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Gary Sinise
Detective Mack 'Mac' Taylor
Melina Kanakaredes
Detective Stella Bonasera
Carmine Giovinazzo
Danny Messer
Anna Belknap
Lindsay Monroe
Robert Joy
Dr. Sid Hammerback
A.J. Buckley
Adam Ross
Danica Patrick
Liza Gray
Guest Star
Antonio Sabàto Jr.
Davi Santos
Guest Star
Marisa Ramirez
Tania Santos
Guest Star
Goof: Flack mentions the town of 'Charlottesville, North Carolina', when he actually should be referencing the city of 'Charlotte, North Carolina', home of the Charlotte Motor Speedway of NASCAR fame. Charlottesville is a city in the Virginia mountains and home to the beautiful Univ. of Virginia flagship campus.
A clipping that features the events that occur in episode 3x24 Snow Day can be seen pinned to Mac's office cork board.
Goof: Davi Santos can be seen breathing on the autopsy table right after he is turned back to laying on his back position.
Mac used to race in he soapbox derby as a child and came third in the Illinois state finals one year. He used to go to the speedway with his father and got Mario Andretti's autograph.
Dwight Bernard: Hey, cool it out there with Gray. This is supposed to be fun, not a competition.
Davi Santos: It's all the same to me.
Connor Wells: Easy for you to say, you don't own the car.
Davi Santos: But I own the spotlight.
Mac: Formula fbz cars are not only built for speed, but safety. They don't explode like this unless somebody makes them.
Mac: Santos received death threats, decided to race anyway?
Flack: Guy's a showman. Didn't want to upset his fans. It's the hottest ticket in the city.
Mac: Hotter than anyone expected.
Dwight Bernard: Davi and Liza Gray, they're both competitors. You put them in anything with wheels, they're gonna drive hard. That's what makes them champions.
Stella: Can you think of anyone who might have been responsible for this?
Dwight Bernard: Davi was the definition of hell on wheels. He'd drive around you, over you or through you to get a win, so he left a lot of enemies in his wake.
Mac: We need a closer look at this car.
Lindsay: That's not going to be easy. Team Cietro is claiming proprietary rights on the car. These racing teams, they pay millions to make sure their competitors never find out what's under the hood.
Mac: Well, that's unfortunate because this car is our crime scene.
Lindsay: Somebody did a bad thing to a good fin cooler.
Adam: You a racing fan?
Lindsay: Anything that rips it up at 200 miles an hour, you gotta respect.
Danny: That's my girl. You grow up in Montana, you love race cars.
Lindsay: Maybe stock car. But open wheel, that's a whole different thing.
Lindsay: You race?
Adam: Playstation.
(Lindsay laughs)
Adam: But... those games are very realistic.
Lindsay: There was no bomb added to this car. The car itself was a bomb.
Mac: I know what the stakes are. New York's been trying to land a racing event for 50 years. There's money and jobs on the line.
Stella: And in this economy, it would be a shot in the arm for the city.
Hawkes: I'm running a textual analysis through the fbi database, from word usage to punctuation to misspellings.
Stella: Well, all comes down to how you cross your t's and dot your i's.
Rick Peters: I can barely change my own tires, much less blow up a car.
Flack: Don't play absent-minded professor with me, okay? I've read your rap sheet.
Rick Peters: I write letters. I don't act on them.
Rick Peters: You ever see a family of four mowed down by a drunk driver? I mean, can you even comprehend that level of tragedy?
Flack: I know about your brother and his family and I know that the driver walked away with a clean bill of health and an open bottle on the passenger seat.
Rick Peters: But what you don't know is that bastard was on his way home from the track, having just attended a race that was sponsored by cietro spirits. What kind of message is that, huh? Fast cars and... and... and hard drinks?
Rick Peters: I'll be honest with you. The idea of blowing up their prized car sky high is lovely, but I don't want to add to their death count.
Flack: What I don't understand is, if you weren't going to do anything to Santos, why send threats?
Rick Peters: Look, I am one individual, okay? If the most that I can do is keep their mascot off the road, that's a victory in my book.
Reece Turnball: I grew up idolizing Davi Santos. He's the reason I drive. I take my role very seriously.
Flack: Well, that's good, 'cause your role just got a lot bigger. With Santos out of the way you're the new face of team Cietro.
