Tuesday, July 29, 2008

The DNA Network

The DNA Network

You gotta love it!!! [The Gene Sherpa: Personalized Medicine and You]

Posted: 29 Jul 2008 06:39 PM CDT

When I open medical news today, I read this headline "Brain Differences Visible In Symptomless Carriers Of Alzheimer's Gene" Sounds fantastic right? Well........ It turns out that the n on this...

[[ This is a content summary only. Visit my website for full links, other content, and more! ]]

Don't forget... [Bayblab]

Posted: 29 Jul 2008 04:49 PM CDT

Don't forget to get your posts in for the upcoming Cancer Research Blog Carnival. /weblog will be hosting the 12th edition (and also designed the slick logo). Submit your posts here. The carnival will appear on August 1. And as always, we're looking for hosts for future editions. Email us at bayblab[at]gmail.com if you're interested.

Bringing science to society [Tomorrow's Table]

Posted: 29 Jul 2008 03:02 PM CDT

Today an article by Andrew Revkin in the New York Times, Science Times, describes the intellectual tussle that occurs when science is testing new ideas -and the public confusion that results from trying to stop the brawl and declare a winner.

"When the work touches on issues that worry the public, affect the economy or polarize politics, the news media and advocates of all stripes dive in. Under nonstop scrutiny, conflicting findings can make news coverage veer from one extreme to another, resulting in a kind of journalistic whiplash for the public."

This has been true for decades in health coverage and global warming. But lately the phenomenon has been glaringly apparent in the debate on crop genetic engineering.

While the public has generally accepted the application of GE for the production of new medicines, some consumers indicate grave unease over the consumption and production of GE food, viewing it as unnatural, potentially unsafe to eat and environmentally disruptive. These questions persist despite broad scientific consensus that the GE crops currently on the market are safe to eat, that gene flow from transgenic crops is similar to that from conventionally bred crops and that biological diversity is enhanced on farms that use GE crops compared to those growing conventionally grown crops.

While these debates- often polarized, non-scientific and political- endure, seemingly with no end in sight, the larger, more important questions such as how can we utilize GE so that it benefits those that need it the most, are ignored. At the same time, the climate warms, humans plow vast amounts of new land each year and children continue to die from malnutrition.

Just as some environmentalists have blamed "energy-dependent industries and the news media for stalemates on climate policy, arguing that they perpetuate a false sense of uncertainty about the basic problem", so are scientists asserting that prolonged debate over basic concepts of genetic engineering are hindering a science that has the potential to broadly, positively affect the environment and the humans that depend on it.

The article poses important questions: How can scientists reach consumers, farmers, and policy decision makers who want to make food choices and policy that will support ecologically responsible farming practices? How can we reach consumers who want accurate information about genetically engineered crops and their potential impacts on human health and the environment? How can we educate those who wish to know more about the food they eat, besides just how to prepare it?

The article notes that scientists are increasingly taking their message straight to the public.

And that is what this blog is about. It is for every shopper who has at one time or another perused the aisles of the local supermarket wondering what labels such as "organic" or "GE-free" really mean for the health of their families and for the future of the planet.

What's in Steve Jobs's Genome? Genetic Information at the Top [PredictER Blog]

Posted: 29 Jul 2008 02:11 PM CDT

Jacob Goldstein of The Wall Street Journal's Health Blog asks "Do Apple Investors Have Right to Steve Jobs's Health Info?" -- Investing is a gamble and gamblers need good information to make the best bets. Publicly traded companies are required (with varied levels of success) by the SEC to be transparent with their books. So, if you're thinking about investing in a company with a very charismatic CEO, you might want to know about the results of his most recent physical exam. As genomic medicine improves one could imagine a future in which "reasonable" investors demand a genome profile too. Goldstein observes that Jobs is both a "swashbuckling entrepreneur" and a pancreatic cancer survivor. I'm unsure how that information would help an investor and I'm fairly certain the science of reading the genomic tea leaves would not be much help, at least not yet.

A Menagerie of Mini Prep Methods [Bitesize Bio]

Posted: 29 Jul 2008 04:40 AM CDT

Over the years there have been a multitude of methods - ranging from the easy to the downright painful - developed for the preparation of plasmid DNA, but I suspect most of us have only used one , or maybe two, of these in our careers.

