing the diagnosis and treatment of everything from cancer to Down syndrom at breaking speed, changing health care forever. Incredibly, only one company--Illumina--is making it all possible. It’s just getting started.
www.forbes.com/sites/matthewherp ... genetic-revolution/

the first three paragraphs:

“When Renee Valint’s daughter Shelby was born in 2000, she seemed weak, like a rag doll. Shelby learned to walk and talk, but she did so slowly, missing developmental milestones. By age 4 she was confined to a wheelchair, and she started using a computerized voice to communicate in the fifth grade. Desperate, Renee took her from Phoenix to the Mayo Clinic in Rochester, Minn. for one last week of tests and discussion with some of the country’s top doctors.

“‘They all put up their hands and said, “We have no idea what’s wrong with her,” ‘ says Renee.  ‘At that point she couldn’t even move. I bathed her, fed her. She couldn’t even swallow. I had to thicken her liquids so she could swallow without choking. It was like a nightmare. That was it. There was nowhere else to go.’

“But then doctors at the Translational Genomics Research Institute in Phoenix used a new technology–DNA sequencing–to look at Shelby’s genes. Based in part on what they found, they guessed that she might respond to the same dopamine-boosting medicines that are given to Parkinson’s patients. Three months later Shelby got up out of her wheelchair. The next day she walked to school, and she hasn’t used the wheelchair since. Now she likes to dance.

My comment:
(a) Illumina (company)
en.wikipedia.org/wiki/Illumina_(company)
(incorporated in 1998; Headquarters  San Diego)
(b) The article deals with business aspect of Illumina. What intrigues me is the science, specifically the DNA sequencing. See the third posting.
(c) Before that, the second posting is about the girl, Shelby Valint.
(d) “As Roche, the Swiss drug giant, noticed Illumina’s unassailability as its own DNA sequencing efforts dwindled to irrelevance.”

454 Life Sciences
en.wikipedia.org/wiki/454_Life_Sciences
(Jonathan Rothberg published a Nature article in 2006 for a competitive sequencing technology; Roche purchase 454 in 2007)
(e) The last paragraph of the Forbes report mentions "Ronaghi, the CTO."  Earlier, the Forbes report states, "Mostafa Ronaghi, an inventor of key DNA sequencing technology and now Illumina’s chief technology officer."  That is what CTO stands for.

choi

Vinodh Narayanan, MD
www.tgen.org/research/research-faculty/vinodh-narayanan.aspx
at Translational Genomics Research Institute (TGen) took up the case. The sequencing found at least a mutation in the DDC gene. (Because no scientific report is published--it is all in newspapers for laypersons, particularly The Arizona Republic--it is unclear what kind of mutation it is.)  DDC is the gene coding “dopa decarboxylase” (also known as “aromatic L-amino acid decarboxylase”)
www.ncbi.nlm.nih.gov/gene/1644
(“The encoded protein catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (DOPA) to dopamine, L-5-hydroxytryptophan to serotonin and L-tryptophan to tryptamine”)

aromatic L-amino acid decarboxylase
en.wikipedia.org/wiki/Aromatic_L-amino_acid_decarboxylase
(section 1 Reactions)
, where you can see the carboxyl group (-COOH) is removed from L-DOPA (DOPA is an acronym) to create dopamine. The dopamine is a neurotransmitter. Presumably the mutation decreases the enzyme function (either by producing less amount, or more likely an enzyme that is less efficient). Instead of prescribing L-DOPA (a mainstay to treat Parkinson’s disease, whose patients produce little dopamine) with its side effects, Dr Narayanan ordered Bromocriptine, whose pharmacology is “a dopamine receptor agonist, which activates post-synaptic dopamine receptors,” according to its pharmaceutical maker.

choi

Professors Shankar Balasubramanian (who was born and raised in India until he went to Cambridge as an undergraduate) and David Klenerman, Department of Chemistry, University of Cambridge invented a revolutionary DNA sequencing methodology, which is cheap and fast. The work was published
Bentley DR et al, Accurate Whole Human Genome Sequencing Using Reversible Terminator Chemistry. Nature, 456: 53-59 (2008)
www.nature.com/nature/journal/v456/n7218/abs/nature07517.html
(a) Click the “full text” in the right column for the text, which will be discussed in (2) below.
(b) The two British professors had formed a biotech company Solexa in 1998 at Hayward, California. Illumina completed the acquisition of Solexa in 2007 and acquired the revolutionary sequencing methodology.