Reece Turnball: I've always wanted to race but not like this. Davi's the last of a dying breed. Most of us are scared as hell when we're behind the wheel. Not him. That's what makes him the best there is.
Flack: Maybe it's me, but I don't get it. You got 80 laps jammed into that tiny car, ten-second pit stops. Where do you go to the bathroom?
Mac: You don't want to know.
Flack: Gotta be nuts. My father calls these things coffins on wheels.
Mac: These drivers push their limits, always on the edge of danger, always chasing a faster time.
Flack: Not me.
Flack: Racing coalition's threatening lawsuits against everybody: NYPD, City Hall.
Mac: It's more about making a profit than making things safe.
Flack: The show must go on.
Stella: Some race teams add compound enhancers to their oil to improve lap times.
Hawkes: And knowing the exact mixture of the additives is where things get interesting. Each team has its own blend. Just like a fingerprint.
Mac: I saw your race in Monaco. Hell of a wreck. Five surgeries. Looks like you've recovered nicely.
Liza Gray: I've worked hard for everything I have and this is no different.
Liza Gray: I send my best to him and his wife.
Flack: That's certainly a different attitude than what you were saying on the rehab trail.
Liza Gray: Don't believe everything you hear. It's just the business of motor sports.
Mac: Yeah, it used to be about the car and the driver and now it's about the brand. Is that right?
Liza Gray: A strong brand is just one more way to stay ahead of the competition.
Mac: I can think of a few other ways.
Liza Gray: Santos has his fair share of enemies both on and off the track.
Flack: Sure, but you're the only one who publicly vowed to go after him.
Liza Gray: It's just racing. It's like Yankees versus Red Sox, Ferrari versus McLaren, Gray versus Santos. It sells tickets.
Mac: Oil trace linked to your team, and a flame-retardant fiber that matches your racing uniform.
Liza Gray: You got all that off a fiber from my uniform?
Mac: It's the business of forensics.
Liza Gray: There's a set of rules and regulations that every team agrees to follow.
Flack: Including the ones who break and enter?
Liza Gray: I just wanted to see his car.
Liza Gray: Davi Santos is a racing icon but ever since the accident, he hasn't won a championship. His times have been getting slower and maybe he lost his edge. But a driver like that, desperation sets in. I heard he started tweaking his engine. So I took some photos. I just wanted to see what I was up against.
Mac: Used to go to the speedway with my father. I got Mario Andretti's autograph one year.
Stella: It's every boy's dream, isn't it? To get behind the wheel of a race car.
Mac: Well, it was my dream, yeah.
Stella: Really?
Mac: Mm-hmm. I raced in the soapbox derby. We came in third in the Illinois state finals one year.
Stella: Little Mac Taylor, the soapbox derby champ.
Mac: It was never about the vehicle. It was a love for speed, pushing a car as far as it could go. That's what it was about. Back then, we all wanted to be Steve McQueen.
Stella: To be young and naive again.
Mac: Yeah.
Stella: But looking at what happened to Santos, it doesn't seem as innocent anymore.
Stella: Mrs. Santos, I'm truly sorry for your loss.
Tania Santos: Yeah. The idea of loss implies that Davi belonged to me in the first place. In a way, his murder is poetic justice.
Stella: Why would you say that?
Tania Santos: Dying was the only way he would have retired.
Davi Santos: Have you heard what they're saying about me? That I lost my edge? How can I retire with that?
Tania Santos: Who cares about them? You made a promise to me.
Davi Santos: Honey... I always come home safely. Haven't I?
Tania Santos: After that crash in Monaco, he wasn't the same.
Stella: In what way?
Tania Santos: His behavior became erratic. I asked him if he was on anything, but he didn't tell me. My husband wasn't great at keeping promises, but it was unusual for him to keep secrets.
Tania Santos: Always knew that Davi's last day on the track would be his last day on earth. But I never thought he'd get taken away from me like this.
Hawkes: Being on painkillers couldn't have made driving a car any easier for Santos.
Mac: I'm guessing that was the reason for his diminished performances. Santos raced with an edge his entire career. Looks like he lost everything trying to keep it.
Danny: Instant turbo boost at your fingertips. Press the button and...
Adam: Boom!
(Danny, Adam, Mac and Lindsay look at each other)
Adam: Sorry. That was... that was your moment. I thought you were gonna... boom... nothing.