At Bitesize Bio, (part of) our life’s mission is to help you build your molecular biology arsenal so in this article I’ll brief you on the different plasmid miniprep methods available. If you have (or have had) a go at any of these, or if I’ve there’s a method I’ve missed, let us know in a comment.

Alkaline Lysis Method (reference here)

This method is based on the lysis and differential denaturation of chromosomal and plasmid DNA in order to separate the two (see here for more detail)

The lysis is performed using a NaOH solution containing SDS. During this step, both chromosomal and plasmid DNA are denatured, but upon neutralization using a high molarity sodium acetate solution, only the plasmid DNA is able to renature and remain soluble whereas the chromosomal DNA forms a complex with cellular proteins, potassium and SDS which is then removed by centrifugation. The plasmid is then isolated by precipitation using isopropanol.

This method creates plasmid generally suitable for most applications, although sequencing would need an extra phenol cleanup to make sure there is no extra soup contaminating your reaction.

Reagents required:

STE (sucrose/Tris/EDTA) solution (optional wash buffer):
8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0). Autoclave and store at 4°C.

GTE (glucose/tris/EDTA) solution:
50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0). Autoclave and store at 4°C.

Alkaline–SDS solution: 0.2 N NaOH, 1% (w/v) SDS, (or 0.1N NaOH if for sequencing)

Neutralisation Solution:
60 mL of 5 M potassium acetate, 11.5 mL glacial acetic acid, 28.5 mL double-distilled H2O. The resulting solution is 3 M acetate and 5 M potassium and has a pH of about 4.8. Store at room temperature, and do not autoclave

Boiling Lysis Method (reference here)

This procedure is quick and reliable for preparing plasmid from small volume cultures. Even though the quality of plasmid obtained from this method is lower than that of alkaline lysis mentioned above, it is sufficient for restriction digestion.

Lysis is performed by treatment with lysozyme (0.5mg/mL final) in STET buffer and heat, which causes the chromosomal DNA to precipitate with the bacterial membrane and other proteins, which is then removed by centrifugation. The plasmid DNA is then precipitated using isopropanol.

This method is not compatible with E. coli strains that produce endonuclease enzymes, unless of course you add the additional step of a phenol:chloroform cleanup.

If you are in love with microwaves, you can also lyse the bacteria using by nuking for 20-25s in STET buffer (reference here) - don't be too in love, things can get quite explosive in microwaves - and then simply centrifuge and precipitate with isopropanol after cooling on ice.

Reagents Required:

STET solution: 8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 5
(w/v) Triton X-100. Filter-sterilize and store at 4°C.

Diatomaceous Earth Method (reference here)

For those of you keen on DIY plasmid prep kits, this one is for you

Make a suspension of 50g diatomaceous earth (Celite) in 500mL dH2O and allow to settle for three hours, then collect the solid and resuspend in at 10mg/mL in the DNA binding solution (see below).

Then working in 0.5mL volumes (scale up as needed), add 1mL DNA Binding solution, shake and leave at room temperature for 2-5 minutes to allow binding. Centrifuge for 10 seconds to pellet the particles, and resuspend the wash buffer containing. Repeat this step, then wash the pellet with 1mL of acetone and dry briefly at 65oC. Elute the DNA by adding 50-200uL and incubating at 65degC for 2 minutes.

Reagents Required:

DNA binding solution: 4M guanidine thiocyanate, 50mM Tris pH 7.0, EDTA 20mM (store in dark, stable for 3 months).
Wash buffer: 50% ethanol, 200mM NaCl, 10mM EDTA, 50mM Tris-HCl pH 7.4

Zwitterionic Lysis Method (reference here)

This is a relatively gentle procedure for lysing the cells and for plasmid isolation that relies on the soft aggregation of cellular proteins which is known to contain a large amount of plasmid DNA (which is supposedly lost in other methods). Contaminants and RNA are simply removed using wash steps, and the plasmid is eluted with TE or water.

Centrifuged cells are re-suspended in Tris (50mM)–EDTA(10mM) buffer (50–10 mM) pH 8.0 and allowed to incubate for at least 1 minute in an equal volume of 0.2M NaOH and 10┬ÁM 3-(dodecyldimethyl-ammonio) propane sulfonate. Prior to centrifugation, 1mL of wash buffer comprising 10mM Tris (pH 8.0) and 10mM NaCl is added to the aggregate, after which the sample is centrifuged at max speed, most of the supernatant is removed carefully (takes some getting used to), and the wash step is either repeated, or the plasmid is eluted with TE or dH2O and the protein aggregate then discarded.