I will explain the work in the following two, the second being the full article of Nature.
(1) Illumina Sequencing Technology; Highest data accuracy, simple workflow, and a broad range of applications. Illumina, undated (under the heading "Illumina Technology: Illumina Sequencing").
res.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf
(Figures 2 to 13)
(a) Fig 2 shows double-stranded DNA shattered to pieces, to which “adaptors” (two kinds: pink and purple) are attached at both ends. The two-stranded DNA fragments are converted into single-stranded DNA (which are all different from one another).  For convenience, I will focus on a specific single-stranded DNA, which I will call X.
(b) Fig 3 demonstrates many different single-stranded DNA fragments AND adaptors are pasted at ONE end to a surface (of what Illumina identifies as a “flow cell”). The other end of X will bend forward to form a bridge with an adaptor.
(c) Figures 4 and 5 show an enzyme (called DNA polymerase) and UNLABELED nucleotides (components of DNA) are added to the flow cell, to change the single-stranded DNA to double stranded DNA.
(d) Fig 6 now convert double-stranded DNA to single-stranded DNA again. The cycle repeat itself a number of times, to create a cluster from a single ORIGINAL single-stranded fragment, as shown in Fig 7. The purpose above is to multiply a single-stranded DNA fragment to a cluster, to amplify fluorescent signals: each cluster is a clone of the ONE and ONLY original single-stranded DNA, who own signal in later steps would be to weak to detect. So a cluster/clone of that particular DNA fragment (“X”) is imperative.
(e) The agents from previous amplification are washed away. Fig 8 starts the cycle of DNA sequencing, the real thing. Now modified nucleotides (all four types: ATCG; to be explained further in (2)), as well as DNA polymerase, are added the flow cell. Laser is directed to the flow cell to excite the fluorescence of the ONE nucleotide NEWLY added to a clone (say, the X). The camera takes a photo of it. The unused reagents are washed away. Chemical reaction is introduced to the modified nucleotide to make its 3’ end available for another round of elongation/DNA synthesis. The reason to immobile the X fragment--and thus the cluster--becomes apparent: the same spot of the flow cell will represent the same cluster of DNA (“X”) THROUGHOUT the DNA sequencing. Eventually the sequence of fragment X will be decided. Billions of such fragments, when overlapped with the aid of software, are combined to make a continuous sequence of DNA.

(2) Return to the Nature publication.
(a) “We describe a massively parallel synthetic sequencing approach that transforms our ability to use DNA and RNA sequence information in biological systems. “

“[P]arallel” because there ar many clusters in each flow cells, all undergoing elongation/DNA synthesis independent of any other cluster in the same flow cell.

(b) “We sequenced DNA templates by repeated cycles of polymerase-directed single base extension. To ensure base-by-base nucleotide incorporation in a stepwise manner, we used a set of four reversible terminators, 3′-O-azidomethyl 2′-deoxynucleoside triphosphates (A, C, G and T), each labelled with a different removable fluorophore (Supplementary Fig. 1a). The use of 3′-modified nucleotides allowed the incorporation to be driven essentially to completion without risk of over-incorporation. It also enabled addition of all four nucleotides simultaneously rather than sequentially, minimizing risk of misincorporation. We engineered the active site of 9°N DNA polymerase to improve the efficiency of incorporation of these unnatural nucleotides. After each cycle of incorporation, we determined the identity of the inserted base by laser-induced excitation of the fluorophores and imaging. We added tris(2-carboxyethyl)phosphine (TCEP) to remove the fluorescent dye and side arm from a linker attached to the base and simultaneously regenerate a 3′ hydroxyl group ready for the next cycle of nucleotide addition (Supplementary Fig. 1b).”  (citations omitted)

Here are the chemical modifications--besides a distinct fluorescent label for each of four nucleotides (ATCG)--of the nucleotides, described in (1)(e) above to ensure only ONE nucleotide is added to “X.”  The modifications also makes sure it is easy to--with a chemical reagent, tris(2-carboxyethyl)phosphine (TCEP)-- cut off the 3’ end of modified nucleotide for a new round of elongation.