text:
Tossing the Afghan COIN
As long as there has been war, as long as one nation has sought to impose its military will on another, there have been insurgents... and there have also been counterinsurgents.
It was the ancient Romans who popularized what has become an oft-imitated means of dealing with recalcitrant and subjugated peoples—the so-called Roman Method of repression to quell the noncompliant. Since those long-ago days, numerous countries and empires (from autocracies to democracies) have used similar counterinsurgency (COIN) tactics. Indeed, the history of COIN is a depressing and unremitting tale of coercion and violence generally aimed not just at armed insurgents but at civilian populations as well.
The United States has not been immune from such conflicts—or their brutality. Counterinsurgent fights were waged at home against Native American tribes and in Central America, the Philippines and Vietnam. These small wars are in some manner the defining element of the American Way of War.
In recent years, however, the US military has not only rediscovered counterinsurgency but reinvented it, first in Iraq and now in Afghanistan. To listen to the American military or, better, to read the Army and Marine Corps counterinsurgency manual, FM 3-24, is to hear a tale of COIN's bloody legacy of coercion and violence magically transformed.
According to this new telling, the key to winning COIN conflicts is in protecting civilians and providing a better future for them—that is, winning hearts and minds via civic action rather than military force. As written in FM 3-24, counterinsurgency fights are population-centric, not enemy-centric, and are defined by an extraordinarily broad array of capabilities, "political, economic, military, paramilitary, psychological, and civic actions."
On the most basic level, the key to COIN victory rests in protecting the population, providing good governance, extending the legitimacy of the host government and gaining the trust of the people. This notion of warfighting has become so internalized in US military thinking that even the past has been rewritten. New assessments of conflicts in Malaya and Vietnam, conducted by COIN advocates, confidently assert that civic action, not coercion—much less forced separation of insurgents from the population—turned the tide of those conflicts. In the case of Vietnam, COIN advocates argue that once the United States turned to counterinsurgency, the direction of the war shifted; and that had the United States had the resilience to see the fight through, the embrace of population-centric COIN would have led the way to success.
Much of this new take on COIN is a result of the US military's experience in Iraq, where, as the argument goes, a focus on protecting civilians and standing up the Iraqi government led to a decrease in civilian casualties. The supposed success of COIN tactics in Iraq, twinned with the 2007 surge of 30,000 US troops, led to the claim that these successes could be replicated in Afghanistan. Indeed, by the fall of 2009 Gen. Stanley McChrystal offered a strategic review of the situation in Afghanistan that concluded that a population-centric counterinsurgency—and only that strategy—could grasp victory from the jaws of defeat.
It is this approach that the United States began using in 2009 and the first half of 2010. Upon being named Afghanistan commander, McChrystal declared in Congressional testimony that it was more important to protect civilians than to kill insurgents. NATO officials even discussed the possibility of creating a new medal for "courageous restraint" for soldiers who take measures to avoid civilian casualties.
Indeed, COIN has become increasingly fetishized in US military thinking, training and guidance. Col. Gian Gentile, a frequent critic of population-centric counterinsurgency, recounts a recent experience listening to a three-star Army general telling West Point graduates that "what they needed to be good at when they went out into the field army—was establishing 'trusting relationships' with local populations." As Gentile acidly notes, "One would have liked soon-to-be-lieutenants told that they must be proficient in their basic branch skills: infantry and armor, basic fire and maneuver with their platoon as part of a maneuver company/team; artillery, fire support; logistics, logistical support; and so on."
The military's focus on COIN has been reflected in more than just rhetoric. When troops were sent into the town of Marja in Helmand province in February, the element of surprise was purposely given away. Military commanders made clear their intention to take the town and their hope that Taliban forces would flee, thus protecting civilians from being caught in the cross-fire.
Under the logic of COIN, such efforts to protect civilians are not only the right thing to do but are essential to military success, because safe and secure civilians will throw in their lot with the government and provide crucial intelligence for use in targeting insurgents. By securing the people, counterinsurgents can win the competition for the loyalty of the population.
This method of placing civilians front and center in military deliberations may on the surface seem quite progressive (indeed, COIN advocates have repeatedly made this argument). But it hasn't worked out that way. The United Nations recorded this past summer that the number of Afghan civilians killed in the first six months of the year jumped by a depressing 31 percent; in southern Afghanistan, where the war's heaviest fighting is taking place, civilian casualties (based on hospital admissions) have increased dramatically. While military officials are quick to argue that these increased deaths are largely the result of insurgent actions (and they are correct), this doesn't mean that local Afghans don't blame the United States and NATO. Recent opinion polling of Afghans suggests they do.
In fact, efforts to protect the Afghan population are failing. According to recent press accounts, insurgents in Kandahar province have carried out a successful campaign of intimidation and violence against local citizens. Writing recently in the Christian Science Monitor, Julius Cavendish reported that Taliban assassinations of officials there have become so pervasive that more than 600 government jobs remain unfilled. The situation in Marja—ten months after US and Afghan troops entered the town in what was billed as a major element of the military's counterinsurgency strategy—also remains challenging.
Beyond the obvious difficulty of making protecting lives a priority in an active war zone, it has become evident over the past year that Afghanistan is a spectacularly poor choice for a population-centric COIN campaign. An effective counterinsurgency relies, in large measure, on a competent and legitimate host country government. Afghanistan has neither. Corruption is so bad that the UN Office on Drugs and Crime has estimated that "drugs and bribes are the two largest income generators in Afghanistan."
Afghanistan's security services are unable to hold areas cleared by US forces, and Afghan police are even less effective and lack the support of a functioning legal system. Making matters more difficult is the continued presence of undisturbed Afghan Taliban safe havens across the border in Pakistan. Finally, there are the Taliban, who have demonstrated a brutal willingness to use violence to cow the civilian populace into not cooperating with the United States, NATO or their own government.
A successful COIN campaign in Afghanistan is predicated on an extraordinary confluence of events: first, militarily reversing the Taliban's momentum without causing significant civilian casualties; second, standing up the Afghan government and security forces so they are able to hold and build on the gains made by Western troops; third, minimizing the impact of Taliban sanctuaries across the border, in part by convincing the Pakistani government to reduce its support for them. And all this must be done while operating under Obama's July 2011 deadline for commencing the withdrawal of US troops.
Achieving these goals demands an enormous political will and outlay of resources. Even by the measure of the military's counterinsurgency doctrine, at approximately 130,000 US and NATO troops and a US outlay of an estimated $100 billion a year, the current effort is underresourced. Achieving these goals would require not only tens (perhaps hundreds) of thousands of more troops; it would require a stated commitment to maintain the fight for years to come. After nine years of war, these are elements that seem increasingly in short supply.
To be sure, many of these arguments have been made for some time by COIN skeptics. But what is most revelatory is that the US military seems to be finally getting the message. In the media uproar over the Rolling Stone article that sank the career of General McChrystal, often overlooked was the recounting of his conversation with US soldiers in southern Afghanistan who complained that restrictive rules of engagement made it nearly impossible for them to do the job they were trained for—killing the enemy. With no sense of irony, McChrystal complained, "This is the philosophical part that works with think tanks...but it doesn't get the same reception from infantry companies."
McChrystal's replacement, Gen. David Petraeus, has adopted a far different approach to the war. While efforts at nation-building continue hesitantly, the biggest change has been in the use of direct military action against the insurgents. Air power, which for a while was minimized because of the risk of civilian casualties, has increased dramatically. According to the blog Danger Room, "The U.S. and its allies have unleashed a massive air campaign in Afghanistan, launching missiles and bombs from the sky at a rate rarely seen since the war's earliest days. In November alone, NATO aircraft launched 850 missions—three and a half times more than the same period in November 2009. Petraeus is also using more Special Operations forces in targeting Taliban commanders; and night raids, which have particularly inflamed the Karzai government, have recently tripled. In the three-month period ending October 21, Special Forces units conducted more than 1,500 operations, killing or capturing 339 insurgent leaders and more than 3,400 foot soldiers. NATO officials now hand out daily updates boasting about the number of insurgents killed or captured.
News reports leaked by military officials to reporters today do not speak of shuras convened, schools opened or corruption battled but instead boast of insurgents eliminated. The shift in tactical approach was perhaps an effort to show "progress" before the White House's planned December review of Afghan policy. But it should also be seen as something else: a more accurate reflection of how counterinsurgency conflicts—even modern ones—are waged. As a COIN theorist and military official said to me, "COIN is a form of warfare and thus involves violence. Don't be fooled by the fact that Petraeus found some useful idiots to make it sound more palatable and humanitarian."
The best evidence of this comes, ironically, from the US experience in Iraq. What led to the decrease in violence there was not an enhanced focus on protecting civilians; it was a confluence of factors: the extraordinary bloodletting in and around Baghdad that led to the forced separation of Sunnis and Shiites into ethnic enclaves; the flight of, by some estimates, 5 million Iraqis who have been internally and externally displaced; the paying off of, and support for, Sunni tribes who took on Al Qaeda in Iraq. And it wasn't just Iraqi-on-Iraqi violence—the number of civilians killed by US troops rose dramatically after the surge and the adoption of supposedly civilian-friendly measures. Four times as many Iraqis were killed by US airstrikes, which rose sevenfold after Petraeus took command in Iraq. While certainly there were US efforts to capture Iraqi hearts and minds, no one can reasonably argue that those measures were decisive.
According to Jeff Michaels, a COIN expert and research associate at King's College in London, "Killing lots of people was a key element of the Iraq surge, and so this [war in Afghanistan] is not too dissimilar. Indeed, too much emphasis has been placed on FM 3-24 rather than the real script they are operating from, which is to say that the Iraq case illustrated a considerable divergence from the theory."
What is happening in Afghanistan is an embrace of the aggressive approach to counterinsurgency once publicly dismissed by FM 3-24 advocates. This is not to suggest that US and NATO forces in Afghanistan have given up on trying to reach hearts and minds. But their embrace of techniques they once argued against is an implicit acknowledgment that the population-centric tactics of FM 3-24 have only marginal effectiveness in a nonpermissive environment like that of Afghanistan today. Like so many counterinsurgents before them, US generals are finding that the carrot is far less effective than the stick.
Their actual approach bears startling resemblance to the smaller-military-footprint counterterrorism strategy outlined by Vice President Biden during last year's strategic review debates. Put aside for now are dreams of state-building in the Hindu Kush or the belief that only by turning the people away from the insurgents can America secure its interests. Instead, military planners have shifted their focus to an end-game strategy of using lethal force to drive the Taliban to the negotiating table.
The shift in emphasis toward a more traditional conflict is compelling evidence of the disconnect between the theory of population-centric COIN and actual US capabilities—and an unstated recognition that FM 3-24 has so far not succeeded. This hasn't stopped COIN advocates from arguing that the shift in military emphasis is all part of the larger COIN effort; after all, they claim, direct military action is a crucial element of counterinsurgency. But these are self-serving and deceptive arguments, intended in part to mask the failure of the military to capture Afghan hearts and minds.
All of this matters for the future of US national security strategy. Much of the rationale for escalation in Afghanistan was based on the story of "success" in Iraq, and in particular on the supposed effectiveness of more population-sensitive counterinsurgency strategies executed there. But what should really be taken away from the US military's experience over the past ten years is not that the United States understands how to fight and win population-centric counterinsurgencies but that counterinsurgencies are as violent and inconclusive as any other conflicts, and that the United States should avoid such wars at all costs.


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Where to watch the Women's World Cup Final Sunday07/03/15Events
Local bars and restaurants are looking forward to an extended celebration of the red, white and blue this weekend.
After the bombs burst in air on Saturday night, folks will be right back out on Sunday to watch the U.S. Women's Soccer Team play to win its third FIFA Women's World Cup.
The Lady Yanks have been battling their way across Canada to get to Sunday's game in Vancouver. It's a rematch against 2011 Women's World Cup champs Japan's Big Pink....
Sesame Street's Maria, Sonia Manzano retires after 44 years on the show07/02/15 Blog
The woman who taught you to sing and count in Spanish is moving away from Sesame Street after four long decades in America's friendliest neighborhood.
Sonia Manzano, 65, who played Maria, will retire from the series she joined just after leaving Carnegie Mellon University in 1971. She was many people's first on screen Latina, and her acting, writing and general awesomeness got her many awards and accolades through the years, says the Washington Post. ...
Disney dumps poop shaped candy from new Animal Kingdom shopping area06/30/15 Blog
Disney's Animal Kingdom isn't selling anymore candies shaped like animal dung at its new Harambe Market shopping area.
The theme park flushed the idea after only two weeks of selling the desserts made of chocolate, peanut butter and caramel, the Orlando Sentinel reports. ...
The Bachelorette, Season 11, Episode 8 -- Tears of a clown06/30/15 Blog
The moment that will define Kaitlyn Bristowe's legacy on The Bachelorette isn't the hook-up with Nick Viall.
It isn't being chosen over Britt Nilsson in the most sexist season opener in Bachelorette history. It won't be the any of the flameouts that happened before the rose ceremony.
We will always remember the moment Kaitlyn cried tears of relief when she realized Shawn B. didn't know she had slept with Nick. She wept with joy, despite supposedly not feeling guilty about having sex with Nick. To those who rushed to Kaitlyn's defense when the world judged her for jumping in the sack with the possible worst person in the world, I ask, if she's free to have sex with whomever she wants, why can't she own that? Why not just say, I was feeling it, I did it, what of it?...
Tampa's Jeff Weldon, an alum of The Amazing Race, joins the cast of Big Brother Takeover06/29/15 Blog
Some people try to strike it rich with a big idea or some undeniable talent.
Jeff Weldon, 27, of Tampa will make do with winning at least one of CBS' game shows.
One of the newest members of Big Brother's 17th season may look familiar to The Amazing Race fans who saw him compete on that show this spring.
Originally from Hampton in Hernando County, Weldon went on his first trip out of the country for The Amazing Race. He told the Times then that he always wanted to travel and he left his job to join the race....
Transgender star Esme Russell spends a life learning to love drag06/25/15 Human Interest
Esme Russell glides through a packed Hamburger Mary's in Ybor City during her second show of Saturday night.
Her skin-tight, flesh-colored tube dress embellished with tiny mirrors reflects the faces of the crowd. She pauses and homes in on a young man who had leaned away during her lip-synch of a Gloria Estefan song.
"What did you think was going to happen if I touched you?" says Esme, 54. "It's not contagious, honey. You can't catch this. You won't wake up one morning and want to put on a dress."...
Alex Newell of 'Glee' talks Coachella, shoes before performing at St. Pete Pride06/25/15Events
Alex Newell doesn't have awesome memories of the summers he spent in the Tampa Bay area visiting family.
"It always rained every day; we'd go out to go right back in," said the actor/singer who lives in Massachusetts. "Every day."
He's hoping for sunnier climes at 7 p.m. Friday when he headlines the outdoor show 27/82: The Concert for St. Pete Pride.
"I've done so many Prides now, I've lost count," he said in a phone interview this week....
Streets will light up for this year's St. Pete Pride parade06/23/15Events
Leave the flashlights at home and don't worry about squinting to see the outrageous costumes and epic dance routines on Central Avenue Saturday night. Parade entrants in this year's St. Pete LGBT Pride Parade have figured out how to do it in the dark.
There will still be plenty happening in the light to support the area's lesbian, gay, bisexual and transgender communities. Friday, Glee star Alex Newell will headline a free concert on Central Avenue. Sunday, vendors from around the Tampa Bay area will peddle their wares to the tens of thousands of visitors and locals expected to attend the Pride street festival. But the rhinestone in the tiara of the parade and block party is from 5 to 10 p.m. Saturday, the parade stepping off at sunset, about 8:30....
The Bachelorette, Season 11, Episode 7- Living down to expectations06/23/15 Blog
You know what's a great way to refute claims that you're a surface-level person who came on The Bachelorette to meet hot guys and hook up?
Sleeping with the guy everyone has been warning you about in his second week on the show.
Kaitlyn Bristowe, you never disappoint. Just when we think this season of The Bachelorette has sunk into the depths of network TV depravity, there you are, smiling with arms full of rocks, ready to take us even lower....
The Bachelorette, Season 11, Episode 6: The Elusive 16th Minute of Fame06/16/15 Blog
This, my friends, is the magic of The Bachelorette.
From the time Ian got out of the limo, editing had painted him as Kaitlyn Bristowe's biggest cheerleader, No. 1 fan and the guy who always had her back in every situation.
In the first episode, he lobbied for her to be The Bachelorette. In Episode 4, he used his last-minute couch rally time to break up team #BrokebackBachelor. When Nick first appeared, he loudly questioned his motives. It seemed like he was protecting a girl he actually liked....
Taylor Swift? Maroon 5? Who will have the song of the summer?06/10/15 Music & Concerts
It's the song you can't escape at your friend's cousin's wedding reception. It's the song you'll hear twice in the car on the way to every cookout, kickback, get-together and beach party, twice at the actual function and three times on the way home. Ubiquity defines the song of the summer, coined by Billboard to give a title to the jam that rules the airwaves and streams between Memorial Day and Labor Day....
Metrocon vendors bring hard-to-get items to anime convention06/09/15Events
Coins will drop all over the Tampa Convention Center this weekend as Tampa Bay anime fans weave through this year's Metrocon.
It's Tampa's largest anime convention, a place for fans of Japanese animation to gather, dress in costume and do some serious shopping. Expanded to four days, organizer Team Dynamite lined up enough voice talent, panels, role-playing games, cosplay contests, parody shows and raves for even n00bs to take a deep dive into the style that has grown a cult following in the past 50 years....
The Bachelorette, Season 11, Episode 5: Friends in low places06/09/15 Blog
Friends don't let friends mack on their girlfriends.
This has been biggest and most fundamental misunderstanding among the remaining men in this season of The Bachelorette.
They turned against Clint because he wasn't friendly. They turned on JJ because he was a bad friend to Clint. They turned on Kaitlyn because she wanted to explore her friendship with Nick. And they will turn on Nick because they don't consider him a friend....
Additions at MOSI will let you ride driverless shuttle, pilot drones and code robots06/05/15Events
Additions to the Innovate exhibit at Tampa's Museum of Science and Industry will allow visitors to ride a driverless shuttle, pilot drones and code robots beginning as early as next week. The intelligent and electric Meridian Shuttle opening June 13 will be the first of its kind open to the general public in the United States, MOSI said. Guests will be able to hop on the shuttle eight at a time in the museum lobby and take a fully computerized spin around the first floor. A docent called a STEAM Punk will ride along with passengers, explain the technology and answer questions, said Megan Haskins, MOSI's marketing director. Starting in July, tech fans will also be able to pilot flying, rolling and submersible drones in MOSI's new Drone Zone. The Idea Zone is also adding a component allowing future inventors to code a robot to explore off-world terrain and navigate a maze. Exhibit organizers say introducing the tech to consumers before it is commercially available gives them the chance to ask questions about the safety and feasibility of driverless vehicles. Admission to the museum is $22.95 adults, $18.95 kids. For more information, visit mosi.org. — Robbyn Mitchell, Times staff writer...
G. Bar in Ybor City closes after sale to Orlando proprietor06/04/15Business
YBOR CITY — Brief-clad, male go-go dancers and sharp-tongued drag queens were only a small part of the legacy of G. Bar, which closed its doors on May 30.
Since its opening nine years ago, the bar at 1401 E Seventh Ave. has hosted countless world-class DJs, dozens of dollar drink nights and a few internationally recognizable pop acts, including Lady Gaga in 2008.
Owners D. Stephen Moss and Steve Donahue decided to sell the G. Bar property in early May, after an Orlando bar proprietor made a generous offer on the space. ...


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Currency Cross Rates
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U.S. Dollar Index Little Higher
July 06, 2015
The September U.S. Dollar Index is trading at 96.60, up 0.31. The U.S. dollar Index is range-bound and lightly choppy. ISM Services ... Read More
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5 Reasons the Fed Will Raise Interest Rates Soon
July 03, 2015
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Jenks safety Steven Parker, who is No. 1 on The Oklahoman's Super 30, said Saturday he will attend Oklahoma State's scrimmage later that day.
Parker has more than 25 offers, including Oklahoma State, and has spent the summer narrowing down his options.
"I'm still kind of neutral," Parker said. "I've definitely narrowed down some schools. I secretly have a top five, but I'm not going to let it get out."
He went on to say he originally planned to commit before the season started, but he has yet to visit each place he wants to visit before committing. Now, Parker's intent is to commit after the season.
Parker was in Norman earlier this week visiting Oklahoma alongside Tulsa Union tight end Carson Meier, who committed to the Sooners in May.
He was going to visit OSU on Thursday, but safeties coach Tim Duffie suggested he attend Saturday's scrimmage.
Fellow Jenks safety Dylan Harding, who is No. 15 on the Super 30, committed to Oklahoma State on July 31.


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Thank you for your question. It likely means that it takes about 3 months for the final results to show after a brazilian butt lift. After fat grafting, some fat cell survive, some do not. Those that don't will slowly, over 3 months, will get reabsorb. Most plastic surgeons will tell their patients that at 3 months, the results is final. Because some cells get reabsorb, a second fat grafting procedure might be required. Another option is to get soft silicone buttock implants, inserted via one small incision within the inter-buttock cleft. Those are permanent and predictable. Combination procedure - fat grafting and buttock implants - is also a great option. See out video! Hope this helps! Best regards, Dr. Marc DuPere, Toronto Plastic Surgeon, Board-Certified, 416.929.9800
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3 month wait-time for the final results in #brazilianbuttlift
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Final Score: Chicago 6, Nashville 3
Chicago, IL (Sports Network) - Troy Brouwer scored the game-tying goal and the go-ahead tally just 40 seconds apart from each other in the third period in Chicago's 6-3 win over Nashville to conclude the home-and-home series.
Dave Bolland also added two goals for the Blackhawks, who avenged the 3-2 shootout loss in the home-and-home set opener and have now won four of their last five contests. Bryan Bickell tallied a goal and an assist while Jonathan Toews also lit the lamp in the win.
Marty Turco made 20 saves for the victors.
J.P. Dumont registered two goals and Martin Erat scored for the Predators, who have dropped two of the last three but have won seven of their last nine. Anders Lindback surrendered six goals on 34 shots in the losing cause.
Nashville took a 3-2 lead at the 3:45 mark of the final frame, but failed to find the back of the net the rest of the way out. The goal came off an offensive zone faceoff win by Chris Mueller. After the quick draw, Dumont stepped up just inside the left circle and rifled the loose puck past an unsuspecting Turco.
However, Brouwer's two-goal rally spelled doom for the Predators.
"When they scored that goal in the third we knew that wasn't going to be enough to stop us," Toews said. "We wanted to repay them for coming back against us yesterday."
Approaching the midway mark of the final period, Brouwer's first goal knotted the game up for a third time. During a power play, Duncan Keith's shot was initially blocked by Lindback's left leg pad, but the rebound was scooped up by an off- balanced Brouwer, who chipped the puck in.
The second goal was all Brouwer as he carried the puck from the left corner and circled around to the high slot where he lasered a wrister just inside the left post.
Two insurance goals then arrived for the home team with 6 1/2 left to play to preserve the win.
Bickell scored the first with a quick flick of the wrists at the top of the crease off a centering pass from Brian Campbell.
Toews' unassisted goal made up for the final score and his 100th career goal.
"We stopped playing our game. They came at us pretty hard," Dumont said. "We have to make sure we learn from it."
The Blackhawks dug themselves in an early hole after Toews was called for a holding penalty only 16 seconds into the game. With time running out of the power play, Erat wristed in a big rebound off a Cody Franson shot to take a 1-0 lead.
Just 4 1/2 minutes later, Bickell controlled a bouncing puck near the left boards and slashed toward the net before he fed a pass through the crease to Bolland, whose tip-in squared the game away.
On yet another power play, Nashville found the back of the net with 5:45 remaining in the opening period. This time it was a Keith holding call that gave the Preds the man advantage, and Dumont hammered home a Shea Weber cross- ice pass to re-gain the edge, 2-1.
Bolland knotted the game once again at two apiece 12 minutes into the second stanza after Tomas Kopecky intercepted a pass in his own defensive zone and shoveled it up to Bolland. He then used some suave stick work to weave around a defender and sweep the puck between the legs of Lindback.
Game Notes
Bolland's goal marks his 100th career point in his 200th career NHL contest...Mueller recorded his first NHL career point off of Dumont's third period goal...Kopecky had two assists in the win...Chicago had four penalties in the first period and ended with six total...Preds defenseman Francis Bouillon sustained an upper-body injury during the first period after taking a hard hit from Brouwer and did not return...The Blackhawks are 16-11-0 as the host this season, while the Predators have a 13-11-1 road mark.


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Marion County ISU Extension is sponsoring three meetings to help boost production for local crop producers and area agi-business.
Keywords: crop, ISU, knoxville
September 30 is the deadline for Marion County farmers to apply for the Noninsured Crop Disaster Assistance Program for their fall 2009 seeded crops.
Keywords: crop, Marion County
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Can You Make It Through This Post Without Having Your Mind Blown?
Are you sitting down? You better be sitting down.
1. Let's start small:
2. Here's something that you never realized:
3. While we're at it, here's some more famous voice actors:
That's Carlos Alazraqui (Reno 911) and Rocko from Rocko's Modern Life, Jessica Walter (Arrested Development) and Fran from Dinosaurs, and James Avery (Uncle Phil from Fresh Prince) and Shredder from Teenage Mutant Ninja Turtles.
4. Here's the right way to eat a cupcake:
5. And cheetos:
6. Now get ready to have your childhood mind blown:
Moving on…
9. DID YOU KNOW? The "YKK" on your zipper stands for "Yoshida Kogyo Kabushikigaisha:"
10. DID YOU KNOW? 3.14 is PIE backwards:
11. DID YOU KNOW? The dot over over an "i" is called a "tittle:"
12. DID YOU KNOW? Cashews grow like this:
13. And pineapples like this:
14. Before we move on, here's the ending to "Inception" explained:
15. Now let's shrink things down. Here's what velcro looks like close up:
16. Here's what chalk looks like under a microscope:
17. And here's what sand looks like under a microscope:
18. Speaking of sand, there are more stars in space than there are grains of sand on every beach on Earth:
Thanks to Carl Sagan for this knowledge.
Let's go deeper:
19. If you shrunk the Sun down to the size of a white blood cell and shrunk the Milky Way Galaxy down using the same scale, it would be the size of the continental United States:
20. Speaking of things that are huge, a Blue Whale's heart is so big, a small child can swim through the veins:
21. There's enough water in Lake Superior to cover all of North and South America in one foot of water:
22. Here's the true size of Africa:
23. And the Pacific Ocean:
24. Let's think bigger: If you dug a hole to the center of the Earth and dropped a book down, it would take 45 minutes to reach the bottom:
25. Here's what Jupiter would look like if it were the same distance to Earth as the Moon:
26. And here's what a sunset on Mars looks like:
Too much? Just breathe:
27. DID YOU KNOW? There are more bacteria cells in your body than actual body cells:
Anywhere from 2 to 9 pounds of them. We're more bacteria than we are human!
28. DID YOU KNOW? John Tyler, the 10th President of the United States, has a grandson that's alive today:
John Tyler had a son, Lyon, when he was 63. Lyon had a son, Harrison Tyler, at 75.
29. DID YOU KNOW? A compressed spring weighs more than a relaxed one:
30. JUST THINK: We know more about the surface of the moon than the bottom of the ocean:
31. Speaking of the ocean, there are more atoms in a glass of water than glasses of water in all the oceans on Earth:
Now let's talk about time:
36. Speaking of time: The Ottoman Empire still existed the last time the Chicago Cubs won a World Series:
37. Keep this in mind, too:
Mind not blown yet? Things are about to get REAL:
38. The Great Pyramid was built circa 2560 BC…
… and Cleopatra lived 69 BC – 30 BC…
… and the first Moon landing was in 1969, AD…
… which means Cleopatra lived closer to the Moon landing than she did to the building of the Great Pyramid.
39. Speaking of the pyramids, they were as old to the Romans as the Romans are to us:
40. Should we keep going? The difference in time between when Tyrannosaurus Rex and Stegosaurus lived is greater than the difference in time between Tyrannosaurus Rex and now:
41. Here's one more: In 1903 the Wright Brothers flew for the first time…
…38 years later, in 1941, the Japanese bombed Pearl Harbor…
… and 28 years later, in 1969, man landed on the Moon.
That's 66 years.
PHEW! That was intense. Here's a picture of a puppy burrito (puprito) to chill you out:
- Greece will decide on Sunday whether they will accept austerity measures as a condition of a bailout that may stave off an imminent financial and economic crisis.
- It took a while, but an English national soccer team has finally defeated Germany in a major tournament. England took third place with a 1-0 win over the top-ranked German team in the FIFA Women's World Cup ⚽
- Chile defeated Argentina on penalties to win the Copa America tournament. It's the first ever trophy for Chile in the 99 years of the Copa ⚽


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I have greatly enjoyed all of these opportunities, as I increase my own personal learning along the way. I hope I can also help you reach your learning goals.
I offer tutoring for English language or literature, all levels; Spanish language, elementary/intermediate; Elementary and middle level subjects (math, reading, writing).
Please feel free to contact me with any questions or to schedule a lesson! Hello, my name is Danielle W. I graduated from the University of Northern Iowa with degrees in TESOL and Spanish. I have worked with many levels of students studying various subjects and enjoyed it immensely. I have tutored elementary ESL… Read more
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Some of the candidates running for Darlington Youth MP: (L-R) Amy Merchant (15), Joe Clark (13), Rachel Gunnell (17), Josh Wakeford (16), Joseph Barker (13, back) and Daniel Brown, (14)
Joanna Morris / / News
The count to decide the town's next Member of Youth Parliament was held in Darlington's council chamber.
Youngsters from across the town hit the polls last week in a bid to determine who will best represent them for the next year.
Loading article content
Six teenagers – aged between 13 and 17 – spent a month campaigning to win a seat in the UK's Youth Parliament, which aims to give support and a voice to young people across the country.
The elected member from Darlington will help their peers have their say on issues in the town for the next two years and will represent the area at a House of Commons debate in November.
The candidates battling it out at the polls were 13-year-olds Joe Clark and Joseph Barker, of Darlington School of Mathematics and Science, Danny Brown, 14, and Amy Merchant, 15, both of Longfield Academy of Sport, Josh Wakeford, 16, of Queen Elizabeth Sixth Form College, and Rachel Gunnell, 17, of Carmel College.
During their campaign trail, the students prepared manifestos, delivered flyers and took to social networking to spread their message.
Issues highlighted by the candidates included alcohol abuse, internet safety, disability awareness, health services and negative stereotypes of teenagers.
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Votes counted to decide Darlington's next member of Youth Parliament
The Northern Echo is getting as bad as Darlington Council.
DOH! - they printed the results yesterday!!!
Danny Brown elected to represent Darlington in UK's Youth Parliament
8:18pm Tuesday 4th March 2014
DOH! - they printed the results yesterday!!!
Danny Brown elected to represent Darlington in UK's Youth Parliament
8:18pm Tuesday 4th March 2014


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Date: 1825-08-19
Description: Second page of the Treaty between the United States and the Chippewa, Sauk, Fox, Menominee, Iowa, Sioux, Winnebago and a portion of the Ottawa, Chippewa, a...
Description: Third page of the Treaty between the United States and the Chippewa, Sauk, Fox, Menominee, Iowa, Sioux, Winnebago and a portion of the Ottawa, Chippewa, an...
Date: 1825-08-27
Description: Map is ink on paper. Map shows plat of the town of Munnomunne, the Fox River, farm lots, and an established highway. The map is signed by "J.D. Doty, Propr...
Date: 1821-08-18
Description: Green Bay treaty, August 18, 1821. The treaty is signed by the chiefs of the six Indian nations for the possession of lands near the Fox River to the Winne...
Date: 1823
Description: In 1823 U.S. Army major Stephen H. Long led an expedition of scientific, trade, and military investigation up the Mississippi River and the Red River of th...
Date: 1827
Description: Drawings of Datura Stramonium, more commonly known by the names jimson weed, devil's trumpet, devil's weed, thorn apple, tolguacha, Jamestown weed, stinkwe...
Date: 1829
Description: Cincinnati, Ohio, Ebr. Martin, engraver.
Date: 1827-06-22
Description: Front page of "Freedom's Journal", Volume 1, #15.
Date: 1821-07-03
Description: Two pages of a letter written by Thomas Forsyth.
Date: 1826
Description: Originally titled "Chippeway Squaw and Child". Portrait of a Chippewa (Ojibwa) woman and her child painted by J.O. Lewis at the Treaty of Fond du Lac in 18...
Description: Drawing of Cassville depicting a horse cart on the river's edge, with men and boats.
Date: 1828 ca.
Description: Increase Lapham's drawing of a Union mondonta (or Unio soleniformis) shell. There are also two views of another shell drawn below and a drawi...
Description: Plat of the Private Claims at Prairie Du Chien.
Date: 1824-08-20
Description: The front page of a letter writen by President Thomas Jefferson to Henry Dearborn.
Date: 1820 ca.
Description: Map of the Northwestern Territories of the United State showing the track pursued by the Expedition in 1820.
Date: 1825
Description: Hand-colored lithograph portrait of Chippewa (Ojibwa) chief Kitch-ee-i-aa-ba, or the Big Buck. Painted at the Treaty of Prarie du Chien (1825).
Description: This plat map is pen-and-ink and shows landowners, the Mississippi River, the Marais de St. Friole, Fort Crawford, the town commons, and the Village of St....
Date: 1821
Description: Engraving depicting the Schoolcraft expedition crossing the Ontonagon River to investigate a copper boulder.
Description: Fourth page of the Treaty between the United States and the Chippewa, Sauk, Fox, Menominee, Iowa, Sioux, Winnebago and a portion of the Ottawa, Chippewa, a...
Date: 1820
Description: Narrative Journal of Travels from Detroit Northwest through the Great chain of American Lakes to the sources of the Mississippi River in the year 1820. Ill...
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Tour Diary
Street Team
Latest Videos
Join the Email List
Free embed music player from ReverbNation.com
Latest CD: "The Red Album"
Get your hot little hands on it at
CD Baby
, or come to an
upcoming concert
Tour Diary
Monday, June 29th, 2015 4:22 PM
COUNTDOWN: 20 MORE SHOWS UNTIL WE GO ON HIATUS! Shows selling out fast so get your tickets early!
RM Tour Diary #261: Something Old and Something New
This week we did a whirlwind tour of the Northeast, with 6 shows in 6 days. We started with two shows at one of our earliest haunts, Caffe Lena in Saratoga Springs. This felt like a homecoming show of sorts, with two sold out shows and rowdy crowds. Thanks to our pal Fred Gillen Jr and wife Peg for hosting us at their lake house- what's not to love about that!?
Thursday we made our way over to Old Saybrook, CT, for our 4th show at the Katherine Hepburn Theater. We love playing this classy theater and have continued to build a great audience at "The Kate", and they were particularly smitten with our opening act Matt Nakoa! We drove north Friday to beautiful Wolfeboro, NH to perform at the Great Waters Folk Festival. We spent one glorious morning by the lake and were sad to leave!
Saturday night we played our first time at new venue The Spire Center in Plymouth this is really a fabulous venue with terrific sound and we were glad to be one of the early acts to stomp around on the stage. Local fiddler & singer Eva Walsh kicked off the evening and tore it up! Sunday found us back down close to home at City Winery- our favorite spot to play in NYC. Opening the show was singer-songwriter Jesse Terry with John Paul on guitar. These are two of the nicest guys you'll ever hope to meet! We're so grateful for all the support and hospitality from these venues over the years!
Next week we'll hit a few more of our favorite spots; The Hamilton in NYC, Sellersville Theater in PA, and a new series up in Clifton Park, NY.
Happy 4th of July!
Molly, Abbie & Laurie


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The Renda Broadcasting Kids Fitness Zombie Chase
The Jacksonville Landing • Jacksonville, FL
Sunday, October 20
What you get
- Zombie Run Entry Package for One
- Choose to Register as a Runner (Ages 5 to 15) or a Zombie (Ages 16+)
- Fun for the Whole Family -- Parents and Grandparents Welcome!
- Proceeds Benefit Shannon Miller Foundation to Combat Childhood Obesity
The perfect way for your kids to get some exercise and ready for the Halloween season? The Renda Broadcasting Kids Fitness Zombie Chase, of course. Held on Sunday, October 20, this event is fun for the whole family: Kids ages 5 to 15 do their best to outrun a throng of infected zombies and avoid being stickered, while those ages 16 and older can put on their scariest Halloween makeup and do the chasing while posing as a zombie. No matter which role your child chooses to take, he or she will still be able to get down to live music and more fun at the post-race party.
$10 ($20 value) for an entry package, including:
• One entry
• T-shirt and medal for ages 5 to 15
• Access to post-race after party
The Renda Broadcasting Kids Fitness Zombie Chase on Facebook
Fine print
*Online registration is required immediately after voucher purchase; registration deadline is October 16REFUND POLICY: This event is nonrefundable. No full or partial refunds are given to customers who miss an event. In the event of a cancellation, your voucher will be fully refunded. • Each voucher is good for 1 person • The deal is valid only for tickets on the date[s] of the event as listed and may not be exchanged or redeemed for any other dates • Entire value must be used in a single visit
The Renda Broadcasting Kids Fitness Zombie Chase
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'No Smoking' Means NO SMOKING!! [Video]
Bernice is the boss at Tremont Towing...in NO uncertain terms! When a rude woman came to retrieve her impounded car, and refused to stop smoking,..
Bernice is the boss at Tremont Towing...in NO uncertain terms! When a rude woman came to retrieve her impounded car, and refused to stop smoking,..
As we get older, our memories seem to deteriorate. Learning becomes more difficult. It's no coincidence, children are the best learners. They take naps.
Stunt Driver Jim Dowdall has been involved in some of the greatest car chase scenes in recent movie history. His work has appeared in the Jason Bourne Movies, the Indiana Jones franchise, and James Bond films. He recently performed a stunt that's never been attempted
Reginald Kenneth Dwight was born 68 years ago today (March 25th, 1947). His "Mum" couldn't possibly have imagined what lay ahead for her little boy. Elton John, as the world knows him, has attempted suicide, sold over 300 million albums, and sold out the legendary Madison Square Garden more times than any musical act in its history. His "Candle in the Wind 1997" is the largest selling single of all time (33 million copies)
The National WWII Museum in New Orleans has just received a 20 million dollar donation. AP reports, Bollinger Shipyards CEO, and chairman, Boysie Bollinger made the announcement at a Tuesday news conference.
They say curiosity killed the cat. I don't buy that. My belief is that knowledge is power. When we stop learning, we stop living. If you don't know about an opportunity, you don't have that opportunity. There's plenty to wonder about in this life.
Former New Orleans Saints safety Darren Sharper is facing multiple charges of rape and drug distribution in 4 states, Louisiana,California, Nevada, and Arizona. He faces two potential life prison sentences in Louisiana alone. A plea bargain has been reached.
I'm a Cancer. That's a "Water Sign." In my case, it really fits. I've always loved the water. I don't generally venture to the "Cement Pond" this early in the year, but something compelled me to put my feet in, and the rest of me...
I'm a fan of history. There's something that gives us a greater sense of who we are. It seems that way to me, anyway. It's interesting to see how customs, fashion and technology have evolved over the years. I'm pretty sure this video wasn't shot using an iphone...
In the words of the late George Harrison, "Little darling, it's been a long cold, lonely winter." Spring is here! Let's usher it in with this joyful rendition of George's classic, "Here Comes the Sun"...
A reporter with the New York Times says time spent with New Orleans Saints & Pelicans owner Tom Benson convinced him the 87 year old patriarch is mentally sound. Ken Belson is a football writer for the Times.Belson told The Advocate he interviewed the Mr. Benson for a total of about two hours in a three day period.
St. Patrick's Day is a BIG deal in Chicago. The city has one of the largest Irish populations in the US. There are parades. Green beer flows, and they turn the river green.


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Poverty can be an overwhelming problem, especially when it affects children. Although many times it's the adults that get the spotlight in advocacy efforts, all too often children and poverty are an even more serious issue. But there is hope! With the proper information and tools, you can make a difference for children and poverty that affects them.
We have tons of resources to help educate and equip you to make a big impact on children and poverty. There are so many ways to get involved ... learn the facts, take quizzes, download screensavers and posters, get free bumper stickers and much more. You can even download sermon outlines to help you share the message with others.
Join us in our efforts to make sure children and poverty have nothing to do with each other.
Every time you tell the Compassion story, children can benefit. That's why we've made it easy to spread the word about what we're doing - and how much children in poverty need help.
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Help spread the word about Compassion's ministry to children in need. Get a variety of resources such as a screensavers, videos, posters, web banners, wallpaper and more.
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Your donation to the Where Most Needed fund will help Compassion better serve children in extreme poverty.
"Thanks for fixing my heart!" - Fatao, a Compassion-assisted child from Burkina Faso


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White Sox 14 in a row!!!
White Sox 14 in a row!!!
"I like my antiques new " - Steve Martin
We just got this from the president of our division at work:
"It's been 102 years since both Chicago baseball teams have played in the post season together! Teddy Roosevelt was President, the average wage for the American worker was 22 cents an hour, coffee was 15 cents a POUND, the American flag had only 45 stars, the population of Las Vegas, Nevada was only 30 and canned beer had not yet been introduced! Yes, it's been a long long time. Congratulations to the White Sox and their fans for the thrilling victory last night. And of course congratulations to the Cubs for winning their second straight Central Division title. Playoffs start tonight and with some luck we will see an all Chicago World Series later this month. Should we be fortunate to see that, I will personally host the Cubs pep rally in the hopes that we see a different outcome in the Series than 1906...when the Sox won the Championship. Of course the Cubs went on to win the 1907 and 1908 World Series after the Sox beat them in 1906. Yeah yeah yeah I know they haven't won it since....everybody is entitled to a bad century once in a while! Let's have some fun with the playoffs...decorate your cube, your office, your car, yourself in blue or black and root on your squad!
Go Chicago....Cubs!"
"All my life I have tried to pluck a thistle and plant a flower wherever the flower would grow in thought and mind." -- Abraham Lincoln
Just for fun, can we go ahead and play the rest of the games in the playoffs?
R.I.P. Cyrano and Roxanne.
Oh yeah. I didn't mean to make it sound like we had skipped to the World Series. Some people at my job say they hope it doesn't happen because if there are any problems it will look bad for our 2016 Olympic chances.
"All my life I have tried to pluck a thistle and plant a flower wherever the flower would grow in thought and mind." -- Abraham Lincoln
Hey PJ - I heard some team won some baseball game today.
Shea Stadium
1964 - 2008


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Ticket Package to LPGA International Crown Golf Tournament
Caves Valley Golf Club • Owings Mills, MD
July 24, 25, 26, or 27
What you get
- The LPGA Tour's Top Pros Compete in an International Tournament!
- Choose from Two Ticket Offers
- Choice of Tournament Date from July 24 to 27
When the LPGA's top golfers gather on the course, anything can happen. Experience the action for yourself with this offer for a ticket to the LPGA International Crown golf tournament at Caves Valley Golf Club from July 24 to 27. This inaugural event pits players from eight countries around the world (the US, the Republic of Korea, Japan, Thailand, Spain, Sweden, Chinese Taipei, and Australia) against one another to crown a country champion. And, with your ticket, you'll catch every drive, chip, and putt of the excitement this summer.
Choose from the following offers:
$25 ($47 value) for a single-day ticket package, including:
• One single-day grounds ticket
• Tournament program
• $10 concession voucher
$49 ($105 value) for a single-day International Pavilion Pass package, including:
• Single-day International Pavilion Pass
• One reserved first tee bleacher seat
• Official tournament program
• $10 concession voucher
• Commemorative lanyard/ticket holder
Fine printREFUND POLICY: This event is nonrefundable. No full or partial refunds are given to customers who miss an event. In the event of a cancellation, your voucher will be fully refunded • Limit 10 per purchaser • Each voucher is good for 1 person; children 17 and under are admitted at no charge to the Grounds and to the International Pavilion with a paying adult holding a ticket • Active duty, retired and reserve military personnel and their dependents will be admitted to the Grounds at no charge with a valid military/dependent ID presented at the gate • The deal is valid only for tickets on the date[s] of the event as listed and may not be exchanged or redeemed for any other dates • Purchaser may change the name of the individual retrieving tickets up to 24 hours in advance of event via the LivingSocial website event details section; no name-changes or alternate pick-ups permitted without notice at venue • Entire value must be used in a single visit
Ticket Package to LPGA International Crown Golf Tournament
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text:
A method of amplifying template DNA by polymerase chain reaction (PCR) in which a single oligonucleotide primer having a restriction site is contacted with the template DNA, whereby the oligonucleotide randomly anneals to a single strand of the template DNA and DNA sequences complementary to the single strand are synthesized. An initial PCR amplification yields synthetic DNA sequences having the oligonucleotide sequence incorporated therein at the 5' end, and a sequence complementary to the template DNA. A second PCR amplification under higher stringency conditions amplifies regions of the template DNA to give DNA fragments having the restriction sites of the oligonucleotide primer. Thereby the method can be used to amplify trace quantities of template DNA of unknown sequence simply and efficiently, which has applications in the construction of DNA libraries of chromosome specific regions and the development of probes for chromosome mapping.
1. A method for amplifying template DNA having an unknown sequence by a polymerase chain reaction (PCR) using oligonucleotide primers comprising a contiguous sequence which consists of SEQ ID No. 1, which method comprises the steps of:
(a) conducting multiple PCR cycles on a first reaction solution containing said template DNA, said oligonucleotide primers, and an amphipathic polymer, each PCR cycle comprising:
(i) denaturing double-stranded DNA into single-stranded DNA, wherein in the first of said multiple PCR cycles the double-stranded DNA is said template DNA,
(ii) annealing said oligonucleotide primers to the single-stranded DNA at 10�-40� C. for 90-150 minutes, and
(iii) synthesizing extension products of said oligonucleotide primers, which products are complementary duplicates of Various regions of the single-stranded DNA,
wherein the amphipathic polymer increases the annealing efficiency of the oligonucleotide primers and single-stranded DNA,
so that a plurality of extended primer molecules are synthesized each having the nucleotide sequence of said oligonucleotide primer at both ends and a nucleotide sequence of each region of the template DNA; and
(b) conducting multiple PCR cycles under stringency conditions higher than in step (a) on a second reaction solution containing the reaction solution produced by step (a) and an additional amount of said oligonucleotide primers, but not containing said amphipathic polymer, thereby amplifying the template DNA as a group of various DNA sequences therein.
2. The method of claim 1, wherein the amphipathic polymer is a polyethylene glycol having a molecular weight in a range of 1,000 to 10,000.
3. The method of claim 1, wherein step (a) the amphipathic polymer is polyethylene glycol present in the range of 10 to 15% and the oligonucleotide primers are present in the range of 1.0 to 1.5 μM in the first reaction solution.
4. The method of claim 1, wherein in step (b) the additional oligonucleotide primers are present in the range of 4.0 to 5.0 μM in the second reaction solution.
5. A kit for amplifying a template DNA having an unknown sequence by the method of claim 1 comprising in combination:
(a) oligonucleotide primers comprising a contiguous sequence which consists of SEQ ID No. 1;
(b) Tag DNA polymerase; and
(c) nucleotide triphosphates.
This application is a continuation of now abandoned application, Ser. No. 07/936,831, filed Aug. 28, 1992.
1. Field of the Invention
This invention relates to a method of DNA amplification. In particular, the invention relates to an improved PCR method of DNA amplification which is useful in a wide range of medical and bio-engineering fields such as the construction of DNA libraries of specific chromosome regions, the isolation of genes originating in specified chromosome regions and probe development.
2. Description of the Related Art
In recent years, rapid progress has been made with molecular biological elucidation of the structures and mechanisms which constitute life and with the accompanying technical applications, and the foundations of genetic engineering and biotechnology have gradually been established.
Using such techniques, various genes which are characteristic of species and individual structures have been identified extending from procaryotes to eucaryotes and much knowledge has been amassed by the analysis of this genetic information. This research is now being extended to the genomic DNA of higher animals, including man.
In order to determine the base sequence of the total human genome, which is said to be composed of 3,000,000,000 base pairs, and to draw up its genetic map, the construction of DNA libraries for specific chromosome regions and the drawing up of physical maps of the chromosomes using individual DNA fragments as indicators (probes) has become an essential process. Furthermore, the physical maps of such chromosome specific regions can be an effective means of identifying the genes responsible for most human genetic diseases which have not as yet been elucidated at the molecular level and for analyzing its structure.
In the past, a chromosome DNA library has been constructed by cloning fragmented chromosomal DNA in a cloning vector, and collecting individual clones to form a library. In this way, in order to study the human genome, specific chromosome libraries constructed from sorted chromosomes or hybrid cells have been used, and, yeast artificial chromosome (YAC) libraries and the like have been prepared and used in the development of new probes for investigating the target gene.
However, in cases where such conventional gene libraries have been used it takes time to obtain the large number of necessary probes for the specific region of the chromosome and, as a result, the need for much effort, time and expense for the elucidation of the target gene cannot be avoided.
On the other hand, methods in which DNA libraries and probes have been prepared by amplifying DNA fragments of physically cut-out chromosome specific regions using the PCR (polymerase chain reaction) method have been suggested in recent years (for example, Ludecke et al., Nature, 338, 384, 1989). The PCR method involves the use of synthetic oligonucleotides complementary to certain base sequences of a template DNA as primers for in vitro DNA synthesis to enable DNA fragments which are more or less the same as template DNA to be obtained in large amounts by repeating a cycle of annealing, extension and denaturation. Automation of this amplification process has led to its application to gene cloning.
However, use of the PCR method requires prior knowledge of the base sequence at least at both ends of the template DNA fragment. Thus in cases where the method is applied to DNA fragments of unknown sequence derived from chromosome specific regions, ligation of the individual DNA fragments with vectors etc. is essential and the template DNA is accordingly subjected to various chemical and enzymic pre-treatments. Consequently, ligation PCR of this type is not only a more complicated operational procedure than the PCR method used for known DNA sequences but the amplification efficiency is also reduced and there is a disadvantage in that many DNA fragments are required for templates for example.
The present invention provides a new method of DNA amplification in which trace quantities of DNA fragments of unknown sequence can be amplified simply and efficiently.
This present invention provides a method of amplifying template DNA by the PCR reaction wherein (a) a single oligonucleotide is contacted with said template DNA whereby said oligonucleotide randomly anneals to a single strand of said template DNA, DNA sequences complementary to the said single strand are synthesized using said oligonucleotide as a primer, and said oligonucleotide also randomly anneals to the complementary strands whereby DNA sequences corresponding to the template DNA are synthesized so that PCR amplification yields synthetic DNA sequences which each incorporate said oligonucleotide sequence at the 5' end, and (b) said synthesized DNA sequences then being subjected to PCR amplification under conditions of stringency higher than in step (a).
Viewed from another aspect, the present invention provides a gene library comprising cloned sequences amplified by means of the above described method.
Furthermore, in a preferred embodiment of the DNA amplification method of the present invention the template DNA and oligonucleotide are annealed for 90-150 minutes in a reaction solution at 10�-40� C. which contains an amphipathic polymer such as polyethylene glycol etc.
FIG. 1 is an agarose electrophoresis pattern (A) of DNA fragments amplified by the method of this present invention and a Southern blot pattern (B) in which human genome total DNA had been used as a probe.
FIG. 2 is an agarose electrophoresis pattern which illustrates the insert size of the λ phage clone in which the DNA fragment amplified by the method of this present invention has been introduced.
FIG. 3 is an agarose electrophoresis pattern of DNA fragments tested with the method of this present invention.
In the present invention, the DNA sequence which is the subject of the amplification is a DNA fragment for which base sequence information has not been obtained (at least, a DNA fragment for which the base sequence information required for conventional PCR amplification has not been obtained), such as a chromosome specific region which has been physically cut out or a DNA sequence which has been cloned for a YAC library etc. for example. In the past, the ligation PCR method as described above could be used in cases where such DNA fragments were PCR amplified, but the method of this present invention is such that DNA fragments of unknown sequence can be amplified very effectively using more or less the same procedure and apparatus as used for conventional PCR without carrying out a chemical or enzymatic pre-treatment of the template DNA like that in the case of the ligation PCR method.
The method of the present invention involves first annealing a single type of oligonucleotide in an arbitrary region of a strand of the template DNA which has been thermally denatured, then extending the DNA chains which are complementary to the template DNA using this oligonucleotide as a PCR primer, and synthesizing the complementary DNA corresponding to a random sequence region of the template DNA. Thus, by repeating the cycle of denaturing, annealing and extension, various DNA sequences having the primer sequence at one end and a sequence complementary thereto at the other end thereof corresponding to a random region of the template DNA may be synthesized (this process is referred to hereinafter as low stringency PCR).
It will be appreciated that the single oligonucleotide also anneals to the complementary strand to enable PCR amplification to take place.
Low stringency PCR differs from ordinary PCR in that it uses as a primer a single oligonucleotide of which the whole sequence is unlikely to be complementary to any sequence of the template DNA. A single primer of 20-30 nucleotides which has a plurality of restriction enzyme recognition sequences and which has at least two bases on the 3'-end which do not form restriction enzyme sites may be used. Thus, since random base sequences exist in the sequence of the DNA chains, the sequence of the 3'-end of the above mentioned nucleotide can provide a PCR primer in any region of the template DNA by annealing to complementary sequences on the template DNA. Furthermore, in cases where the above mentioned oligonucleotides are used as primers, the various DNA sequences which are ultimately amplified have a plurality of restriction enzyme cleavage sites at both ends and ligation to a vector etc. can be carried out easily. The synthetic nucleotide comprised of the base sequence 5'-TAGATCTGATATCTGAATTCCC-OH3' (SEQ. ID NO. 1 referred to hereinafter as BVE22cc) can be cited as an actual example of such an oligonucleotide. BVE22cc has within the sequence comprised of 22 nucleotides recognition sequences for the restriction enzymes BgIII, EcoRV and EcoRI (AGATCT, GATATC, GAATTC respectively) and it has a continuous sequence of cytosine (C) at the 3'-end. The DNA sequence which is amplified by means of the present invention can be used directly as a probe for the screening of genome libraries for example, and in this case it is also possible to use oligonucleotides which do not include a restriction enzyme recognition sequence.
However, as mentioned above, the low stringency PCR of this present invention involves the use of an oligonucleotide of which the complementary sequence for the template DNA is small and so the efficiency of the annealing of the template DNA and the primer is low.
Thus, with the method of the present invention it is recommended that the annealing reaction of the template DNA and the oligonucleotide in the low stringency PCR is carried out over a long period of time. That is to say, whereas the annealing reaction in ordinary PCR is of the order of 1-2 minutes, in the present invention it is set at 90-150 minutes and the opportunity for the template DNA and the primer to approach and anneal is increased.
Furthermore, this annealing reaction is preferably carried out at low temperature. Ultimately, with ordinary PCR, the template DNA and the primer are annealed in a reaction solution at 50�-60� C., but in such a temperature range the free primer and template DNA in the reaction solution are subject to the effects of thermal vibration and so mutual annealing is difficult even when they approach. Hence, the temperature of the reaction solution during the annealing reaction is set to a low temperature of 10�-40� C. and the effect of thermal vibration is minimized.
Moreover, in the method of the present invention an amphipathic polymer such as polyethylene glycol (PEG) or a polysaccharide such as glucose or sucrose for example may be added in order to increase the annealing efficiency of the template DNA and the primer. Ultimately, these amphipathic polymers increase the viscosity of the reaction solution and suitably suppress the Brownian motion of the free template DNA and primer and increase the unification potential, providing conditions which are suitable for annealing. Moreover, these amphipathic polymers have no chemical action on the DNA molecule and no effect on the denaturation and extension steps in the PCR.
These means of increasing the annealing efficiency in low stringency PCR can be adopted suitably either individually or in various combinations according to the added amount and molecular weight of the oligonucleotide which is used for the primer and/or the template DNA which is the subject of the amplification for example.
Next the various DNA sequences synthesized by the low stringency PCR mentioned above are amplified without changing the sequences by means of PCR under different conditions (referred to hereinafter as high stringency PCR). In this case, the DNA sequence which is the subject of the amplification, as aforementioned, has the primer sequence at one end and its complementary sequence at the other end and so the oligonucleotide used in the low stringency PCR is used for the primer. Moreover, if the primer is of a single type and a plurality of templates (DNA sequences) is excluded, the high stringency PCR is such that template DNA is amplified according to the number of cycles in accordance with more or less the same general principles as the conventional PCR. Furthermore, the conditions for denaturation, annealing and extension can be set in the same way as for conventional PCR, or the respective conditions can be modified somewhat and carried out in a number of stages.
The various DNA sequences amplified in this way may be used as probes for genetic screening for example since they include random sequence regions of the template DNA and they are comprised of the quantities required for the various genetic operations, or they may be cloned into a vector to establish a gene library.
Such DNA sequences may be further characterized following amplification by sequencing.
Examples are described below and the DNA amplification method of the invention is described in practical terms, but of course the invention is not limited to the cases indicated below.
Moreover, in the following examples the high stringency PCR is carried out in two stages, changing the conditions, and so the low stringency PCR is indicated as the 1st PCR and the two types of high stringency PCR are indicated as the 2nd PCR and the 3rd PCR.
Chromosome DNA recovered from two metaphase chromosome 4 50-100% fragments (ie. fragments of the distal half of the short arm of human chromosome 4) was synthesized and amplified using the method of the present invention.
First of all, for the 1st PCR, 2 μl of the 1st PCR solution was added to a tube which had been filled with 1 μl of pickup solution (composition as indicated in Table 1) which contained chromosome DNA fragments and a cycle of denaturation (90� C., 5 min), annealing (22� C., 120 min) and extension (50� C., 20 min) was repeated six times using a DNA thermal cycler (made by Perkin-Elmer-Cetus). Furthermore, at this time the temperature was raised gradually from 22� C. to 50� C. over a period of 20 minutes from annealing to extension in each cycle. Moreover, the reaction solution composition used in the 1st PCR was as shown in Table 1.
Next, after completing the 1st PCR, 6 μl of the 2nd PCR solution (same composition as in Table 1) was added to the reaction product (3 μl) to make a total of 9 μl, and the 2nd PCR was carried out by repeating the cycle of denaturation (92� C., 1.5 min), annealing (50� C., 1 min) and extension (72� C., 2 min) eleven times. However, on the first cycle only, the denaturation was carried out for 5 minutes at 92� C.
Finally, for the 3rd PCR, 51 μl of the 3rd PCR solution was added to the 2nd PCR product (9 μl) to make a total volume of 60 μl and a cycle of denaturation (92� C., 1.5 min), annealing (55� C., 1 min) and extension (72� C., 2 min) was repeated thirty times. Moreover, the composition of the 3rd PCR solution was as shown in Table 1.
This accumulation of DNA chains for which the chromosome DNA amplified by the three stage PCR was the template was analyzed using the agarose electrophoresis method and the Southern blot method using full-length human genomic DNA as a probe. Moreover, as a comparative example for this analysis, amplification with the same method as described above was also carried out with 1 pg and 10 fg of RsaI digested human genome DNA (positive control) and metaphase chromosome DNA which had been completely sublimed by laser irradiation and with no template (negative control).
The results were as shown in the agarose electrophoresis pattern (A) and the Southern blot pattern (B) of FIG. 1.
Moreover, in FIGS. 1 (A) and (B), Lane 1 shows the state of amplification of the DNA of metaphase chromosome 4 p 50-100% fragments, Lane 2 shows the state of amplification of the metaphase chromosome DNA which had been completely sublimed, Lanes 3 and 4 show the states of amplification respectively of the 1 pg and 10 fg RsaI fragments of human genome DNA, and Lane 5 shows the state of amplification in the case where no template DNA was added. Furthermore, in FIG. 1 (A), Lane 6 shows the electrophoresis pattern for the Pst I fragment of the λ phage DNA (reterred to herein after as λ/Pst I) used as a size standard.
As is clear from FIG. 1, in the method of the present invention the DNA fragment of unknown sequence cut out from the chromosome specific region (chromosome 4 p 50-100%: Lane 1) was amplified with good efficiency in the same way as the RsaI fragment of human genome DNA (Lanes 3 and 4). Moreover, the average sizes of the respective amplification products were about 500 bp for the human genome DNA fragment, and a smaller size of about 300-400 bp for the chromosome DNA fragment, implying that there is some mechanical or chemical damage during chromosome preparation. Furthermore, faint bands were obtained on amplifying the sublimed chromosome (all burned down metaphase: Lane 2) and in the absence of a template (no template: Lane 5), but these were thought to be due to inadequate sublimation treatment (laser irradiation) of the chromosome or to originate from some contamination in the manipulation process for example. In any case, there is a clear difference on comparing the amplification efficiency in the presence of the proper template DNA.
The PCR products of the chromosome DNA obtained in Example 1 were each cloned in a λ phage vector and a library was built up.
First of all, the DNA was extracted from about half of the 3rd PCR solution in Example 1 by extraction with phenol/chloroform and chloroform/isoamyl alcohol, and by ethanol precipitation, and this DNA was treated with EcoRI to create cloning sites on both ends. After removing the very small DNA fragments by ultrafiltration, the fraction of amplified DNA (50-100 ng) was introduced and ligated to the EcoRI sites of λ phage gt11 which had been pre-treated with phosphatase and this was packaged in vitro and used to infect E. coli Y1090r cells. As a result 5�106 clones were obtained.
Next, 68 clones were selected randomly from this λ phage library and the inserts characterized. That is to say, the insert was amplified by means of conventional PCR taking each clone as template DNA and using a primer of the same sequence as the EcoRI part of the λ phage and the sequence adjacent thereto, and the size was analyzed by means of agarose electrophoresis.
The agarose electrophoresis patterns for 20 of the 68 types of insert analyzed are shown in FIG. 2. The results showed that the insert size of the 68 clones analyzed was distributed over about 100-1500 bp, and this is in close agreement with the size distribution of the amplified chromosome DNA in Example 1 (see FIG. 1). Furthermore, when these inserts were hybridized with human genome DNA (EcoRI or HindIII fragments), 40-60% of the clones were confirmed as being a human unique sequence, about 10% were confirmed as being a high frequency repeat sequence and 20-30% were confirmed as being a low-intermediate frequency repeat sequence.
The optimum concentration range of the primer and/or amphipathic polymer in each PCR was tested.
10 fg of RsaI digested human genome DNA was amplified in the same way as described in Example 1. The 1st PCR was carried out with a PEG concentration of 0, 8 or 12% in the reaction solution and with a primer (BVE22cc) concentration 0.2, 0.5 or 1.0 μM. After completing six PCR series, one sixth of each reaction solution was subjected to electrophoresis on 1.5% agarose, stained with ethidium bromide and the respective reaction efficiencies were analyzed.
The results of this experiment were as shown in the agarose electrophoresis patterns (1st PCR) of FIG. 3. Moreover, in FIG. 3 (1st PCR), the unnumbered leftmost lane shows the λ/PstI used as a size standard, and Lanes 1-3 show the states of amplification of the DNA fragment (10 fg) with a primer concentration of 1.0 μM and PEG concentrations of 0, 8 and 12% respectively, while Lanes 4-6 show the states of amplification of the DNA fragment (10 fg) with a PEG concentration of 12% and primer concentrations of 0.2, 0.5 and 1.0 μM respectively. As is clear from FIG. 3 (1st PCR), the efficiency of the DNA chain synthesis increased as the PEG concentration was increased when the primer concentration was set at 1.0 μM (Lanes 1-3), and the efficiency of the synthesis increased as the primer concentration was increased when the PEG concentration was set at 12% (Lanes 4-6). However, although not shown in FIG. 3 the synthesis of DNA chains was also observed in cases where no template DNA was present with a PEG concentration of more than 15% or a primer concentration of more than 3 μM. Consequently, it was adjudged that when BVE22cc is used for the primer and PEG is used for the amphipathic polymer in the low stringency PCR of this present invention, a PEG concentration in the reaction solution set within the range 10-15% and a primer concentration set within the range 1.0-1.5 μM is desirable.
Next, the solution which contained 12% PEG and 1.0 μM primer from among the various reaction solutions used in the above mentioned 1st PCR was treated to a 2nd PCR using the same method as described in Example 1 and then it was submitted for a 3rd PCR. The 3rd PCR was carried out with the adjustment of the primer concentration in the reaction solution to 3.0, 4.0 or 6.0 μM.
The results obtained were as shown in FIG. 3 (3rd PCR).
Moreover, in FIG. 3 (3rd PCR), Lanes 7-9 show the states of amplification when the primer concentration was set at 3.0, 4.0 and 6.0 respectively. As is clear from FIG. 3, of the three primer concentrations tested, the amplification efficiency with 6 μM primer was the best. However DNA chains which had a smaller average size than the DNA chains synthesized at the other primer concentrations (3.0 or 4.0 μM) or in the 1st PCR (PEG 12%, primer 1.0 μM) were amplified at this concentration. Consequently, it was adjudged that in the high stringency PCR a primer concentration set to some 4.0-5.0 μM in the reaction solution in the final stage is desirable.
TABLE 1______________________________________ReactionSolution Composition______________________________________Pickup 10 mM Tris-Cl(pH 8.3), 0.1% SDS,Solution 0.5 mg/ml-1 Protease K1st PCR 10 mM Tris-Cl(pH 8.3), 75 mM KCl, 2.25 mM MgCl2,Solution 0.0015%(w/v)gelatin, 0.75%(v/v)Nonidet P-40, 0.75%(v/v)Tween 20, 15% Polyethylene Glycol #6000, 1.5 μM BVE22cc Primer, 300 μM each dNTPs, 0.075 unit Taq DNA Polymerase2nd PCR 10 mM Tris-Cl(pH 8.3), 50 mM KCl, 1.5 mM MgCl2,Solution 0.001% Gelatin, 200 μM each dNTPs, 0.15 unit Taq DNA Polymerase3rd PCR 10 mM Tris-Cl(pH 8.3), 50 mM KCl, 1.5 mM MgCl2,Solution 0.001% Gelatin, 4.7 μM BVE22cc Primer, 200 μM each dNTPs, 1.25 unit Taq DNT Polymerase______________________________________
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 1(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 bases(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE:(iii) HYPOTHETICAL:(iv) ANTI-SENSE:(v) FRAGMENT TYPE:(vi) ORIGINAL SOURCE:(A) ORGANISM:(B) STRAIN:(C) INDIVIDUAL ISOLATE:(D) DEVELOPMENTAL STAGE:(E) HAPLOTYPE:(F) TISSUE TYPE:(G) CELL TYPE:(H) CELL LINE:(I) ORGANELLE:(vii) IMMEDIATE SOURCE:(A) LIBRARY:(B) CLONE:(viii) POSITION IN GENOME:(A) CHROMOSOME/SEGMENT:(B) MAP POSITION:(C) UNITS:(ix) FEATURE:(A) NAME/KEY:(B) LOCATION:(C) IDENTIFICATION METHOD:(D) OTHER INFORMATION:(x) PUBLICATION INFORMATION:(A) AUTHORS:(B) TITLE:(C) JOURNAL:(D) VOLUME:(E) ISSUE:(F) PAGES:(G) DATE:(H) DOCUMENT NUMBER:(I) FILING DATE:(J) PUBLICATION DATE:(K) RELEVANT RESIDUES IN SEQ ID NO:(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:TAGATCTGATATCTGAATTCCC22__________________________________________________________________________
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Hotel Villa De Pasquale, Aeolian Islands
- Free buffet breakfast, free WiFi in lobby, and free parking
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- 16 guestrooms
- Outdoor pool
- Rooftop terrace
- Free ferry terminal shuttle
- Bar/lounge
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- 24-hour front desk
- Air conditioning
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- ATM/banking services
What's around
- In Lipari Historic Centre
- Lipari Castle (0.6 mi / 1 km)
- Museo Archeologico Regionale Eoliano (0.7 mi / 1.1 km)
- Contrada Diana Archaeological Park (0.6 mi / 0.9 km)
- Cathedral of St. Bartholomew (0.7 mi / 1.1 km)
- Marina Lunga (0.8 mi / 1.3 km)
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- Standard Double or Twin Room
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- Year Built 2004
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The Very Best of the Beach Boys
No Trouble For Kelly's 'Double'
June 06, 2007 12:00 AM EDT R. Kelly easily scores his sixth No. 1 album on The Billboard 200 as "Double Up" debuts with 386,000 units ... Jason Aldean nabs a top 10 debut on the big chart and a No. 1 entry on Top Country Albums with his second album, "Relentl ...