Liam's Plasmid Method

Ok, so this hasn't been published, and no I haven't tested it, but I'll bet a pair of old socks that it works like a bomb. I thought whilst writing this piece, that a combination of these methods that is quick, cheap and not too dirty would be helpful. I have also added a tweak using arginine in the lysis buffer that has been reported to give better quality DNA. So this is what I've come up with:

1, Spin down your pellet and resuspend pellet in 1/3 the original culture volume with STE buffer, vortex to resuspend and wash, then spin down again at maximum speed.

2. Resuspend by vortexing your pellet in 3/10 the original culture volume with buffer 1.

3. Add an equal volume of L-arginine lysis buffer. This can be left for longer than the normal 5 minutes with no problem (but not obviously overnight), mix well, but do not vortex. This high, but controlled pH solution should result in less denatured plasmid and prevent co-precipitation with protein and peptidoglycan fragments, resulting in better restriction digests later.

4. Neutralise the solution with an equal volume of 5M potassium acetate (pH 4.75) (Lithium and Sodium acetate work as well, provided they are made with equimolar quantities of acetate salt and acetic acid)

5. Spin the lysate down at maximum speed for 10 minutes then transfer the supernatant new tube and the precipitate the plasma DNA with 0.8 volumes of isopropanol which can then either be chilled at -20degC for 15 minutes, or spun straight away at max speed

Reagents Required:

Buffer 1: 50mM Tris, 10mM EDTA pH 8.0, 100ug/mL RNase A

L-arginine lysis buffer: 1% SDS solution containing 0.5M L-arginine (pH 11.7)

Neutralization solution: 5M potassium acetate (pH 4.75)

The L-arginine lysis buffer itself can be used as a substitute for the lysis buffer in commercial column-based methods (remember to recycle the columns!) and will give an increased yield and a better quality DNA

Although there is a small cost addition with the use of the L-arginine buffer, the amount of time and expense saved by eliminating failure of enzyme reactions and sequencing makes it worth it.

Please let me know if this method works for you, and yes, I will be trying it in the next few weeks and will post comparative results right here - so watch this space!

Symbiotic microbes induce profound genetic changes in their hosts [Think Gene]

Posted: 29 Jul 2008 03:29 AM CDT

Andrew: Microbes are everywhere, so a coping mechanism to survive in a world of bacteria seems evolutionary necessary. We traditionally consider our “anti-non-self ” immune system as that coping mechanism, but organisms may have also evolved a system of stable microbial coexistence. How? By inter-species gene regulation.

This is particularly interesting to me because it shows how genomics isn’t about closed, procedural, self-describing systems. Rather, genomics is contextual, recursive, and non-deterministic, like how DNA is part of the phenotype described by the genotype described by that DNA. Even better, the confusion isn’t limited to a single organism’s genome: here, squid genomic expression is modified by symbiotic bacteria to create an stable environment for the microbes and a novel phenotypic advantage for organism. So, evolutionary selection may act not only on what potential phenotypes a genome describes itself, but how that genome may be expressed as influenced by the expression of other organisms’ genomes and the environment.

…And you want to go back to “genes determine traits?” Boring!

Though bacteria are everywhere — from the air we breathe and the food we eat to our guts and skin — the vast majority are innocuous or even beneficial, and only a handful pose any threat to us. What distinguishes a welcome microbial guest from an unwanted intruder?

Research from the University of Wisconsin-Madison suggests the answer lies not with the bacteria, but with the host.

A study appearing online this week in the Proceedings of the National Academy of Sciences may help reveal what sets a platonic relationship apart from a pathogenic one. In the paper, researchers from the UW-Madison School of Medicine and Public Health and the University of Iowa identify a slew of microbe-induced genetic changes in a tiny squid, including a set of evolutionarily conserved genes that may hold the secrets to developing a mutually beneficial relationship.

“Interactions of animals with their microbiota have a profound impact on their gene expression, and to create a stable association with a microorganism requires a lot of conversation between the microbe and the host,” says UW-Madison medical microbiologist Margaret McFall-Ngai, senior author of the new study. (more…)

The challenge of promulgating bear facts. [Omics! Omics!]