Dwight Bernard: Davi's erratic behaviour and his refusal to stop driving not only endangered himself but everyone around him. Something had to be done.
Mac: And you decided that something was sabotaging his engine.
Dwight Bernard: Guys like Davi understand and respect one thing, and that's losing their edge. I wasn't trying to kill him, Just throw him off his game.
Dwight Bernard: I'm not trying to make excuses; I deserve whatever punishment that I get. But I've been around cars my whole life and I'm here to tell you there is no way that what I did caused that car to explode.
Tania Santos: We both loved Davi so much, and we were angry with him for so long, for being selfish with his life. It just seems like a cruel joke that our attempt to save him led to his death.
Connor Wells: I recognized an opportunity and as a businessman, I acted on it.
Mac: Opportunity? That's what you call murder?
Connor Wells: I thought I was looking out for the best interests of my company. Life without racing is worse than death for guys like us.
Stella: You are so loving this. A little wish fulfillment?
Mac: Yup. I just thought maybe you'd like to see what it's like behind the wheel.
Stella: Oh, me, huh? Not you?
Mac: You just better bring your A-game, Bonasera.
Stella: I always do, Taylor.
Music Featured:
Trouble Comes Running by Spoon.
Despite featured in the opening credits, Robert Joy does not appear in this episode.
Danica Patrick, who plays Liza Gray, is actually a professional race car driver who makes her acting debut in this episode. Liza is the only woman who has won an IndyCar race.
Antonio Sabato Jr, who plays Davi Santos, has a lot of experience behind the wheel as well.
Rick Peters, who plays Josh Weaver, also appears in the CSI: Miami episode Killer Date and in the C.S.I. one Scuba Doobie Doo.
Kevin Kilner, who plays Dwight Bernard, also appears in the CSI: Miami episode Just One Kiss.
Marisa Ramirez, who plays Tania Santos, also appears in the CSI: Miami episode Hurricane Anthony.
Erik Eidem, who plays Reece Turnball, also appears in the CSI: Miami episode Backstabbers.
Original International Air Dates:
Canada: February 10, 2010 on CTV.
United Kingdom: April 17, 2010 on Five.
Czech Republic: June 5, 2010 on AXN.
Spain: June 22, 2010 on AXN.
Turkey: July 11, 2010 on CNBC-e.
Sweden: August 16, 2010 on Kanal 5.
Norway: September 15, 2010 on TVNorge.
Slovakia: December 13, 2010 on JOJ.
Germany: December 20, 2010 on Vox.
Finland: June 1, 2011 on MTV3.
Mac: I got Mario Andretti's autograph one year.
Mario Gabriele Andretti (1940-) is one of the most sucessful American racing drivers of all times. He won races in Formula One, IndyCar, World Sportscar Championship, NASCAR and also in midget cars, sprint cars, and drag racing.
Mac: Back then we all wanted to be Steve McQueen.
Terrence Steven McQueen (March 24th, 1930 – November 7th, 1980) was an American actor and racer. Before becoming well known on the big screen, he supported himself by competing in both motorcycle races. He loved to do most of his driving stunts and even got to design and patent several pieces for race cars.
Flack: The show must go on.
The Show Must Go On is a song by Queen, featured on their 1991 album Innuendo.
Lindsay: We are putting Humpty Dumpty back together again but not everything fits.
Humpty Dumpty is a character from a well-known nursery rhyme that traditionally goes as follows:
Humpty Dumpty sat on a wall,
Humpty Dumpty had a great fall.
All the king's horses and all the king's men
couldn't put Humpty together again.
Danny: Alright Speed Racer, calm down.
Speed Racer is the English adaptated name for the Japanese manga and anime character Mach Go Go Go. He is a young boy who aspires to become a professional racer despite his father's opposition.
Lindsay: You race?
Adam: Playstation.
The PlayStation or PS is a 32-bit fifth-generation video game console which was first released by Sony Computer Entertainment in Japan on December 3rd, 1994. It was the first console and handheld game device in the PlayStation series.
S 9 : Ep 17
Aired 2/22/13
S 9 : Ep 16
Aired 2/15/13
S 9 : Ep 15
Aired 2/8/13
S 9 : Ep 14
Aired 2/1/13
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Christians living with epilepsy
This a group where you can share your faith in Jesus Christ openly. We come to pray, support, share, care for each other as we are called to as the body of Christ. PLEASE: No other discussions will be communicated here....There are other groups where u can join 4 that, but please not here. Iron sharpens iron here, listening welcome, love received
Members: 15
Latest Activity: Jun 16, 2014
This group does not have any discussions yet.
I just redicted my life to Jesus. it feels so right this time.
I think it is harder for my wife to live with my epilepsy. But God has been good to us in many ways. All our bills are taken care of, etc. But it finally cost my career as an RN and that is hard. Hoping I can pray for all here. I find that others have a hard time understanding the illness itself, but worse of all is the other problems it causes, brain dysfunction (at least with me) and other problems.
Hello Group I'm new here. I been having epilepsy from age 11
I am happy to find a group that is letting others know that Jesus is in Your life.
I am so thankful that God is working beside me. I recently came out of the hospital. it was last sunday when I got to go home. I was upset at first that I wasn t able to go to church but I found a service on channel 5. Thank you lord for always being there. and letting my dad see the holy sprit through me. as Jesus continues to work though me every day.
Bless you all
Cindy we are so glad that God almighty saw you through your seizure on yesterday. We know how going to the ER can be. He is always there when we need Him!! We are here for u...pray your weekend will be better.
I am so thankful to the lord for helping me through a on and off seizure yesterday. and not having to go the ER.
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The Crystal Coast Tourism Authority says gas prices are actually drawing drivers to the beach, and thousands are expected this holiday weekend.
Gas prices peaked earlier this month at almost $4.00 a gallon. The average gas price now in North Carolina is $3.73 a gallon. That's $1.00 more than last year, but 12 cents less than the peak in May. AAA estimates 865,000 North Carolina drivers will hit the road this weekend, which is 3% more than last Memorial Day.
However, most trips will be short ones. Many people plan to stay close to home and head to local beaches- including the Crystal Coast. Some hotels were close to being booked Thursday, while restaurants scheduled more staff for the upcoming holiday. Crystal Coast beaches have easy road access for many nearby states, making it a popular destination for a weekend getaway.
"We are basically a drive destination and people come from anywhere from 2 to 6 hours away." said Crystal Coast Tourism Authority Elizabeth Barrow.


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Images: Mississippi, Alabama team for sling load training [Image 3 of 8]
Photo by Staff Sgt. Scott Tynes
Mississippi Army National Guard Sgt. Shawn Bounds, of Forrest, Miss., signals Soldiers of the 117th Field Artillery Battalion of the Alabama National Guard to board the CH-47 Chinook for transport with their 155th howitzer to a new location May 5 at Camp Shelby Joint Forces Training Center. Mississippi's Company B of the 1st Battalion, 111th Aviation Regiment, partnered with the 117th for sling load training. (U.S. Army National Guard photo by Staff Sgt. Scott Tynes, 102nd Mobile Public Affairs Detachment)
This work, Mississippi, Alabama team for sling load training [Image 1 of 8], by SSG Scott Tynes, identified by DVIDS, is free of known copyright restrictions under U.S. copyright law.
Date Taken:05.05.2014
Date Posted:05.06.2014 16:01
Photo ID:1318719
Size:2.64 MB
Location:CAMP SHELBY, MS, US
Hometown:FOREST, MS, US


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Saturday News, October 19
Galles Selected As "Employee Of The Month"
(Le Mars) -- Le Mars Chamber of Commerce has honored the October Employee of the Month.
This time the honor is given to Barb Galles, an employee of Ameriprise Financial Services.
Michael Ruhland nominated Galles for the award, saying she does a terrific job every day.
As the only employee, Barb wears many hats and yet is able to prioritize her daily tasks and
deliver exceptional results. Whether it is tracking business being processed at the home
office or following up on beneficiary changes, Barb continues to monitor various activities
on an ongoing basis. And our clients really appreciate the job she does. Often times Barb
is the individual that the clients will depend upon for follow up service issues. Barb's
occupational responsibilities are challenging but also very rewarding. Barb sees the fruits
of her labors being rewarded by the appreciative clients that she has helped serve for
nearly 12 years. She greets the clients with a friendly smile and she always conveys a
positive attitude. Our congratulations to Barb Galles for being named the October Employee
of the Month.
Nilles Wins Poppy Poster Contest
(Le Mars) -- Aubree Nilles, the 7th grade daughter of Lee ('94) and Angela Nilles, was
recently notified that she had the "Best Poppy Poster in Class III in the Central Division"
at the "2012-2013 National Poppy Poster Contest" held in Phoenix, AZ. She initially created
the poster for the Le Mars American Legion Auxiliary Unit #241, where she won the local,
district, and state contests before going on to compete at the national level.
Aubree created her poster in her Gehlen Catholic 6th grade art class. She depicted a bald
eagle head surrounded by four extra-large poppy flowers. The poster reads "He Always
Stands, Never Falls." Aubree says "Since the beginning of 4th grade, she has tried to excel
in my artwork. Aubree says she used the bald eagle in her poster design because it is a
symbol of the United States that shows strength and courage. The poppy flower represents
freedom for me. She says she is proud that God gave her the ability to be creative."
Sioux City Man Rejects Plea Offer For Shooting Woman
SIOUX CITY, Iowa (AP) - A Sioux City man charged with murder in the 2012 shooting death of
a woman has rejected a plea agreement that would have let him avoid a potential lifetime prison sentence.
35-year-old Fernando Jaimes-Martinez was offered a chance to plead guilty to second-degree murder and be sentenced to 50 years in prison. If he's convicted of first-degree murder, Jaimes-Martinez would face a life sentence.
Through an interpreter Friday, Jaimes-Martinez rejected the offer, telling Judge Jeffrey Poulson, "I'm not going to accept 50 years."
Jaimes-Martinez is accused of firing a gun into a crowd on Sept. 22, 2012, and killing
27-year-old Karla Gonzalez.
Besides first-degree murder, he's charged with intimidation with a dangerous weapon,
willful injury and being a felon in possession of a firearm.
Investigation Into Robberies Leads To Arrests And Gun Siezure
(Sioux City) -- The last of nine suspects was recently sentenced in federal court in a criminal conspiracy involving weapons trafficking, robbery and drugs in Sioux City and South Sioux City. The robberies included Seoul Foods in South Sioux and Sarge's Mini Mart in Sioux City. U.S. Attorney Tim Duax says the suspects were prosecuted under the Hobbs Act.
Listen to
Duax says a total of 33 guns were seized in the case, which started with an investigation that led to the arrest of David Johnson.
Listen to
Around 20 grams of crack cocaine was also seized.
The suspects were sentenced from five to over thirty years in federal prison for their roles in the crimes and ordered to pay restitution to their victims.
Iowa State Patrol Receives Grant For Driving Simulators
(Le Mars) -- The Iowa State Patrol has been given a gift consisting of a computer driving
simulator. State Farm Insurance made it possible through a grant, and according to Trooper
Vince Kurtz, the highway patrol hopes to use the simulator for education purposes.
Listen to
Kurtz says the driving simulators are more than just a computer game.
Listen to
The State Patrol trooper says the driving simulator can be adapted to any particular driving
experience and conditions.
Listen to
Kurtz says the driving simulator will be available for anyone, but it is targeted towards the beginning driver.
State Tourism Office Hosting Photo Contest
DES MOINES, Iowa (AP) - The state tourism office is hosting a photo contest to find the
cover of the next Iowa Travel Guide.
The tourism office says people have until Sunday to upload a photo on Travel Iowa's
Facebook page. The top three photos will be selected and then the public will get to vote on
a winner.
The winning photo will be announced Nov. 12.
A similar competition last year drew 600 photo entries. Jessica Rilling, of Cedar
Rapids, took the winning image, which was featured on the 100,000 printed copies of the
guide and digitally on www.traveliowa.com. She did not receive any financial compensation
for the photo.
A recent study showed that Iowa tourism generated $345 million in state tax receipts in
Civil Rights Commission Reaches Settlement With Apartment Complex
DES MOINES, Iowa (AP) - The Iowa Civil Rights Commission says it has reached a settlement
agreement the owners and builders of a newly constructed Grimes apartment complex to make
numerous changes that will comply with handicapped accessibility requirements.
The commission says it checked Meadowlark Place Apartments, which includes four
apartment buildings with 30 apartments each to ensure it met requirements of the Iowa Civil
Rights Act and the Federal Fair Housing Act.
The inspection found 325 deficiencies including inadequate space around kitchen islands
for wheelchairs, bathroom faucets too close to the walls, doors requiring too much force to
open, and hallways within units that are too narrow.
The agreement means the property Hubbell Apartments; Hubbell Construction Services, and
Hubbell Property Management must make required improvements along with the architect Wells &
Casino Tycoon Steve Wynn Donates $25 Million To University Of Iowa
IOWA CITY, Iowa (AP) - Las Vegas casino tycoon Steve Wynn says he gave $25 million to the
University of Iowa after becoming convinced that its researchers were leading the world in
the search for a blindness cure.
Wynn tells The Associated Press that researchers "are knocking on the door" of a
discovery that was once unthinkable.
He spoke in an interview after the university celebrated the naming of the Stephen A.
Wynn Institute for Vision Research during a ceremony Friday afternoon.
The chairman of Wynn Resorts Ltd. says that when he learned he had a genetic defect
that would slowly compromise his vision decades ago, "there was no hope." He says he now
believes that scientists will one day be able to restore vision by using stem cells to
replace diseased cells.
Maquoketa Middle School Teacher Selected As "Teacher Of The Year"
MAQUOKETA, Iowa (AP) - A Maquoketa (muh-COKE'-ih-tuh) middle school teacher is Iowa's 2014
teacher of the year.
Gov. Terry Branstad announced Friday that Jane Schmidt won the award. Schmidt, an
eighth-grade literacy and language arts teacher at Maquoketa Middle School, has worked in
education for 33 years.
Besides teaching in the classroom, Schmidt is a teacher mentor and began a new role
this fall in which she works on curriculum and gauges the effects of teaching practices
across the school district.
Schmidt says teaching has "always been my passion, and it's always given me purpose."
The finalists were Aaron Maurer, of Bettendorf Middle School; Kari Murray, of Carlisle
High School, Jon Parrott, of Urbandale Middle School; Kristi Wickre, of Smouse Opportunity
School in Des Moines; and Elaine Wolf, of Central Campus in Des Moines.
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How to Make Politics Interesting in a Fictional Setting
Say the word "politics" and people often run screaming away, even–or especially–when that's in the pages of a fiction book or the context of a role-playing game. This allergy to politics may be very understandable, given how it's conducted here in our modern times, but the concept itself is something else again.
It taps into into the power dynamics present in every group and society, and this is the motherlode for rich plotting and character challenges.
This world-building post of mine uncovers some essentials of power dynamics that lurk behind the interactions of politics. It includes specific questions writers should ask themselves that will help get a handle on how to use all this creatively in their fictional setting.


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Tucson man killed after truck collides with utility pole
TUCSON, AZ - A motorist is dead in Tucson after his pickup truck collided with a utility pole.
Tucson police identified the victim as 46-year-old Ken Josephson.
Police say the truck Josephson was driving jumped a curb and struck a wooden utility pole about 6:30 a.m. Friday near the intersection of North Country Club Road and East 3rd Street.
Josephson was alone in the vehicle.
He was taken to a Tucson hospital, where he later died of his injuries.
Police say it's unclear why Josephson veered off the road and officers are continuing to investigate the cause of the fatal crash.


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Compatible with OS X, Windows, Linux and Android devices, Google Music is able to manage the music we want to climb the Google cloud to hear from wherever we want. We have our music library on a hard drive to take it into the cloud, we can upload up to 20,000 songs completely free. Google Music sells ... and also offers exclusive In the Google Music Store find, finally, interviews and extras bands present. Let your friends hear the same thing you once ... how to jailbreak iphone
The main novelty is the long-anticipated opening of the iTunes Store from Google itself. Comes integrated with Android Market and sell albums and songs in MP3 format at 320 kbps quality.
It will offer free and exclusive songs. Among those announced today, giving the concert in Madrid to present his latest Coldplay caramelito, several unpublished direct Pearl Jam concerts and also never heard of the Rolling Stones.
Much had been said integration with Google+ and this comes in the form peculiar. Each user can send music purchased on Google Music Store to your circles on Google+ and there your friends can hear each song in full once. No, this is not Spotify + Facebook, but it's something.
Google has negotiated with three of the four major labels: Universal, EMI and Sony Music (of course Warner) for the content of your store. And also has the support of the indies, to a catalog of over 13 million songs and a thousand labels. Google Music Artists Hub: The Bandcamp of Google


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Adding it up
There are a lot of numbers and formulas involved when you're convicted of a crime. For example, when I pled guilty to 18 U.S.C. 666(a)(1)(B), a corruption charge, I became a 17. The judge assigned that number to the crime I'd committed, then a chart told the judge what the sentencing parameters should be. I got two years in federal prison and had to pay $5,100 in fines.
When the doors clanged shut, I became an inmate with 067 as part of the long string of numbers on my uniform. Those three digits identified me as being from Eastern Pennsylvania. It was certainly a change from being identified as 75, the number I wore proudly on the football field as a Wolverine and a Carolina Panther in the NFL.
I was imprisoned 500 miles away from my two kids. As a single father, that was the worst of it. I could have been incarcerated closer to my family, and there was probably a formula for where they placed me - I just never learned it.
I was released after 19 months, but I have three years of probation ahead of me. That's just shy of the number of years I played for the NFL Raiders - the Los Angeles and Oakland versions both - and almost as long as I played football at U-M.
I was a two-time All-American at Michigan, won four Big Ten championships, and I played in three Rose Bowls. I played for the NFL for seven years total. When I went into politics in Pennsylvania, serving as county commissioner, I was elected twice. Not far into my second term, I was already thinking about running for Congress or state office, even governor.
Things were good. Which begs the question: What's the formula, the equation, the number you can assign to a mistake so big that it changes your whole life? How do you add up the toll it takes?
I am at a loss as to how to tally the shame I feel about letting my family and former U-M coaches and teammates down by committing a corruption felony. I played under the great Bo Schembechler, who placed integrity above everything. To have tarnished what he stood for will haunt me forever.
There is no way to subtract the guilt I feel for having let down my community. Not that subtraction is the answer, necessarily, otherwise how would the lessons I've learned mean anything? I might not be able to assign a number to the things I've realized, the contrition I feel, but I can change. I can move forward and be better. I can help other people learn from the mistakes I've made.
There is no number left except perhaps zero. I am starting over. I am humbled and sorry and I did all this to myself. But zero is not a negative. Zero is where I start from here. Zero is after all, what the scoreboard reads before the game begins.
Reprinted courtesy of LSA Magazine, University of Michigan College of Literature, Science, and the Arts
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Here in our little slice of Ozarks heaven, we are most fortunate to be granted the privilege of greeting every new day with a lyrical symphony of sound that has the ability take our breath away. Yet, there are those rare occasions when the sweet symphony becomes more like a raucous…Continue
Added by Jill H on April 5, 2012 at 7:35am — No Comments
Before we have another power outage and towns run out of water, we need to encourage people to develop a backup water system of our own. We discovered that our town of Seymour has backup generators for an emergency that could pump water for about 3 days until the fuel supply is exhausted. They have no backup plans for a very long emergency. Therefore we need to take care of ourselves without waiting for the government.
We picked up a…Continue
Added by Lincoln B. Justice on October 23, 2010 at 2:10pm — No Comments
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Venezuela's oil diplomacy is past its sell-by date
Crackers in Caracas
The Bolivarian Republic still gives about $9 bln a year in free crude to the likes of Cuba. Even ignoring domestic shortages of staples, that's enough to cover, say, a year of debt service. With economic cracks widening, President Maduro could start by cutting off this largess.
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I have a class which is written for a single thread with no methods being synchronized.
class MyClass implements MyInterface{
//interface implementation methods, not synchronized
But we also needed a synchronized version of the class. So we made a wrapper class that implements the same interface but has a constructor that takes an instance of MyClass. Any call to the methods of the synchronized class are delegated to the instance of MyClass. Here is my synchronized class..
class SynchronizedMyClass implements MyInterface{
//the constructor
public SynchronizedMyClass(MyInterface i/*this is actually an instance of MyClass*/)
//interface implementation methods; all synchronized; all delegated to the MyInterface instance
After all this I ran numerous amounts of test runs with both the classes. The tests involve reading log files and counting URLs in each line. The problem is that the synchronized version of the class is consistently taking less time for the parsing. I am using only one thread for the teste, so there is no chance of deadlocks, race around condition etc etc. Each log file contains more than 5 million lines which means calling the methods more than 5 million times. Can anyone explain why synchronized versiuon of the class migt be taking less time than the normal one?


