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Step 1: The Parts
Step 2: Putting the rack together - The Rack
Nothing to it!... Just thread two of the pipes into the matching 1/2 inch inputs in the two "T' connectors.
Step 3: Putting the rack together - Bang em in the ground
Take the back of your axe or perhaps a heavy mallet and drive the pipes with their connectors into the ground about a foot. These obviously need to be spaced on either side of your intended campfire location and can't be farther apart than the length of the horizontal pipe (I used a 48 inch pipe but you can employ longer pipe if you like).
You'll notice that while the pipes are 1/2 inch in inner diameter, the straight-through inputs of the T-connectors are 3/4 inch in inner diameter. This allows the pipe to slide through the connector.
Step 4: Putting the rack together - The Hangers
Snap a carabiner or S-hook onto each end of the two lengths of chain.
Step 5: Putting the rack together - The Hangers
Slide one carabiner/chain end onto the pipe. Repeat for as many chain hangers as you've chosen to employ.
Step 6: Attach your Ovens and Light the Fire!
Adjust the height as you deem necessary by passing the loose end of the chains through the pot bails and snapping the carabiner to the appropriate chain link. It's easy to modify the height of the pot above the fire and control temperatures as necessary! Enjoy! - "Outdoor Ed" Livesay
One question though. I've cooked on an open pit like this several times before, and always on a 4' X 1' fire pit under 1/2" cold rolled fire irons. They always felt solid. My top bar will be 5' and I'm going to be hanging two 16" Dutch ovens and a couple coffee pots on the top bar. The DO's are around 40 lbs a piece when empty. How solid is the half inch pipe? I don't want to get everything going just to have it collapse in the fire. I was thinking of stepping up to 3/4" and 1" fittings.
Any thoughts?
Hmmm... Good question. Just to err on the safe side, I'd say that going to 3/4" pipe would certainly do the trick! Thanks! Ed
Thank you so much. This is exactly what I was looking for. I am trying out a one month project where all dinners will be cooked outside weather permitting. I am curious to see the cost savings over using my old indoor stove that cost a fortune to run. Plus I have a few garbage bins of scrape oak, pine and cherry to use up. This will save me a lot of money. Thanks
Oh, and did I say it ? Thanks again :)
- i see you pound the uprights about a foot in the ground.
For an all round oven rack - you could use the same priciple of screwing the piping together but go for an A-frame style on each end.
No pounding of the uprights in the ground and might be a bit more stable all round.
I'm just thinking of if it rains and the ground soaks and your pots/ovens are full - the wind starts blowing - pots / ovens start swinging - you may find your next meal on the ground being eaten by the critters.
Plus with an A-frame style you can go with lighter weight piping for the uprights.
another idea just looking at it - to prevent the pots chain from slipping you could use say 10" pieces of piping - with a coupler 1.5"nipple - then coupler then 10" piece and the same again to make up the horizontal support (top pipe) the nipple in the middel of the couplers would prevent the chain from slipping side to side or down the pipe - say if you tip the pot/oven to one side - like scooping out that last deliscious drop of campifire stew...?
just some thoughts... but nice job and one last question...
So... when is dinner being served?
Come on down... we'll drink breakfast together!
I also like the suggestion to add tee-connectors to the bottom of each leg. To avoid having to stake the connectors to the ground, though, you could run another length of pipe through each of the bottom tee-connectors to make the whole thing stable and free-standing.
Step 3 - Perhaps putting a piece of wood on the pipe before whacking it with the back of an axe might be a tad safer for the metal. Black pipe tends to crack or shatter when struck repeatedly. If this critical point breaks we'd starve. ;)
Another thought - if you put Ts at the bottom of the vertical pipes you might not need to pound this in the ground. You could anchor the inverted Ts with a few stakes made out of rebar bent in a U shape.
I did vote for your instructable.


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In an interview with The New York Times, Google's CEO says we should focus on the potential benefits of technology.
This much is clear: Things are getting frothy out there. But does the stock market's reaction to Twitter's IPO mean another tech bubble is forming?
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Users of the site for sharing geotagged photos are upset Google is migrating to its Views service instead, and now the founders want Google to reverse course.
The new wearable and pair of smartphones have given a jolt to the tech industry. CNET looks at the reverberations for Apple's rivals and partners.
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Real Pets, Not Real Celebrities.
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You love animals? You also love Michael Jackson, Audrey Hepburn, Charlie Chaplin and the Simpsons? If you nodded at both the two questions, we bet you must love these special Pet Presents launched by the British-based company Takkoda.
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Killing an indoor herb plant during the winter is almost like a rite of passage for gardeners. So don't worry if your rosemary croaked in the past. The problem is most likely your house, not you. Low indoor humidity and light levels have knocked off more than a few indoor herbs, especially when combined with overzealous watering. Experts often recommend growing herbs under artificial light, but who wants a shop light hanging in their living room? So we came up with the perfect compromise: concealing a fluorescent light inside a bookcase. This technique allows you to give your herbs plenty of light, and it's a pretty handy place to store, say, your back issues of Organic Gardening!
What you'll need:
- A bookshelf, preferably with adjustable shelves at least 18 inches deep and crown molding or other trim that you can conceal the light behind
- A fluorescent light fixture that fits inside the bookcase
- A drill
- Screws
- An automatic light timer
1. Drill a hole in the back of the case near the top for the light fixture's cord.
2. Place the bookcase upside down on the floor, fit the light fixture into the bookcase, and attach it to the top with screws.
3. Turn the case upright and put it in the desired location. Fill a shallow tray with pebbles or crushed glass and set your herb containers onto the tray. Adjust the top shelf so that the herbs will be about 1 or 2 inches below the light. Place the herbs under the light fixture and fill the tray of pebbles with water, which will slowly evaporate and improve the humidity around your plants.
4. Plug the light into the timer. Set the timer so that the herbs receive 14 hours of light and 10 hours of darkness each day. Allow the herbs' soil to nearly dry between waterings and hold off on fertilizer until the plant begins to grow vigorously in spring.


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July 2, 2015
A convicted drug dealer, charged with shooting a man at a local gas station last August, will not be going to prison.
It took Bay County jurors about 20 minutes to find 24-year-old Canom "Candyman" Williams not guilty of aggravated battery Tuesday afternoon.
Williams allegedly shot a man named Arthur Collier on August 28 at the Cannon Oil gas station in the 1500 block of Martin Luther King Jr. Blvd.
Collier claimed Williams opened his car door, put a 9-millimeter pistol to his thigh and fired. The bullet shattered Collier's femur bone. Despite the wound, he drove himself to a local hospital for treatment.
Apparently the jury didn't buy the story and acquitted Williams.
Williams previously been charged with possession of cocaine with intent to distribute and possession of narcotics paraphernalia. Both of those charges are from 2000.
In 2001 he faced charges of battery, resisting arrest with violence, witness tampering and aggravated assault. In all, he served 22 months in jail.
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Many of our customers come to us seeking a solution to one or more of the following problems:
- "Rotten Egg" Smell
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If you are experiencing any of these problems, Clearwater Systems is ready to help. For a free home water analysis, please fill out the form below and click submit when you're ready. Your local Clearwater Systems professional will be in touch within 24 business hours to schedule your free drinking water analysis appointment.


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Essay - Human Need for Privacy Environmental Psychology is the Study of...
Human Need for Privacy
Environmental psychology is the study of the interaction between the physical world and human behavior. In recent years, the topic of privacy has been a key aspect of this field ***** study, as research reveals that it directly relates to a sense of well being and control. A supportive physical environment has been proven ***** contribute to a successful soci*****l, private ***** work life, and privacy is a ***** f*****ctor in creating a positive environmental setting.
Why We Need *****
There are many theories as to why people need *****. Many psychologists believe that people need to maintain personal space between themselves and others to avoid overstimulation. ********** to Scott (1993), when people ***** too close to one another, they are bombarded with too many social or physical stimuli.
Some *****ers argue ***** people ***** to maintain personal space ***** avoid various stressors associated with very close proximity. This group says that when ***** space is inadequate, ***** have feelings of arousal.
Altman (1975) views personal space as a boundary regulation mechan*****m needed to meet desired levels of ***** and group privacy. According to Altman, ***** is an interpersonal boundary process by which people control interactions with *****.
Through variations in the extent of *****ir private space, people make sure ***** their desired and achieved levels ***** privacy are consistent. If it is impossible ***** control ***** regulate these boundaries so that the desired level of privacy is met, negative effects and coping are the result.
When ***** purposely invade other people's privacy, the s*****uation often involves ***** consequences for both parties. Felipe ***** Sommer (1966) conducted a ***** experiment *****t a 1,500 bed mental institution in which a stranger approached lone patients at a distance of six inches and sat down. If ***** participant attempted to move away, the stranger *****d so ***** to maintain a close proximity. The majority of the group showed signs of discom*****t and many got up and left.
***** a sep*****rate experiment ***** Felipe and Sommer (*****), female students were *****ing at a large table with six chairs on either side of the table, with ***** least two empty chairs on either side of each subject, and one opposite. *****re were several experimental conditions, including:
The experimenter sat next to the ***** participant and moved his chair nearer to hers (close).
Sat ***** seats away from her, leaving ***** chair in between them (distant).
Sat three ***** away (distant).
***** ***** across from her (control).
***** *****s of this study were:
Left within 10 minutes
Still working after half-hour
Privacy Factors
Privacy can be broken down into the following classifications: Kaplan (1982)
Solitude: total visual privacy;
Intimacy: privacy, within a small ***** unit, such as a family at home a*****;
*****: privacy, ***** a social *****, where one is able to observe without feeling observed; people merging with the environment;
*****: a psychological barrier against intrusion, allowing people to be ***** within gro***** situations.
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Oberndorf Scholarship Application Due
Friday, May 30, 2014
Recently, many Phi Theta Kappa members across the country have been impacted by severe weather and natural disasters. To provide as many students as possible with the opportunity to complete the Oberndorf Lifeline to Completion Scholarship, the application deadline has been extended to Friday, May 30, at 5 pm CDT. No late or incomplete applications will be accepted after May 30.
The Oberndorf Lifeline to Completion Scholarship helps Phi Theta Kappa members overcome unanticipated financial barriers preventing the completion of a student's first college credential, an associate degree or certificate, while enrolled in the two-year college.