Posted: 28 Jul 2008 10:05 PM CDT

I had an opportunity this evening to briefly review the impact of DNA research on the taxonomy and conservation of Ailuropoda melanoleuca which also made me reflect on the frustrating struggle of scientific fact to struggle to the public forefront. Put more simply, we had a bedtime discussion of pandas, DNA, relatedness & poop.

As I've mentioned before, some innocent parental actions resulted in the strong imprinting of pandas on my greatest genetics project, so that our house is now filled with various likenesses of the great bicolor Chinese icons. That I can see only 3 where I am sitting now is surprising -- and partly reflects the fact it is dark outside. We have numerous books on giant pands and the school & public libraries have supplied more, and tonight a new little book from Scholastic arrived mysteriously on TNG's pillow. He was eagerly reading it when he came to the fateful passage "It says they're not bears!". But 'The Boy' knows better, and he knows why.

This is a recurring theme in panda books. For a long time the taxonomic placement of pandas was a matter of great dispute, with some assigning them bearhood, some placing them with raccoons, and some allotting pandas a unique clade. A related question concerned the affinity of giant pandas for red pandas and red pandas with the other carnivores. Finally, in the late 1980's the problem yielded to molecular methods, with the clear answer that pandas are bears, albeit a the root of the ursine tree.

What's surprising is how slowly this information has moved into the world of children's books. Of course, the public & school libraries often have books which predate the great resolution, so they are forgiven. Some explain that pandas are bears, but fail to give the evidence. And a few have caught up. But this Scholastic book wasn't one of them, despite having an original copyright solidly after the molecular studies AND a bunch of professors listed as advisors.

Given that TNG is so fond of pandas, and it is no secret, there are those (often adults) who will attempt to dissuade them in their bearness. So I've tried to coach him in how to go beyond simply asserting that they are bears, but explaining why science classes them so. And for an eight year old, he can give a pretty good 1-2 sentence summary.

Which leads us to scat. He merges the two a bit, certainly because of the affinity of his age group for matters excretory (which, of course, his cunning father considered in introducing this topic!). A key question in panda conservation is how many are in the wild. Between their secretive habits and dense bamboo forest habitat, it is difficult to spot a panda in the wild, let alone make a census (nevermind those questionnaires!). So, as with many wild animals, DNA from panda scat is a convenient way to track individuals, and with this tracking the estimate of the number of pandas has shot up -- from the really depressing (to panda fans) ~1500ish to perhaps about a thousand more -- still in grave peril as a wild species, but a thousand more pandas napping in the woods is something to cheer. Unfortunately, the items on pandas in kids magazines & kids sections of newspapers still often quote the older figure.

A similar sort of experiment came up as an item of controversy earlier this year. There are many things I find admirable about John McCain (which is not synonymous to say I'm voting for him -- I haven't decided & I won't tell once I do!), but his pandering about a bear issue earlier this year wasn't one of them. In his fight against congressional earmarks (a good thing), he had singled out a study in Yellowstone National Park's which was sampling DNA from grizzly scat. Amongst his assaults on this study was the question asked loudly of what good this would do beyond setting up a bear dating service. Now, on the one hand I think scientists should be carefully thinking why this important study is apparently being funded by earmark and not peer review. But it is truly sad when you can explain population sampling to an eight year old, but not to someone older than his father who wishes to run the country. Yes, bear counting isn't quite on the same scale as some of the other great scientific issues which are being discussed this election year. But, given that the source behavior of that study is often cited as a benchmark for veracity ("Does a bear..."), it wouldn't be a bad one to get right.

reporter gene statistic [Reportergene]

Posted: 27 Jul 2008 02:31 AM CDT

According to my site meter, in the first 2008 semester, Reportergene received visits mainly from US, and most of them were from California. However, US visitors were speed-readers: they spend about 58 seconds on site, while Norvegians stay more than 4 minutes. 10% of visitors come back again at least three times, and two companies really involved in reporter assay development showed their loyalty at the top ten (according to network IP locations). Comparing biological sciences sites of similar size, Reportergene benchmarks more visits (4x) and more pageviews (3x).

Together, the data show that there is a hidden niche of people interested in functional reporter-genomic and my goal for 2009 is to pull out this community. Does anyone would help me?

No comments: