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Nanopore DNA Sequencing
mindpixel writes: "Harvard scientists have concieved a revolutionary technology for probing, and eventually sequencing, individual DNA molecules using single-channel recording techniques. The technique essentially pulls a single strand of DNA through a nanopore, reading off the individual bases electrically. The technique could allow for decoding of a person's genome in hours instead of years." While the sequencing in hours instead of years is something that's pretty darn cool, our holdup in using this data is actually now what the genes are, and how they interact. That will still take years for us to figure out.
Actually, I'd say "decoding" is just a journalist term. I've never heard anyone who actually does sequencing or bioinformatics refer to sequence analysis as "decoding".
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This would make real DNA Fingerprinting a reality.
Get arrested, give a blood sample. It'll only take a few hours to verify who you are. None of this "probably" stuff, they'll have YOUR sequence on file, and there won't be any doubt (unless you have an identical twin).
I think gels are used for very small samples of DNA. About 500 base pairs is the limit for a gel. With 72 channels on the gel that is nowhere near the number of base pairs in a chromisome.
The faster better technique is electrostatically driven capliary tubes. They suck the sample through a microcapliary and shine a laser on it as it passes by. Generally the same 72 channels in parallel as the gel method - something about legacy analysis software as well as the plates that hold the samples and the robots which manipulate them. Kind of like the gauge of the railroad tracks used to carry space shuttle boosters being determined by the wheel spacing of the wheels on a roman chariot which was determined by the space required by two side by side horses.
we have the most advanced transportation system in the world having a major design parameter determined by the width of a roman horses ass.
Z
enough is too much
The article mentions 3 billion bases in less than 2 hours. That comes to
(3/2) x 10e9 / (3.6 x 10e3) bases/sec
= 416667 bases/sec.
So you would need a sustained writing speed of about 400 kilobytes/sec, or if you compress it into 4 bases per byte, say 100 kilobytes/sec. to write to about 3 GB (or 730MB compressed) of disk space.
You could fit it onto an IBM Microdrive attached to your Palm!
You are both right and wrong with your post, and your enthusiasm for learning is encouraging.
You're correct: The protein folding ploblem is is hard, in the worst (best?) sense of the word. That is to say, NO, there have been no interesting advances in the field; All that has been done so far, more or less, is to throw some processor cycles at the problem. There have some good results from this aproach, but no new insights or understanding of the problem.
This would be a good project for an interested and motivated student looking for a trip to Stockholm.
With an understanding of protein folding, we could rapidly discard the current: "piss on the porch, see if the cat licks it up," era of biotech, and actually do some engineering. Proteins are, to put it mildly, amazing: they are nanomachines that work, sometines with such elegance that when you see what is really going on it is enough to take your breath away as you try to yell Holly Sh*t and wonder if you are living your life right.
Designing proteins to functional specifications would be highly interesting, i.e., one of the simplest scenarios: "Uh, how about we have it arange the glucose carbons in a cubic crystal latice and let the O and H diffuse out as water?"
Today, we can pretty much string together any arbitrarilly long sequence of amino acids we want, but they are just so much fried egg until the folding problem is solved. When, and if, that day comes, our world is going to change in surprising ways. The implications reach far beyond medicine and agriculture.
You're a bit wrong: DNA folding is not, by itself, a barrier to our understanding of the genome. Actually, we have a pretty good understanding of how the whole transcription translation thing works, with a pretty solid understanding of splicing, targeting, etc., even some understanding of promoters, enhancers, and so on, we just don't know, for the most part, what the hell the products of this process (proteins) are doing.
Usual biotech-speak is "sequencing" and "mapping," the latter being figuring out which genes are where in the raw sequence data (AGCCTGATC ...) which comes from the former.
"Decoding" is a useful but too-broad term; it covers mapping and everything that comes afterward, such as figuring which of the mapped genes will express when, and to what degree, and how the proteins thus expressed will fold, and how they'll interact with each other. Once you've got all that, you have Mastered The Secrets Of Life Itself.
There is, to put it mildly, a lot of work to be done here. Job security for bioinformaticians.
The correlation between ignorance of statistics and using "correlation is not causation" as an argument is close to 1.
What will it be used for?
Immortality, my friend. Immortality.
The honolulu(sp?) technique used in cloning (done with mice - an incredibly difficult subject for cloning because the eggs cycle so quickly), unlike the faulty technique used with Dolly (which involved starving the nucleus-donor cell, and then using an electric shock to cause it to merge with the nucleus-free egg), has fertility rates that are rapidly approaching those of normal mice, and little to no genetic damage per breed. Yet, at around 5 generations of clones (cloning a clone of a clone of a clone of a clone), we start to see premature aging. Why? Because, as DNA lives, it slowly mutates. As must mutations are bad, it steadily poisons the mice's genetics.
The key is in a DNA backup.
Digital data doesn't corrode. It can be verified, backed up, copied, you name it. With the recent production of a completely DNA-synthesized fruit fly, the possibility approaches that we can completely re-create an individual's DNA strand. Then, when cloning organs for the individual, we use the backup DNA, not DNA from the person themself, as that DNA has become slightly corrupted overtime.