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Small and Medium Enterprises (SMEs) can access longer-term financing through capital markets even if they do not qualify to the listed on the stock Exchange, financial experts have said.
Speaking on Tuesday during the launch of the findings of a study titled: 'Leveraging Capital Markets for SME Financing in Rwanda,' the executive director of the Capital Markets Authority, Robert Mathu, said that SME's need not list on the Rwanda Stock Exchange in order to access longer-term funds. "There are other avenues such is issuing of bonds," he said.
The study, a joint effort of the African Development Bank (ADB), the Government of Rwanda and the Capital Markets Authority, is a contribution to measures aimed at improving access to finance by small enterprises.
Small and medium Enterprises are generally considered the "pulse" of Rwanda's economic activities because they employ the largest portion of the country's workforce.
While several initiatives have been put in place to support SME's in their search for longer-term funding, there are still some challenges mainly related to the nature of the businesses.
The biggest challenge is that commercial banks continue to shun SMEs because most of them have no business plans, don't proper books of accounts and have no cash flow projections among others issues. As a result, banks find it hard to lend to business whose entire existence may be dependent on an individual.
The ADB resident representative for Rwanda, Negatu Makonnen, said that the study is aimed at "complementing the bank's lending operations by sharing lessons of experience from other countries where capital markets and related financial instruments have been used to finance SMEs."
Success stories have been cited from Ghana, Korea, South Africa and Brazil.


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Summer Commencement Ceremony 2015
- Family & Education
- 285 W. Huntington Drive Arcadia, CA 91007, US
Silicon Beach LA's Weekly Unwind At The Bungalow (Donate Any Amount To Register)
Silicon Beach LA
Friday, January 4, 2013 from 5:00 PM to 7:00 PM (PST)
Mingle and talk shop- in a super casual setting- with other founders, fans, financiers, and faces from here in #SiliconBeachLA. No panel, no opening/closing speech, no open bars, and definitely no hors d'oeuvres. Just friends, colleagues, a bar, and a view of one of #SiliconBeachLA's greatest assets...[hint is in the name]
We're using HereOnBiz, a local #SiliconBeachLA area company's networking app that you can download in the app store. When you arrive, check into the event; "Silicon Beach LA's Weekly Unwind At The Bungalow" to see the full attendee list with their LinkedIn profiles. Saves you from having to work the room blind and we use it all the time, and yes we gotz the rhymes!!! (we couldn't help that last one)
And we're using TapShare, another local #SiliconBeachLA area company's social photos app that you can download in the app store. When you arrive, check into the event; "Silicon Beach LA's Weekly Unwind At The Bungalow" to share your own photos and see others taken by everyone else. Shared mobile/social photo album for the event = PURE WIN
And finally, we're using ParkMe, another local #SiliconBeachLA area company's mobile parking space location app. There is a main structure for parking just south of Wilshire on 2nd street, unless you prefer to valet for $5-10 at the door.
Your Personal Tour Guide To The LA Tech & Startup Community.
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


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CANADA seems set to join the US in reneging on the Kyoto Protocol on climate change unless it gets key concessions, which the European Union adamantly opposes.
Canadian Prime Minister Jean Chrétien told a meeting of EU ministers in Madrid last week that he was "not in a position to ratify until some elements are clarified". These elements are believed to be Canada's demand to gain credits under the protocol for exporting hydroelectric power and natural gas to the US.
Canada claims that the gas will replace coal in US power stations. Because burning gas produces less carbon dioxide than burning coal, that would reduce the US's contribution to global warming, it says.
Just six months ago, the Canadian government joined every industrialised nation bar the US in signing up to the protocol, which includes detailed provisions covering carbon credits. But it has since come under heavy pressure from its ...
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Photo by Romulo Yanes
YIELD: Serves 4
ACTIVE TIME: 30 minutes (Includes making the adobo)
TOTAL TIME: 2 1/2 hours (Includes making the adobo)
Make the adobo:
Heat a comal, griddle, or heavy skillet over medium-low heat, and toast the chiles 2 or 3 at a time, turning them over and pressing down on them with tongs frequently, until the chiles are fragrant, about 1 minute per batch. Soak the chiles in enough cold water to cover until they're soft, about 30 minutes. Drain and discard the soaking water.
Put the vinegar and beer in the blender jar with the chiles and the remaining adobo ingredients. Blend until smooth, at least 3 minutes, adding a little water if necessary to puree. If you like a silky, smooth texture, strain the adobo through a medium-mesh sieve. Set aside 1/2 cup of the adobo to marinate the steak, and keep the rest in an airtight container in the refrigerator for up to five days or in the freezer for up to one month.
Make the steak:
Pat the steaks dry, season them with the salt, then coat them generously with adobo. Let them marinate in the refrigerator for 1 to 2 hours.
Heat a grill or grill pan over medium-high heat. Lightly oil the grill or grill pan and cook the steaks, 3 to 5 minutes on each side for medium-rare, depending on their thickness. Let the steaks rest for 5 minutes before slicing for tacos or serving as whole steaks.
Adapted with permission from Truly Mexican by Roberto Santibañez with JJ Goode, (C) 2011 John Wiley & Sons, Inc.


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Gatling Named to Naismith Midseason Top 30 List
Feb. 12, 2014
CLEMSON, S.C. – Just two days after being named to the Wade Watch List, senior center Markeisha Gatling of the No. 10 NC State women's basketball team (21-3, 8-2 Atlantic Coast Conference) has been named to the 2014 Naismith Trophy Women's Midseason Top 30 List.
Gatling has had a fantastic senior season for NC State, averaging 17.2 points and 6.7 rebounds per game. She has held the highest shooting percentage in the country since Dec. 22, 2013, and has scored in double figures 23 straight times.
The Naismith Trophy presented by AT&T is awarded annually to the men's and women's college basketball players of the year.
Each year, the Atlanta Tipoff Club's Board of Selectors chooses the 50 pre-season "Naismith Watch" list candidates as well as the mid-season team of 30 candidates. The Naismith voting academy narrows that group down to four finalists who will make up the final ballot. The voting academy, comprised of leading basketball journalists, coaches, and administrators from around the country, bases its criteria on player performance throughout the season.
NC State will next be in action on Thursday, Feb. 13 at Clemson. Due to winter weather in the area, a tip-off time is still being determined.


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about.me – dancing to the beat of my own drum
Helen Ross bio
Australian writer, poet, children's author, educator, workshop presenter.
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My Very Close Shave tale on Gabrielle Bryden's blog
Talented writer, poet and blogger, Gabrielle Bryden, recently wrote of her close shave with an aquaplaning bus. OMG! Subsequently, Gabrielle expressed an interest in receiving any close shave poems or stories from regular readers of her blog and people she … Continue reading
Weekly Photo Challenge: Threes
This week's 'The Daily Post' photo challenge is threes. So my photos are based on the 'Three Picture Stories' (storytelling with images) that I posted about last Tuesday. All these photos were taken at my local park, except the last … Continue reading


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Is hay a pollutant?An outspoken cattleman, at odds with the Environmental Protection Agency, says the agency has declared hay to be a pollutant, at least in his own case. Mike Callicrate, a Kansas cattle feeder and member of the Ranchers-Cattlemen Action Legal Fund, United Stockgrowers of America, also says the EPA is working to drive small- and mid-sized feedlots out of business for the benefit of meatpackers and big corporate feedlot operators.
By: Jonathan Knutson, Agweek


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1 User Response
Well, my one year lease is almost up and I will not be re-newing. The place is okay, but I've live din better. Parking is horrible, I'm always afraid someone will hit or ding my car. The walls are paper thin, I hear my neighbors arguing day and night. The bug situation is the main reason I am leaving though. The day I went to make toast and found ants all over my kitchen was a day I will never forget. I told management they but two combat bait traps in the kitchen, that was their solution. Well, the ants just moved futher into the apartment making their way into the bathroom. I woke up one morning to find a nest in the bathroom cabinet, they were all over my toothbrush. Thankfully winter came and I have not seen the ants since, however, the spiders, the 1000 legged sliverfish, and countless black bugs still reside in the place. I don't dare bring family or friends over. If you enjoy insects then you will like willow crest, if not take my advice keep looking. God forbid I ever see a roach, management will have a lot to answer for.
I'm also have the ants problem, and decide to leave. They let me pay for one and a half rent penalty!!!!!Such a bad management, I don't know whether I can get my security deposit back.
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Vikings sign tackle Carter Bykowski
EDEN PRAIRIE, Minn. (AP) The Minnesota Vikings have placed linebacker Michael Mauti on injured reserve and signed offensive tackle Carter Bykowski off San Francisco's practice squad. Coach Mike Zimmer said Wednesday that Mauti needed minor surgery on his knee. The second-year player from Penn State has played almost exclusively on special teams. Bykowski had been on the practice...
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WATCH: Minnesota's Michael Mauti Absolutely Crushes Tampa Bay Bucaneers KR Trindon Holliday
Trindon Holliday is a tiny man (5'5" and a 166 pounds), but he is a fast-as-heck tiny man. When he gets up a head of steam and has a little territory to work with he can really make some magic happen. There is just one problem. As small as he is when the opposition puts a helmet on him it looks really, really bad.Like it did Sunday when Michael Mauti of the Vikings cover team got...
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Mauti getting exposure at each linebacker position in Zimmer's defense
MANKATO, Minn. -- Despite the Minnesota Vikings trying to identify a middle linebacker, Michael Mauti was moved from his familiar spot in the middle to the outside in coach Mike Zimmer's 4-3 defense. First, Mauti was tried at the strongside spot. Then he was moved to the weakside. The moves would seem to indicate Mauti, the second-year player out of Penn State, was out of the running...
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The popular young professionals profile column "Keeping Our Eye On" has been a fixture of the MBJ for several years now and we're just getting started.
In 2013 alone, we've profiled 20 and 30-somethings in the fields of accounting, law, public housing, employment staffing, television, education and many others.
The columns have been a success story on both sides. It helps acquaint our publication with the rising stars in Mississippi's best and brightest companies and sectors while giving our subjects a platform to talk about their career and offer advice and fun facts to fellow young professionals.
As the summer approaches, feel free to drop me a line if you or someone you know would be a good fit for this column.
Also, feel free to pass the word along to family and friends and your church and civic/professional organizations.
We are interested in "keeping our eye on" all careers and industries. Thanks for reading!
Stephen McDill
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(601) 364-1041


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Image Source: swamplot.com
A fuzzy video is annoying as much as it is problematic. If you encounter this knid of problem, the forst thing to do is to buy a new miniDV tape, and check if the fuzzy display is still there. MiniDVs usually disintegrate and are rendered useless in a number of years. But if the problem is still there, you might have a bigger problem at hand. this is a very common problem for all camcorder owners. Most especially when you are livin gin a country that is humid most often. The problem is FUNGUS. Yes, fungus. Fungus grows in humid places. It grows inside your camera lens. One sign to tell its fungus is you'll see something knida spidery growing inside the lens. Store your camcorder in a cool dry place. Put some silica gel packs to fend off fungi attacks.


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Many schools and local businesses closed a little early Tuesday for in preparation for winter weather moving through Mississippi.
In the event of school closings, the Oktibbeha County School District will utilize its alert messaging system, which sends messages to parents' cell phones, home homes and e-mail.
The Starkville School District will make a decision regarding school closing early Thursday morning and will inform parents through the Alert Now system and the local news stations.
All parents should tune into the local news station Thursday morning for the latest in school closings.
According to the Associated Press, the winter storm forecasts call for up to five inches of snow in northwest Mississippi and an inch or less around Atlanta — enough to snarl traffic in a region with few salt trucks and plowing equipment.
Winter storm warnings were issued for an area stretching from northern Louisiana to Georgia.


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I posted this picture of an astronomically high price for a bag of fake pennies earlier. It reminded me of this piece by me also involving the penny.
All original images and writing © 2008 - 2015 The Looking SpoonCopyrighted images are used in a satirical and non-commercial context
The Looking Spoon is a right-wing, right-of-center, conservative blog that focuses on humor, satire, art, and general commentary. Posts are about the latest news in politics and current events covering topics such as politics, Republicans, Democrats, liberal and progressive lunacy. Other topics include as politicians such as Barack Obama, Nancy Pelosi, Harry Reid, Barney Frank, Al Gore and pop culture. All posts aim to be funny, witty, sarcastic, satirical, humorous, and biting while striving to also be sharp, incisive and insightful from a right-of-center point of view in an effort to make thelookingspoon.com one of the top conservative humor and satire sites on the web.


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Skin Tightening at Lubbock Laser Clinic
Sagging skin and wrinkles on your face are some of the most obvious signs of age. Until recently, the only ways you could fight the effects of time on your facial appearance involved injections or surgical facelifts, with results that too often resulted in an obvious, unnatural appearance.
Skin Tightening from Lubbock Laser Clinic offers you another safe & effective option. Our Skin Tightening treatment uses clinically-tested infrared and radio frequency (RF) energies to stimulate healthy collagen remodeling inside your skin & to tighten sagging skin. Unlike treatments that paralyze or mechanically tighten your skin, our Skin Tightening stimulates natural, healthy processes inside your skin that make it look and act younger and firmer.
You can achieve tighter, younger looking skin after as few as three skin tightening treatment sessions. Wrinkles will start to fill in, and sagging skin tightens up and return to its original contours. The benefits of Skin Tightening treatments can last as long as two years.
How does Skin Tightening work?
The infrared and radio frequency (RF) energies produced by our customized systems penetrate the surface of your skin to heat the collagen inside of it and to promote collagen remodeling over time, while immediately promoting skin tissue tightening as a result of deep heating. The treatment head actively cools the surface of your skin to minimize or eliminate any heat-related discomfort during the skin tightening treatment process. The cooling factor of the treatment head is adjustable to each individual patient's heat sensitivity.
Collagen is a key component of your skin's elasticity and firmness. Once collagen remodeling is promoted by Skin Tightening treatments, skin becomes naturally more elastic and firm, filling in wrinkles and bringing sagging areas back closer to their original contours.
Skin Tightening Safety
Unlike injections or plastic surgery, Lubbock Laser Clinic Skin Tightening is non-invasive and does not involve any breaks in the surface of your skin, so it does not present an infection risk or require any recovery period.
Your Laser Specialist will ensure that any concerns are identified before treatments begin to ensure your safety and comfort throughout the skin tightening process.
Skin Tightening Post-Treatment Care
As with any dermatologic procedure, your results will vary slightly based on your individual skin conditions and your post-treatment skin care.
For a free consultation, call our Lubbock location at 806-793-7700.
Our Services
Why Choose Us
- Free consultations
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Select Menu > Office > Converter.
The converter has limited accuracy, and rounding errors may occur.
Convert measurements
Select Menu > Office > Converter.
Scroll to the type field, and select Options > Conversion type to open a list of measures. Select the measurement type to use (other than currency) and OK.
Scroll to the first unit field, and select Options > Select unit. Select the unit from which to convert and OK. Scroll to the next unit field, and select the unit to which to convert.
Scroll to the first amount field, and enter the value to convert. The other amount field changes automatically to show the converted value.
Set base currency and exchange rate
Select Menu > Office > Converter.
When you change base currency, you must enter new exchange rates because all previously set exchange rates are cleared.
Before you can make currency conversions, you must choose a base currency and add exchange rates. The rate of the base currency is always 1. The base currency determines the conversion rates of the other currencies.
To set the exchange rate for the unit of currency, scroll to the type field, and select Options > Currency rates.
Scroll to the currency type, and enter the exchange rate you would like to set per single unit of currency.
To change the base currency, scroll to the currency, and select Options > Set as base currency.
Select Yes > Done to save the changes.
After you have set all the necessary exchange rates, you can make currency conversions.


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It's time for The Masters Tournament, golf's most prestigious title, get tickets >
Top 5 Games of Last Week (8/4/14 - 8/10/14)
I can't. I can't do it. I just can't put preseason NFL games on here. But I also can't abide a month straight of just MLB games, so I'm changing things up. #5. Little League World Series: Pennsylvania vs. Delaware At 5'4, weighing in at 105 pounds, it'sssssssss Mo'ne Davis! Without a doubt, the story…Read more


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Whoa hyperbole
Whoa hyperbole
Nobody else thought it sounded like crap? Out of tune singing n shit.
Goddamn Letterman is awful.
Last edited by Phantasma Del Mar; 01-28-2014 at 12:22 PM.
I know Daft Punk is very meticulous with their decisions regarding music, but I'm wondering if winning these awards are getting people to push them for a tour?
Good luck to us trying to get tickets for them if they tour again...it'll be like LCD Soundsystem @ MSG for every tour date.
Supposedly this is real
i cant open this link at work... please describe the just of the article for those with internet blocking. Fucking MUSIC category...
Supposedly it's a collaboration with Daft Punk and Jay Z.
My guess is that it is 100% fake.
May 15-17 - Psycho California
June 6 - The Rolling Stones
June 10 - Melvins
June 20 - Blur
June 24-28 - Glastonbury
July 18 - Death Grips?
July 26 - Faith No More
September 25 - Kraftwerk
Pitchfork is confirming that it is real.
Although no information is available about when it was recorded or if it will see release, "Computerized" seems to have been made before the Samsung-sponsored days of Magna Carta Holy Grail: "Start macking on my BlackBerry," Jay raps, "She got jealous/ I was tapping on my BlackBerry."
There have been rumors that Kanye West was somehow involved in the making of the song, but we can confirm that he was not.
It's so incredibly terrible. I wish it was fake.
5/23 Purity Ring, BRAIDS - Stubb's
5/30 Kate Tempest - Holy Mountain
6/05 Courney Barnett - Mohawk
6/06 Metallica - X Games
6/16 First Aid Kit - Stubb's
7/18 Death Grips - Mohawk
8/01 Gorilla vs Bear 5 - Mohawk
11/6-8 Fun Fun Fun - Auditorium Shores
07, 08, 09, 10, 12.1, 13.1, 14.1, 15.1


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Goldman Sachs Group Inc (GS) Financial News - Bank of America Leads Jump in Default Swaps on U.S. Lenders
Goldman Sachs Group Inc (GS)
Bank of America Leads Jump in Default Swaps on U.S. Lenders
2011-08-08 11:01:25Quote: A Goldman Sachs Group Inc. research team led by economist Jan Hatzius said in a note to investorsthat "S&P has indicated that it would not immediately downgrade any banks or broker dealers in relation to a sovereign downgrade†although "Moody's has indicated that it might in the event that it downgraded its U.S. Sovereign rating.†Swaps Prices Contracts on Charlotte, North Carolina-based Bank of America, which rise as investor confidence deteriorates, gained 24 basis points to 232 basis points as of 8:35 a.m. in New York, according to broker Phoenix Partners Group. Swaps on New York- based Morgan Stanley jumped 22.9 to a mid-price of 221.6, the highest since July 2010, prices from data provider CMA show. Goldman Sachs Group Inc. (GS)JPMorgan Chase & Co. (JPM)Wells Fargo & Co. (WFC) rose 10 to 120, according to Phoenix. Contracts on Citigroup added 17.3 to a mid-price of 179.8, CMA prices show. The contracts pay the buyer face value if a borrower fails to meet its obligations, less the value of the defaulted debt. A basis point equals $1,000 annually on a contract protecting $10 million of debt. email@example.com@bloomberg.net firstname.lastname@example.com ...
Open whole article (external link)
Other Financial and Stock Market News concerning Goldman Sachs Group Inc
Cash Greater Than Net Debt And 50% Discount To Tangible Book Value: Are US Banks Next Safe Haven For European Money? Exclusive Interview With 38 Year Banking Valuation Expert (2011-08-09 07:52:14)


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8th September 2012
Rowan Williams: Church 'wrong' about homosexuality but Cameron has embarrassed us on gay marriage 68
11:42 AM — In an interview ahead of his upcoming retirement from the role of Archbishop of Canterbury, Dr Rowan Williams has said that while the church was "wrong" not to have advocated gay equality, Prime Minister David Cameron was wrong to embarrass the Church of England over the issue of equal marriage.
4th September 2012
Lynne Featherstone moved from equalities role in UK Government reshuffle 48
7:26 PM — Liberal Democrat MP Lynne Featherstone is to leave her post as the government's equalities minister. It has been reported that she will move to a new role at the Department for International Development, while the Home Office will loose responsibility for the equalities agenda in government.
23rd August 2012
Bishop of Buckingham: 'Church should get its head around gay relationships and be @Out4Marriage' 68
1:04 PM — The Bishop of Buckingham has become the most senior member of the Church of England to join the Out4Marriage campaign to introduce equal same-sex marriage in the UK. But a spokesman for the Church says that his message of equality is not the official view of the Church of England.
20th August 2012
Poll suggests churchgoers less likely to vote Tory in light of PM's support for marriage equality 34
2:23 PM — A new poll suggests that many churchgoers in the UK are increasingly unlikely to vote Tory owing to David Cameron's promise to legalise marriage equality prior to the next election.
Video: Boris Johnson comes @Out4Marriage and says marriage is a 'happy state' 53
1:31 PM — The Mayor of London Boris Johnson has become the most high-profile Tory in the country to release a video message in support of the global campaign for equal marriage.
17th August 2012
Pressure piles on Boris over @Out4Marriage video promise 35
7:27 AM — The Mayor of London Boris Johnson is facing renewed calls to honour a commitment to record an Out4Marriage video after London Assembly Member Tom Copley submitted a formal question.
12th August 2012
Equalities Office to look into Boris Johnson's @Out4Marriage video pledge 59
11:29 PM — After months of failing to produce a video in support of the Out4Marriage campaign, London Mayor Boris Johnson's inaction is now being looked into by the UK Government's Equalities Office.
5th August 2012
Catholic Bishop: Government should make incest legal if it really believes in equality 191
11:23 AM — A senior Catholic Bishop in Scotland has stunned equal rights campaigners by suggesting that if the Scottish Government truly believed in equality it would make incest and polygamy legal.
26th July 2012
Video: Dan Savage says marriage is already 'about love, not gender' 3
10:31 AM — Author, columnist and founder of the 'It Gets Better Project' Dan Savage has rejected arguments that gay people are seeking to 'redefine marriage', saying it has already effectively gender-neutral.
25th July 2012
Anti-abortion activists say David Cameron will 'pay' for same-sex marriage policy 49
5:24 PM — Following Prime Minister David Cameron's speech reaffirming his commitment to allowing gay couples to marry, anti-abortion activists the Society for the Protection of Unborn Children (Spuc) says he will get punished at the general election for his pro-equality stance.
23rd July 2012
Video: Dean of St Albans believes God 'is Out4Marriage too' 56
2:16 PM — Dr Jeffrey John, the gay Dean of St Albans, has said God would be in favour of government plans to allow gay couples to marry and said the Church of England's opposition "doesn't deserve to be listened to".
16th July 2012
Video: Equal marriage film seeks votes for national awards 6
11:41 AM — Makers of the equal marriage short film Homecoming, which was released earlier this year to support government plans to legalise gay marriages in England and Wales, are appealing for help promoting the film to show in cinemas nationwide.
24th June 2012
Anti-gay marriage group 'Keep Marriage Special' says UK Government will legalise incest next 163
3:12 PM — A new campaign against same-sex couples marrying, Keep Marriage Special, has claimed in glossy leaflets that the next logical step will be legalising incest and polygamy. Out4Marriage has branded the group's claims "extremist and confused".
20th June 2012
Video: Deputy Prime Minister Nick Clegg says equal marriage no longer a matter of 'if' in @Out4Marriage video 45
10:35 AM — The Deputy Prime Minister and leader of the Liberal Democrats, Nick Clegg, has reiterated his support for marriage equality between gay and straight couples in England and Wales, affirming that it is no longer a question of 'if' the move will go ahead.
17th June 2012
Anglican Mainstream: Gays marrying will lead to children being taught about 'eating human faeces' 180
8:59 AM — Writing about her letter to her MP, Dr Lisa Nolland, lay minister and blogger for Anglican Mainstream, has claimed that legalising same-sex marriage will lead to children being taught risky sexual practises in school, including "eating human faeces".
16th June 2012
Home Office Minister Nick Herbert: Churches should be allowed to marry gay couples 39
10:53 AM — Nick Herbert the Minister for Policing at the Home Office and Ministry of Justice has said that churches that wish to solemnise marriages of gay couples should be allowed to do so. He said the Church of England's current position has made him feel "distant" from his own faith.
3rd June 2012
Video: Australian celebrities launch a high-profile campaign for equal marriage 10
5:37 PM — As the Out4Marriage campaign gathers momentum in the UK, celebrities in Australia have lent their voices to a high-profile equal marriage campaign, launched today.
30th May 2012
Video: Oona King comes @Out4Marriage to redress 'outrageous' marriage inequality 8
6:31 PM — Labour politician Oona King has become the latest to film a video for the Out4Marriage campaign, saying her support is inspired by the fact that her parents' mixed-race marriage would have been illegal in some parts of the US at the time they wed.
29th May 2012
Video: Shadow Chancellor Ed Balls calls for equal religious marriage in @Out4Marriage video 24
1:08 PM — The Shadow Chancellor, Ed Balls, has joined the Out4Marriage campaign with a video message calling on the government to allow religious bodies to marry gay couples if they choose, inspired by his late, gay uncle.
28th May 2012
Video: Shadow health secretary Andy Burnham supports equality with @Out4Marriage film 14
6:23 PM — Shadow health secretary Andy Burnham has become the latest politician and one of the most senior Catholic figures in British politics to record an Out4Marriage video.


text:
The present invention provides substrates having a plurality of micro-locations on its surface. Each micro-location has an effective dose of an ion beam treatment such that the plurality of the micro-locations exhibit an affinity to a compound that is different from the affinity of the remainder of the substrate surface to that compound. The substrates of the invention can be utilized to form microarrays of biological molecules, such as oligonucleotides or peptides. Such microarrays can find a variety of applications. For example, they can be employed in large scale hybridization assays in many genetic applications, such as mapping of genomes, monitoring of gene expression, DNA sequencing, genetic diagnosis, and genotyping of organisms.
1. A bioanalytical microarray, comprising:
a surface with a plurality of micro-locations, wherein the plurality of micro-locations is in discrete regions of the surface and forms a pattern, and each of micro-locations has a dose density of implanted ions in a range of about 1012 to about 1017 ions/cm2 such that the plurality of micro-locations has a surface energy different from the rest of the surface and functions as potential energy locations, and exhibits a affinity for a compound which is different from the remainder of the surface, and
a plurality of macromolecules confined to the micro-locations.
2. The microarray of
3. The microarray of
4. The microarray of
5. The microarray of
6. The microarray of
7. The microarray of
8. The microarray of
9. The microarray of
10. The microarray of
11. The microarray of
12. The microarray of
The present invention provides substrates on which a microarray of molecules, such as a microarray of oligonucleotides, can be formed, and further provides methods for generating such microarrays.
Microarrays of hundreds or thousands of biological compounds are widely utilized for biological analysis. Tiny droplets, each containing a different known reagent, usually distinct polynucleotide or polypeptide biopolymers, such as, known DNA fragments, can be deposited and immobilized in a regular array on a solid substrate, such as a glass microscope slide. The array of reagents can be exposed to an analyte solution containing unknown target sequences, for example, complementary DNA (cDNA) fragments, pre-labeled with fluorescent or radioactive chemical labels. Under certain conditions, binding reactions or hybridizations occur between a polynucleotide sequence in the array and a complementary sequence in the analyte. Subsequent optical or radiosensitive scanning procedures can be used to determine those position(s) in the microarray at which hybridization reactions have occurred, thereby identifying the complementary compounds present in the solution.
Various methods for immobilizing synthesized oligonucleotides or peptides on a substrate surface via chemical reactions are known. Such methods typically attach linking groups to a substrate surface at pre-defined locations, and connect known nucleotide or amino acid sequences to these linking groups, in order to create microarrays. One drawback of such methods is that the oligonucleotides or peptides in the microarrays are immobilized in particular configurations, which may inhibit optimal interactions between target sequences in test samples and the immobilized oligonucleotides or peptides.
Another known method of generating microarrays utilizes agarose gel pads placed at predefined locations on a flat surface. In one application of such microarrays, oligonucleotides of a known length and sequence are placed within each gel pad along with a test sample containing the target sequence. Each gel pad provides a three-dimensional environment in which the oligonucleotides and the target sequence in the test sample can react.
In many of the known methods for forming microarrays, the interactions between the immobilized molecules and their complementary counterparts can be hindered by the presence of linking groups, and/or the specific orientations of the immobilized molecules. Further, even in microarrays that utilize three-dimensional gel pads, although the oligonucleotides are not immobilized, the interactions between the oligonucleotides within the gel and the test sample are not optimal because they occur within the confines of a gel pad rather than in an aqueous solution.
In addition, conventional microarrays have limited densities largely as a result of the shortcomings of the methods utilized for generating them. For example, fixation of reagents on a substrate to form a microarray can require the presence of large bulky protective groups that often limit the density of the array. Likewise, when agarose gel pads are employed for forming a microarray, there is an upper limit for the density of gel pads on a substrate.
Accordingly, a need exists for microarrays in which optimal biological reactions can occur between reagents deposited in, or on, a micro-location of the array and a target molecule in a test sample. A need also exists for microarrays having higher densities of micro-locations suitable for containing reagents for biological reactions.
In one aspect, the present invention provides a bioanalytical microarray that includes a surface with a plurality of micro-locations in discrete region thereof, each of which is subjected to an ion beam treatment such that the location has a differential affinity for a compound than the remainder of the surface. The microarray further includes a plurality of macromolecules confined to the micro-locations.
The ion beam treatment can include, for example, implanting an effective dose of an ion, e.g., argon or nitrogen, in each micro-location such that the micro-location has an affinity for one or more selected compounds that is different than the affinity of the remainder of the surface, i.e, the portion of the surface not treated with an ion beam, for those compounds, or alternatively, each micro-location can be subjected to an ion beam assisted deposition (IBAD) process to selectively modify one or more of its properties.
The invention can be practiced on a variety of surfaces to form micro-locations thereon having modified surface properties. Such surfaces can include, for example, glass, silicon, nylon, an amino or carboxy activated plastic, cellulose, or a silane-coated glass, GaAs, GaN, metal-coated glass, such as silver or gold-coated glass. An ion implanted at a micro-location is typically selected based on the desired modification to the surface properties of that micro-location. Any ion that is amenable to implantation in a given surface, and leads to a desired surface modification can be utilized. Such ions include, but are not limited to, nitrogen, oxygen, argon, carbon, fluorine, chlorine, hydrogen, and helium. Further, more than one ion type can be implanted at a micro-location to modulate selected surface properties of that micro-location. The modified surface properties can include, for example, different wettability, i.e., hydrophilicity or hydrophobicity, or ionic binding relative to the remainder of the surface.
In a related aspect, the dose of the ion implanted at a micro-location is selected to be a range of about 1012 to about 1017 ions/cm−2. Since the area of each micro-location will be typically smaller than a square centimeter, the actual dose of ions at each location will typically range from about 107 to about 1014 ions per micro-location. The micro-locations formed on a surface in accordance with the teachings of the invention can have a wide range of densities. In general, the density of the micro-locations is selected based at least in part on the desired application. In many biological applications, the practical density for the micro-locations will be determined by the limitations of the reagent deposition process rather than the methods of the invention. It is expected that liquid droplet depositions can achieve a density of the micro-locations in a range of about 1000 cm−2 to about 50000 cm−2, although higher densities may also be attained. Array densities of up to about 104-106 cm−2 or higher can be obtained by utilizing the methods of the invention.
A variety of macro-molecules can be utilized for forming a bioanalytical micro-array according to the invention. Such macro-molecules include, but are not limited to, DNA and RNA nucleotide sequences, amino acid sequences, peptides, cell extracts, antibody epitopes and the like.
In other aspects, the invention provides a method for identifying the presence of a target molecule (i.e., an analyte) in a test sample. The method includes a step of providing a surface with a plurality of micro-locations formed in discrete regions thereof, where each micro-location has an effective dose of an ion implanted therein such that the location has a differential affinity for a compound vis-ŕ-vis the remainder of the surface. A probe is deposited at each micro-location to generate a plurality of bound probes, each probe itself being capable of binding or otherwise reacting with the analyte. A test sample is then applied to these probes. The combination of the test sample and the probes is incubated under conditions that allow a target molecule, if present, to interact with at least one of the plurality of the probes. A detection signal from each micro-location is monitored such that an alteration in the detection signal at a micro-location indicates the presence of a target macro-molecule at that location, thereby identifying the presence of a target macro-molecule in the test sample. For example, the probes can comprise oligonucleotides having a detectable label, for example, a fluorescent marker, when the target molecule is a nucleic acid molecule.
In another aspect, the invention provides a method for identifying a suspected mutation in a target molecule in a test sample by providing a surface with a plurality of micro-locations formed in discrete regions thereof. Each micro-location has an effective dose of an ion implanted therein such that the location has an affinity for a compound that is different from the affinity of the remainder of the surface for that compound. A probe having a known sequence is deposited at each micro-location to generate a plurality of probes. Each probe has a known sequence that corresponds to a portion of the target sequence without a mutation (i.e., wild type sequence). A test sample containing a target molecule with a suspected mutation is applied to the probes, and the combination of the test sample and the probes is incubated under conditions that allow a target molecule with a suspected mutation to interact with the plurality of the probes. A detection signal from each micro-location is monitored. An alteration in the signal detected from at least one micro-location can thereby indicate the presence of a mutation in the target sequence.
Further understanding of the invention can be obtained by the following description of various embodiments of the invention and the accompanying drawings.
So that the invention may more readily be understood, certain terms are first defined:
The term "micro-location", as used herein, refers to a localized area on a substrate surface in which a dose of an ion has been implanted. A micro-location may have any convenient geometrical shape and/or surface topolography, e.g., circular, rectangular, elliptical, wedge-shaped, conical, and the like. Further, a micro-location may be formed as a flat portion of a substrate surface, or alternatively, it can have a three-dimensional structure e.g., a well, a cavity, or depression, raised regions, etches, trenches, or the like.
The term "surface", as used herein refers to any material that is rigid or semi-rigid onto, or into which ions can be implanted or deposited using standard ion implantation techniques. Preferably, the surface is substantially flat, although curved surfaces can also be utilized in the present invention. Examples of suitable surfaces include, but are not limited to, glass, nylon, amino or carboxy activated plastics, cellulose, and the like. Other suitable surfaces can include, for example, surfaces of semiconductor substrates, such as, silicon (Si), gallium arsenide (GaAs), gallium nitride (GaN), and metal-coated glass, such as silver or gold-coated glass. A surface, as used herein, can also have a multi-layer structure, for example, a silane-coated glass surface.
The term "discrete regions", as used herein, refers to physically separate locations on a surface. Each micro-location exists in a discrete region of the surface.
The term "ion beam treatment," as used herein, refers to any treatment of a surface location by utilizing an ion beam, such as, implanting ions in a surface location or depositing ions on a surface location by exposing that location to an ion beam, or alternatively, depositing atoms or molecules on a surface location or implanting atoms or molecules in a surface location by subjecting that location to an ion beam assisted deposition process.
The term "effective dose", as used herein, refers to a concentration of implanted ions that can alter one or more surface properties of a location in which the ions are implanted. The effective dose may vary according to different ion types utilized for implantation. For example, an effective dose of nitrogen may be different than an effective dose of argon to produce a selected change in the surface properties of a portion of the substrate. Suitable effective dose ranges of implanted ions can be from about 1012 cm−2 to about 1017 cm−2.
The term "differential affinity", as used herein, refers to the difference between attractive or repulsive forces, exerted by one portion of a substrate surface and another portion of a substrate surface on compounds, macromolecules, or particles. As discussed in more detail below, in some embodiments of the invention, a plurality of micro-locations formed on a substrate surface in accordance with the teachings of the invention exhibit a differential affinity relative to the remainder of the substrate for selected substrates, compounds, macromolecules, or particles. The term "differential affinity" can also indicate a difference in the hydrophilicity or hydrophobicity between a plurality of micro-locations and the remainder of the substrate surface.
The term "macromolecules", as used herein, refers to large molecules such as biological molecules, for example, DNA, RNA, polypeptides, carbohydrates, antibodies, antibody epitopes, lectins and the like. The biological macromolecules can be naturally occurring macromolecules such as those isolated and purified from a subject. The term "macromolecules" is also intended to include chemically synthesized molecules such as polymers, oligonucleotides, and the like.
The term "nucleic acid molecule", as used herein, refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) polymer in either single or double stranded form, and unless otherwise indicated, it encompasses known analogs of natural nucleotides which function in a similar manner as naturally occurring nucleotides, such as those with allelic variations.
The term "hybridize" or "anneal" as used herein refers to the physical interaction between complementary regions of two molecules, such as protein-protein interactions and nucleic acid-nucleic acid interactions. In particular, the term "hybridize" refers to interactions between two molecules under hybridization and washing conditions that allow complementary regions of the two molecules to remain engaged. The term "hybridization conditions" refers to temperature, solute concentrations and polarity of a given hybridization medium which provides detectable differences in hybridization rates between two oligonucleotides, each having between about 8 and 30 bases, and having at least one non-complementary, i.e., mismatch, base pair relative to one another.
Typically, the hybridization conditions are stringent conditions that are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. Another example of high stringency conditions is hybridization in 50% formamide, 5×SSC, 25 mM potassium phosphate buffer (pH 7.4), 5×Denhardt's, and 50 μg/ml denatured salmon sperm DNA for 4-12 hours at 37° C., which is followed by hybridization for 12-24 hours at 37° C. and washing in 2×SSC containing 0.1% SDS, at 55° C., or an equivalent thereof.
One example of low-stringency hybridization condition is with 25% formamide, 5×SSC, 25 mM potassium phosphate buffer (pH 7.4), 5×Denhardt's, and 50 μg/ml denatured salmon sperm DNA for 4-12 hours at 37° C., which is followed by hybridization for 12-24 hours at 37° C. and washing in 2×SSC containing 0.1% SDS, at 42° C., or an equivalent thereof. See e.g., Sambrook, et al. Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, New York (1989), hereby incorporated by reference. These conditions can be modified as appropriate for use with specific microarrays.
The term "complementary", as used herein, refers to the topological compatibility or matching together of interacting surfaces of a target molecule and its probe. For example, the term "complementary" refers to nucleic acid-nucleic acid base pair interactions. For example, pairing interactions between two DNA molecules involves the bases adenine (A), guanine (G), thymine (T), and cytosine (C), such that A-T, and G-C pairing. The term "complementary" also includes peptide-peptide interactions, for example, a peptide probe that has an amino acid sequence which is an epitope for an antibody target molecule is complementary to that target molecule.
The effective dose of the implanted ions at each micro-location can be in a range of about 1012 to about 1017 ions/cm2. More preferably, the effective dose can be in a range of about 1014 to about 1016 ions/cm2. Further, a variety of ions, such as argon, nitrogen or oxygen, can be utilized for implantation at surface positions of a substrate corresponding to the micro-locations. In general, the type of the implanted ion and the implanted dose are selected based on the desired modification of the surface properties of the micro-locations. One such modification can relate to the wetability of the micro-locations when exposed to aqueous solutions. For example, a dose of nitrogen ions in a range of about 1015 to about 1016 ions/cm2 can be implanted at selected surface positions to provide a plurality of micro-locations which are more hydrophilic than the remainder of the substrate surface. Alternatively, a dose of fluorine ions in a range of about 1012 ions/cm2 to about 1017 ions/cm2 can be utilized to create micro-locations that are more hydrophobic than the remainder of the surface.
The density of the micro-locations 14, that is, the number of micro-locations per unit area, can vary in accord with the intended use of the substrate 10. For example, in some embodiments of the invention, the surface 12 can include a few thousands micro-locations per square centimeter. In other embodiments, the density of the micro-locations can be in a range of about 1000 to about 100,000 per square centimeter. In yet other embodiments, the density of the micro-locations can be in a range of about 1000 to about 10,000. The surface area of each micro-location can also vary over a wide range in accord with the intended use. For example, each micro-location can have a surface area in a range of about a few micrometer square to about tens of micrometer square.
One method of the invention utilizes ion bombardment of selected positions on a substrate to implant an effective dose of a selected ion at these positions, thereby forming a plurality of micro-locations, such as those described above, on the substrate surface. More particularly, with reference to
A variety of different substrates and masks can be utilized to practice the invention. For example, a plurality of micro-locations can be formed on a silane-coated silicon substrate in accordance with the teachings of the invention. Other suitable substrates can include, but are not limited to, glass, nylon, amino or carboxy activated plastics and cellulose, semiconductor substrate surfaces, such as, silicon, gallium arsenide, gallium nitride, and metal-coated glass, such as silver or gold-coated glass.
Further, a variety of masks can be utilized to selectively expose different portions of a substrate surface to an ion beam. For example, the exemplary shadow mask 16 can be formed of silicon dioxide (SiO2). In another embodiment, a mask formed of silicon oxide can be deposited on a silicon substrate, for example, by utilizing chemical vapor deposition (CVD). The silicon oxide mask can then be patterned by employing a number of known methods, such as photolithography, to generate a plurality of openings therein. The substrate is then exposed to a beam of ions, such as nitrogen, to create micro-locations at those positions on the substrate which are in register with the openings in the mask. In other embodiments, resist materials employed in photolithography can be patterned to form masks suitable for use in the present invention. One advantage of such masks is that they can allow generating micro-locations, via ion implantation, with very high densities.
With continued reference to
The micro-locations formed on a substrate in accord with the teachings of the invention can have a variety of geometrical shapes, and can also have different three-dimensional structures. For example, the micro-locations can be flat surface portions. Alternatively, the micro-locations can be formed as physical wells created in the substrate, and implanted with a selected ion. As an exemplary illustration,
As discussed above, each position on the substrate surface in which an effective dose of a selected ion is implanted, i.e., each micro-location, differs in certain surface properties from the rest of the substrate. For example, the micro-locations can be more hydrophilic than the rest of the substrate. That is, the difference between the surface energy of each micro-location and the rest of the substrate can allow each micro-location to act as a potential energy well for a selected compound, e.g., water.
In some embodiments of the invention, a selected number of micro-locations are formed on a substrate surface by implanting one ion type, e.g., nitrogen, in certain discrete regions of the substrate while other micro-locations are formed on the substrate surface by implanting another ion type, e.g., argon, in other discrete regions. This results in generating a substrate having two types of micro-locations. The ions can be selected such that the two types of micro-locations have different surface properties relative to one another and also relative to the remainder of the substrate surface. For example, one set of the micro-locations can be formed by implantation of nitrogen ions and another set by implantation of argon ions. Further, in some embodiments of the invention, more than one ion type is implanted at each discrete region of a substrate surface corresponding to a micro-location.
In another embodiment of the invention, ion implantation is utilized to form one or more strips on a substrate surface having a different surface property, e.g., an increased hydrophilicity, relative to the remainder of the substrate surface. For example,
A dose of a selected ion, such as, nitrogen, can be implanted in portions of the substrate surface that are aligned with the openings 30, for example, by bombarding the masked substrate with a beam of nitrogen ions. The implantation of the selected ion in these substrate portions results in modification of one or more selected properties of these portions, thereby generating strips of the substrate surface having modified surface properties. For example, strips of the substrate surface having an increased hydrophilicity can be generated in this manner.
Ion beam surface treatment techniques that can be utilized to produce a bioanalytical array of the invention are not limited to ion implantation by exposing a surface location to a beam of the ion being implanted. For example, in some embodiments, an ion beam assisted deposition process (IBAD) can be employed to cause selected atoms to be embedded within and/or upon a surface location. For example, an IBAD process can be employed in which an ion source can accelerate ions into selected portions of a growing film formed on a surface by physical vapor deposition. The resulting collisions mix the film atoms with the surface and cause the film atoms to be embedded in and/or on the selected portions of the surface that are subjected to the ion beam. U.S. Pat. No. 5,520,664, herein incorporated by reference, provides further details regarding IBAD process and apparatus therefor.
The differences in the surface properties of the ion beam treated locations, e.g., ion-implanted locations, and the remainder of the substrate surface can be utilized in a variety of applications, as discussed in more detail below. In one embodiment, an array of macromolecules, such as, an array of oligonucleotides, can be formed on a substrate surface of the invention at the positions of the micro-locations. Such an array can be formed, for example, by depositing a droplet of an aqueous solution containing a macromolecule at each of a plurality of micro-locations, which are designed to be more hydrophilic than the rest of the substrate surface. The high hydrophilicity of the micro-locations inhibits the spread of the droplets to the remainder of the substrate, and hence effectively confines the droplets to the micro-locations.
Microarrays of biological macromolecules, formed in accord with the teachings of the invention, can be utilized in a variety of different applications. For example, microarrays of immobilized nucleic acid sequences can be employed in large scale hybridization assays in many genetic applications, such as mapping of genomes, monitoring of gene expression, DNA sequencing, genetic diagnosis, and genotyping of organisms. Such microarrays can also be utilized to identify optimal therapeutic protocols for early and rapid detection of a variety of disease states.
In some embodiments, microarrays can be created on a substrate surface by depositing pre-synthesized probes (e.g., oligonucleotide probes or peptide probes) onto the micro-locations. Oligonucleotides or peptides can be synthesized, for example, by employing an automated nucleotide or peptide synthesizer, such as the Applied Biosystems Instruments (ABI) Synthesizer (See e.g., U.S. Pat. No. 5,734,018 and U.S. Pat. No. 6,063,571). The deposition of the probes on the micro-locations can be accomplished by employing any standard technique, for example, utilizing robots for deposition onto the micro-locations.
In one application, a microarray of probes deposited at micro-locations of a substrate surface of the invention can be utilized to detect the presence of a target macromolecule in a test sample. When the target macromolecule is a nucleotide sequence, the probes can be oligonucleotide probes having different nucleotide sequences. The oligonucleotide probes typically include a region of nucleotide sequence that can hybridize under stringent conditions to at least about 12 or 15, preferably about 20 or 25, and more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a target nucleotide sequence. An oligonucleotide probe can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length, and can be prepared, for example, by utilizing enzymatic ligation reactions known in the art. The oligonucleotide probes can be prepared from naturally occurring nucleotides, such as those that increase the biological stability of the molecules. Alternatively, the oligonucleotide probes can be synthesized using an automated nucleotide synthesizer, such as an ABI synthesizer.
Microarrays of oligonucleotide probes can be employed for sequencing-by-hybridization (SBH) on genomic DNA fragments or other nucleic acid molecules. Microarrays of oligonucleotide probes can also be used to detect mutations in a target nucleotide sequence for which the normal or "wild-type" nucleotide sequence is already known. Mutations or polymorphisms associated with phenotypic changes having clinical significance in human patients may also be investigated by utilizing a micro-array of probes on a substrate of the invention. For example, the cystic fibrosis transmembrane conductance regulator (CFTR) gene and P53 gene in humans have been identified as the location of several mutations resulting in cystic fibrosis or cancer, respectively (See e.g., U.S. Pat. No. 6,309,823). In such applications, each micro-location can have a different oligonucleotide probe deposited thereon, thereby creating a microarray with a plurality of different oligonucleotide probes. A test sample with a target nucleic acid can be added to the array of oligonucleotide probes to hybridize under conditions that allow complementary sequence interactions to take place. The hybridization pattern can reveal the identity of all target nucleic acid sequences that are complementary to one or more probes. Both the presence of a target nucleic acid and its sequence can be discerned from the hybridization pattern.
Where the microarrays are used to detect mutations or polymorphisms, a number of oligonucleotide probes corresponding to regions of the known nucleotide sequence of the wild type gene, e.g., the CFTR gene, can be generated. Each probe with a specific sequence can be placed in a separate micro-location. A test sample containing, for example, a CFTR gene with a suspected mutation, can be applied to the microarray of probes. The presence of the polymorphism or mutation can be detected based on comparison of the illumination pattern of a microarray incubated with a gene suspected to have a mutation with e that of a microarray incubated with the wild type gene. An alteration in the detectable signal in a particular micro-location indicates a polymorphism or mutation in a particular region of the gene corresponding to the probe sequence at that microlocation. The sequence of the polymorphic or mutated gene may be discerned by combining the sequences of the oligonucleotide probes from each micro-location that produces a detectable signal.
The hybridized target sequences can be detected by utilizing any method that provides a detectable signal. For example, the hybridized target sequences can be detected by utilizing one or more detectable marker labels or chemicals, such as dyes, fluorescent chemicals, radioisotopes, or other markers. Such detection methods are well known in the art. For example, Gold et al. in U.S. Pat. No. 6,242,246 B1, incorporated herein by reference, describes a detectable system that monitors a change in fluorescence, or a change in a physical parameter, such as electrical conductance or refractive index, at each location on a biochip.
In one embodiment, a hybridization pattern is discerned by scanning the surface of a micro-array to detect the presence of the detectable markers at one or more of the micro-locations forming the micro-array. For example, the detection of a fluorescent signal at a micro-location can signify the presence of a target nucleic acid sequence that is complementary to at least a portion of a probe at that location. In another embodiment, a hybridization pattern is discerned by scanning the surface of the microarray to detect the absence of a detectable marker at one or more micro-locations forming the micro-array.
One embodiment of the invention utilizes a detection method that employs an energy transfer pair of markers, such as those involved in fluorescent resonance energy transfer (FRET) assays. FRET is a useful assay for detecting interactions between molecules (See e.g., Heim et al., (1996) Curr. Biol. 6:178-182; Mitra et al., (1996) Gene 173:13-17; Selvin et al., (1995) Meth. Enzymol. 246:300-345). As used herein, "FRET energy transfer pair" includes a first member which is a donor molecule, and a second member which is an acceptor molecule. The donor and acceptor molecules can form a fluorescence resonance energy transfer relationship when they are in close proximity of one another.
A FRET assay measures a change in fluorescence caused by a change in the distance separating the donor molecule (e.g., a first fluorophore) from an interacting resonance energy acceptor molecule (either another fluorophore or a quencher). The mechanism of FRET energy transfer pair interaction requires that the absorption spectrum of one member (donor molecule) of the pair overlaps the emission spectrum of the other member (acceptor molecule). (See e.g., Stryer, L., "Fluorescence Energy Transfer as a Spectroscopic Ruler," Ann. Rev. Biochem. 1978, 47: 819-846 ("Stryer, L. 1978"); Biophysical Chemistry part II, Techniques for the Study of Biological Structure and Function, C. R. Cantor and P. R. Schimmel, pages 448-455 (W. H. Freeman and Co., San Francisco, U.S.A., 1980) ("Cantor and Schimmel 1980"), and Selvin, P. R., "Fluorescence Resonance Energy Transfer," Methods in Enzymology 246:300-335 (1995) ("Selvin, P. R. 1995")).
In FRET, a donor molecule (e.g., a fluorophore) absorbs radiation at a first wavelength and emits radiation at a second, typically longer, wavelength. The acceptor molecule or quencher, which is proximate to the donor molecule (the FRET range is reportedly about 10-100 Å) and has an absorption spectrum that overlaps the emission spectrum of the donor molecule, absorbs some or most of the energy emitted by the donor molecule. If the acceptor molecule is a fluorophore, it re-emits the energy at a third, still longer wavelength. If the acceptor molecule is a quencher, it releases the energy as heat.
Commercially available fluorophores and their derivatives can be employed to label proteins or nucleotides. The fluorophores can be either synthetic, relatively low-molecular organic compounds (e.g. fluorescein and rhodamine and derivatives thereof, and lanthanide chelates) or proteins (e.g. phycoerythrin, and green fluorescent protein (GFP)). Various parameters of the fluorescence emitted by the fluorophores can be measured, such as, fluorescence intensity, life-time, polarization. These can also be used to monitor and quantitate the FRET phenomenon.
Examples of fluorophores, such as fluoresceins include, but are not limited to 6-carboxyfluorescein (6-FAM), 2′,4′,1,4,-tetrachlorofluorescein (TET), 2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED) and 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
A quencher moiety is a moiety whose absorption spectrum at least partially overlaps the fluorescence spectrum of a donor molecule such that the fluorescence of the donor molecule is substantially absorbed, that is, quenched by the quencher moiety, when the moiety is in proximity of the donor molecule during fluorescence resonance energy transfer (FRET). Examples of quenchers include, but are not limited to, rhodamine dyes such as tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX); DABSYL, DABCYL, and cyanine dyes such as nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds and the like.
One example of a FRET energy transfer pair as disclosed in Matayoshi et al. 1990, Science 247: 954-958, and incorporated herein by reference, includes DABCYL as a quenching moiety and EDANS as a fluorophore. The absorption spectrum of DABCYL has a high degree of overlap with the emission spectrum of EDANS.
Fluorescent nucleotide analogs can be introduced into a oligonucleotides during synthesis (Jameson & Eccleston (1997) Meth. Enzymol. 278: 363-390). In another embodiment, fluorescent dyes can be attached to oligonucleotides via reactive groups on the DNA and the dye (Langer et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78: 6633-6637; Waggoner (1995) Meth. Enzymol. 246: 362-373).
To label proteins, amino acids with reactive side chains can be introduced at strategic points into proteins by protein engineering techniques (see, Buckle et al. (1996) Biochemistry 35: 4298-4305; de Prat-Gay, (1996) Protein Engineering 9: 843-847). These reactive side chains can be further fluorescently labeled by techniques known in the art (see, e.g., Haugland, "Handbook of Fluorescent Probes and Research Chemicals" (1996) Chapters 1-3, 7, 15, 18).
The microarrays can be composed of one member of a probe pair labeled with one member of a FRET energy transfer pair. A "probe pair" consists of first and second members of oligonucleotides that are completely complementary to each other. The first member of the probe pair can have a first member of the FRET energy transfer pair (e.g., donor molecule) attached at one end (e.g., the 3′ end), while the second member of the probe pair can have the second member of the FRET energy transfer pair (e.g., an acceptor molecule) attached at one end (e.g., the 5′ end). When the two members of the probe pair anneal to each other, the first and second members of the FRET energy transfer pair are brought into close proximity. When the members are illuminated by light having an appropriate wavelength, the FRET phenomena occurs between the FRET energy transfer pair, and a detectable signal is generated. In another embodiment, the second member of the FRET energy transfer pair is a quencher, and when the two members of the probe pairs anneal to each other, the fluorescence is quenched by the quenching moiety.
In one embodiment, a first set of oligonucleotide probes are pre-synthesized and labeled with a first member of an energy transfer pair of markers, e.g., a FRET energy transfer pair of markers. This first set of oligonucleotides is disposed at each micro-location to create a microarray of oligonucleotide probes. A target DNA can be added to the microarray of probes, and be allowed to hybridize under conditions that permit complementary sequence interactions to take place. A second set of oligonucleotide probes that are complementary to the first set of oligonucleotides probes can be synthesized and labeled with a second member of the FRET energy transfer pair of markers. The second set of oligonucleotide probes, can be introduced into each micro-location that contains the corresponding oligonucleotide probe. The second set of probes will be free to bind to their complements at each location where a binding reaction with a target DNA has not already occurred and competitive binding interactions will result in the displacement of non-complementary target DNA sequences from the first set of oligonucleotide probes at locations where weak hybridization matches have occurred. Thus, the second set of complementary oligonucleotide probes, labeled with the second member of the FRET energy transfer pair, will anneal to the first set of oligonucleotide probes everywhere except at the locations where the target DNA has strongly hybridized to a particular oligonucleotide probe of the array.
When the first and second members of the energy transfer pair are fluorophores, FRET will occur and produce a detectable fluorescent signal. When the second member of the energy transfer pair at a micro-array position is a quencher, it will quench a fluorescent signal emitted from the first member, when in close proximity of the first member, thereby eliminating the signal at that position in the micro-array. Thus, the absence of a FRET signal at a micro-location can be indicative of the lack or partial complementarity of the target with the probe at that location.
Further manipulation of complementary complexes may also take place on the microarray. For example, an oligonucleotide probe disposed at a micro-location, to which a target nucleic acid sequence has hydridized, can act as primer sequence for further amplification reactions. The target nucleic acid sequence acts as a template, along which an extension reaction, initiated, e.g., by standard PCR procedures and reagent, occurs. The amplification reaction conditions, such as temperature, ionic strength, and pH, can be selected so as to permit a polymerase reaction to take place by applying thermally stable polymerase, nucleotides, and other necessary reagents. The amplification product may incorporate a detectable marker label.
In other embodiments, microarrays of the invention can be used to determine antibody epitopes. This involves the use of primary antibodies, i.e., those which bind to an epitope (e.g., a rabbit anti-protein antibody), and labeled secondary antibodies, i.e., those which bind to a primary antibody (e.g., a goat anti-rabbit antibody linked to a detectable label, See e.g., U.S. Pat. No. 5,891,656). In these embodiments, small regions of a primary antibody, for example, regions of about 6 amino acids in length, can be synthesized and deposited in each micro-location. This creates a microarray of hexapeptides which contains at least one epitope for the primary antibody, that will allow high affinity binding between the hexapeptide and the primary antibody. The primary antibody is deposited over the hexapeptide array and the peptide-primary antibody interaction is allowed to occur in a first incubation step. After the first incubation step, the peptide-primary antibody complex is exposed to a fluorescently labeled secondary antibody that recognizes, and binds to the primary antibody in a second incubation step. Following the second incubation step, the surface of the microarray is scanned for fluorescence signals, for example, by utilizing a fluorescent microscope, to reveal bright and dark areas on the microarray. Each bright area of the microarray indicates a hexapeptide that is an epitope for the primary antibody.
The presence or lack of a fluorescent signal at micro-location can be detected by directing light from a light source, such as a laser light source of the type well known to those of skill in the art, to that micro-location. The light incident on the micro-location can cause the emission of a fluorescent signal from that location that can be detected by a detector appropriately placed over the substrate. Alternatively, such a detector may detect no fluorescent signal signifying a lack of fluorescence at that location.
A detector typically detects the intensity of fluorescent light emitted from molecules disposed on the substrate as a function of position. In one embodiment, the detector is a CCD array of a type commonly known to those skilled in the art (See e.g., U.S. Pat. No. 6,310,189, incorporated herein by reference). The CCD array can be used in conjunction with an x-y translation stage, in which a light source, and the detector are all operably connected to a computer or equivalent for control of the device and data collection from the CCD array. In operation, the microarray is appropriately positioned by the translation stage. The light source then illuminates the substrate surface, and intensity data is gathered by the computer via the detector.
Data can be collected as an image data file and then outputted for display. The output from the data collection system can be, for example, in the form of an array indicative of fluorescent intensity versus location on the microarray.
A microarray according to the invention provides a number of advantages. For example,
Further, the upper surface of the sample confined in the well 34 is less curvilinear than that of the sample confined in the conventional cell 32. This results in a better signal to noise ratio when fluorescence signals are utilized in hybridization experiments.
Another advantage of a substrate having micro-locations according to the invention is that such a substrate is easy to manufacture, is durable and can be re-used for different experiments. In addition, such a substrate permits confinement of an aqueous sample at the micro-locations, thereby allowing solution phase chemistry to be performed at these locations.
Moreover, as discussed above, the micro-locations of the invention can be formed as flat portions of a substrate surface. This in turn results in forming substantially flat sample surfaces at these locations. Such flat sample surfaces can advantageously enhance the signal-to-noise ratio in fluorescence detection methods.
Yet another advantage of the invention is that it allows providing a higher density of micro-locations on a substrate surface than conventional microarrays. For example, as described above, the methods of the invention can be utilized to generate micro-locations on a substrate surface with a density in a range of about 100,000 to 1000,000 cm−2, which is substantially higher than the micro-location densities obtainable by conventional techniques.
Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. For example, methods other than ion bombardment of a substrate that can effect ion implantation in the substrate can also be utilized to practice the present invention. All U.S. Patents and other references noted herein for whatever reason are specifically incorporated by reference. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.
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