Digitizing DNA strands is a key to immortality.
That is why this is important.
-= rei =-
P.S. - I vote for genetically altered population. I certainly hope we see that day soon when people can make choices on whether or not they want their children to have to suffer.
"This may be presumptuous..." "That's my favorite kind of 'This'."
I was thinking that this method could also be used to sequence proteins - a process which is now done using an automated process which can only produce (correct me if I'm wrong, and if I am I'll eat a bug) maybe 30 "letters" of sequence. Compare this to many hundreds at a run from DNA sequencing. If proteins could be sequenced hundreds of amino acids at a time, you could sequence a whole protein in one run. This would be better than the current method, where fragments are sequenced, and then the overlaps are compared to piece together the whole sequence.
Freedom: "I won't!"
It looks to me that there is a big gap between the idealized graph above, which shows clearly the different nucleotides, and the actual data they have gotten which shows blocks of 30 purines and 70 pyrimidines. Can they really distinguish between adjacent nucleotides or are they so close physically they will just crowd through and blur together?
And will they be able to tell A from G (both purines) and C from T (both pyrimidines)? I don't have the charges handy but I think C and T are pretty close.
Also, DNA breaks very easily. No way are you going to be able to pull a whole chromosome through at once. If they get just 100 bases at a time, will that be useful?
It's great the we might be able to do sequencing in a matter of hours rather than years, but the real question is, what does that get us?
Every drug company has a Genomics division these days, to analize the existing data from the Human Genome Project. Now that new data can be gathered at such increadible speeds, are we any closer to improving the quality of life based on this work. Probably not, and the cause is a double edged sword.
The problem is the restrictions through international treadies and government regulation, on gentic engineering of humans. Don't get me wrong, I'm not in favor of such modification of the human genome, howeer, this leaves only one recourse. They can create medications that the sufferer of a genetic defect can take every day for their entire lives to prevent the ocurrance of an illness that they are genetically predisposed to. This is a boon for drug companies. If they can generate long term revenue streams by creating medications which reduce the chances of developing illnesses to which certain people are genetically predisposed to, and clain that they are doing this, instead of developing ways to repair a gene at birth - not because it's more proficable to do it this way but - because this is the only avenue they're allowed to pursue due to federal and international regulations against messing with the human genome; then who are the regulations truly serving? the population, or the drug companies?
Along the same lines, there will always be countries which are not signatories to the afore mentioned international regulations - in which drug companies can deelop the gene theropies which could truly benefit sufferers of gentic diseases and defects. That said, there will always be a black market for these theropies, once deeloped.
The question becomes which is a better world to live in: one where we have a drug dependant population, or one where we have a genetically altered population.
At this point I'll conclude my analysis because any further speculation will lead to the realm of Gattica style science-fiction. There is, however a great deal to consider...
--CTH
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That was my first take as well, but then when I looked through the references, I found that many feasibility questions seem to be resolved already. For instance, I read the main page and thought, "Sure, but how do you transport the strand through the nanopore?" Then I checked the first reference listed, and what do you know: "We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane."
The final system may still be largely conceptual, but it's by no means blue sky. I tend to be a techno-skeptic but this work impresses me.
The page sounds to me like a breathless plea for lots of venture capital funding.
This is grossly unfair. The language and style are well within the normal bounds for scientific papers. The word "revolutionary" is appropriate for a technology that would do years of work in hours. And in case you didn't notice, it's not private research -- it's being done at The Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University. What interest would a university laboratory have in "venture capital"? If they later spin it off into private industry for product development, then they might go for venture funding, but it simply makes no sense to do so now. There's a big difference between research sponsorship and venture funding.
Tim
It must be a bit like climbing mount Everest the hard way and while you are sitting at the top eating your Kendal Mint Cake, someone rides up the access road on a bicycle.
So what was the point of spending several hundred million doing the job the hard way? Oh they filled a gazillion patents on the sequences the read out. And there I thought you had to invent something to get a patent.
Looking for an Information Security student project suggestion?
Try http://dotcrimeManifesto.com/
Just to correct a few inaccuracies. Firstly it hasn't been completed yet. Celera Genomics decided to claim they'd finished it, which spurred the public project to also claim the same. In reality both had only 90 - 95% of the consensus sequence and only then at "draft" quality.
Secondly, I feel it's wrong to claim that Celera were the people to "complete" it. Celera used their own data in conjunction with the public data, and yet they have (more or less) comparable results in terms of coverage, number of contigs, quality and so on. Personally I feel that this is like admitting that their own work doesn't add anything new to the public effort - ie they failed.
The bottleneck with sequencing at the moment is in the "finishing" process - tidying up the results to produce highly accurate answers. This is largely caused due to the randomness of the shotgun sequencing approach. As it's effectively solving a jigsaw puzzle from lots of randomly cut pieces of DNA, in some places you'll get lots stacking up and in others you'll find none. It is unrealistic (not to mention expensive) to keep increasing the coverage so that everywhere gets covered by the random shotgun process.
Instead a fixed depth (only 3 or 4 fold in the draft sequences) is used followed by directed sequencing where the user, or an automatic program, analyses the data set and chooses experiments to perform (primer walking typically). The graphs I've seen of draft vs finished data show quite well how the finishing is lagging behind.
However this new strategy, not being random, will greatly reduce the amount of finishing needed. However note that at present they are using it for probes rather than full scale sequencing. It has great potential, but looks to be years away from replacing the current work.
A similar technique is DNA sequencing using scanning-tunneling microscopes and atomic force microscopes. Here is a Google search, and here is an article from 1992.
Harvard scientists have concieved a revolutionary technology
This sounds exactly like late-night infomercials that invariably say things like "our scientists [actors in white lab coats conspicuously walking around behind the one being interviewed] have devised a revolutionary new formula that will make you lose weight without dieting or exercising!"
That is, if the people selling something describe it as "revolutionary" themselves, it isn't. If it really is revolutionary, we'll hear about it in other places. The HP-35 from the slide rule article -- that's revolutionary.
So while this may be a significant improvement, I'd change the prose if I was them.
It will not replace conventional sequencing technology, unless it can beat the now pretty cheap cost. Conventional sequencing is based on labelling the individual DNA bases with a different flourescent dye, and running the DNA through a gel which seperates the DNA according to size: As each base runs through the gel, it goes past a laser/detector which can detect the specific DNA base (A,T,C or G) at that position. Due to gradual impovements to this technique over the last 20 or so years (originally it employed radiation, rather then flourescence) the speed, sensitivity and cost has decreased dramatically. For example, the human genome project started in ernest about 10 yrs ago. Celera Genomics, using modern technology (and alot of financial backing, and the fact they are a subsidiary of the people who make sequencing machines,) competed the genome in a matter of months. The increase in DNA sequencing capacity puts moore's law to shame.
For example, our lab could process around 100kb (thats KiloBases guys!) of data a day, but we never even touch this with our machine. No need, and the same stands for many small-medium research labs. Alot of people like us will probably stick with conventional sequencing technology for a long time (it works well, is high enough throuput, cheap & easy).
However, the are some exciting applications with single strand sequencing. For example forensics. Also, it allows the oppotunirty of sequencing RNA (this is the "messenger" which passes the "important" part of the DNA message to the ribosomes, which then "compile" a protein - the stuff which actually does things, like an enzyme or structural component). Sequencing RNA is exciting, as currently you have to convert the RNA back to DNA (which can cause problems) and then sequence that.
Another obvious application for this would be very high throuput sequencing which would be employed by the major sequencing centres. Yes, i know we already have the Human Genome, but a fashionable idea at the moment is comparative genomics. This is very much taking biology back to its roots (i.e. like Darwin and Wallace comparing the morphological characteristics of certain species and infering adaption), but at a molecular level. This will yield amazing insights with discoveries having important implications from medicine to evolution. In fact I think the general public & media will soon be bored of this. Each week it will be a new genome being announced; mouse, chicken, rat, pufferfish, rice, corn, dog, cat, cow, chimp......
Hey, dont knock electrophoresis mate. It is the basis of PCR resolution, Southerns, Northens, SSCP, conventional sequencing methods plus a multitude of other applications. Furthermore, refinements to this approach (read: capillary electrophoresis) have supplied one of the major advances to sequencing methodology in recent years, unlike the technology we are (were?) discussing.
Who's GNOME did we sequence anyway
Good Question. Apparantly, they took DNA from around 100 (i forget the exact figures here) US citizens of various sex/ethnicity, picked 7 out of the hat, and sequenced portions of each (most from a single, unidentified individual - although if nanopore technology comes to fruition, i reckons we can track him) down
p.s. thats one of my favourite all-time techno-typo's: the Human Gnome Project. Almost as good as sequencing my ARS (a yeast thang....)
But it is a place to start.
Side note:
while looking up the Finnish Language pages for this comment, I came across this tidbit: That Finnish has "no equivalent of the verb to have". This has interesting philosophic implications in the history of open source, etc.
Check out the Vinny the Vampire comic strip
"It is a greater offense to steal men's labor, than their clothes"
I am a zoological systematist, working in entomology.
The human genomes was sequenced by taking lots of DNA, cutting it up randomly sequencing the random pieces of cut up DNA.
In my field, we work with much smaller amounts of DNA. Sometimes I only have a single specimen of a tiny insect, or unique material (from rare or extinct species) to try and get some DNA out of. In older material, DNA is usually degraded and many times we end up with nothing but a destroyed or damaged specimen.
With small amounts of DNA to begin with, we have to amplify (PCR) single genes or regions by using general primers, which means that they don't only fit on the insect DNA, but fungi and human DNA too, making contamination of your material very real risk.
If this technology turns out to work on a larger scale, it's amazing news for me and my collegues.
The nanopore technolgy sequences single moleculer, which means the PCR step becomes unneccesary! This means that we can get sequences from specimens with severely degrades DNA, and we don't have to be as afraid of grinding up rare material in hope of getting sequences.