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It occurred to me that we hadn't posted any of the sweet audio goodness from Novembers release of The Non-Profit Narrative in audio format.
So here's the Overview, and the Intro.
Remember, if you want to find out who gets it in the end.. (Was it Colonel Mustard with the rope in the drawing room?) You'll have to pick it up on Amazon, iTunes, or Audible.
Enjoy! (You can download these tracks for FREE!)


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|Número de publicación
|Tipo de publicación
|Número de solicitud
|Fecha de publicación
|6 Mar 2001
|Fecha de presentación
|28 May 1998
|Fecha de prioridad
|31 Mar 1993
|También publicado como
|Número de publicación
|Robert Teoule, André Roget, Thierry Livache, Christelle Barthet, Gérard Bidan
|Cesionario original
|Cis Bio International
|Exportar cita
|BiBTeX, EndNote, RefMan
|Citas de patentes (3), Citada por (20), Clasificaciones (20), Eventos legales (4)
|Enlaces externos: USPTO, Cesión de USPTO, Espacenet
This application is a Continuation of application Ser. No. 08/525,506, filed on Sep. 22, 1995, now pending, now U.S. Pat. No. 5,837,859, which was filed as International Application No. PCT/FR94/00354, filed on Mar. 30, 1994
The present invention relates to the binding of nucleic acids to an electrically conductive polymer (ECP).
In a great many techniques commonly used in biology, for example the synthesis or hybridization of nucleic acids, oligonucleotides are covalently bound at their end to a solid support. Various supports have been used for this purpose: paper, nylon, glass, silica, polystyrene, polyacrylamide, etc.
At the present time, many teams are researching into the production of supports bearing a large number of oligonucleotides with various sequences, arranged according to a preestablished arrangement, in order simultaneously to perform various reactions (hybridization on a support for example).
Thus, this approach has been proposed, for example, in order to facilitate the sequencing of nucleic acids.
Various oligonucleotides arranged in columns and rows on microsurfaces (oligonucleotide matrices on a support) have been proposed in order to sequence nucleic acids [LYSOV et al., Proc. USSR Acad. Sci., 303, 1508-1511, (1988); KHRAPKO et al., FEBS Lett. 256, 118-122, (1989); KHRAPKO et al., DNA Séquence, vol 1, 375-388 (1991); BAINS & SMITH, J.Theor.Biol. 135, 303-307, (1988); CHURCH & KIEFFER-HIGGINS, Science 240, 185-188 (1988); SOUTHERN, PCT application WO89/10977 (1989)]. The method is based on the hybridization of target DNA or RNA chains on a set of oligonucleotides. In theory, the presence or absence of a sequence in the target nucleic acid may be determined by the hybridization observed on the microsurfaces under defined rigorous conditions.
As regards the in situ synthesis of polynucleotides or polypeptides, by combining the methods of chemical synthesis on a solid phase, photolabile groups and photolithography, FODOR et al. [Science, 251, 767-773 (1991)] have succeeded in synthesizing 1024 peptides on a grid (square, side length 100 μm). These peptides were obtained by simultaneous and parallel syntheses, using photolithography masks and photolabile protecting groups for the peptide synthons. A dCpT dinucleotide was prepared in situ, using thymidine which was 5′-protected by a photolabile protecting group (5′-nitroveratryl thymidine). The light was directed by a photolithography mask and a deposit in a checked pattern with a side length of 100 μm was obtained.
MASKOS & SOUTHERN (Nucleic Acids Res. 1992, 20, 1675-1678) performed, under a microscope, the in situ synthesis of four different oligonucleotides on a glass slide.
Hitherto, the techniques used for the directed deposition of oligonucleotides use either manual deposition (which cannot be used on the industrial scale), or photolithography techniques, which require the use of "masks" and are, moreover, difficult to apply with nucleic acids, which are photolabile.
The aim of the present invention is to obtain novel supports and novel processes for binding oligonucleotides, which do not have the drawbacks of the processes proposed in the prior art.
With this aim, the inventors have had the idea of using electrically conductive polymers as a binding support.
The inventors are now able to bind stably, and via a covalent bond, nucleotides and oligonucleotides to an electrically conductive polymer, and thereby to obtain novel copolymers.
The subject of the present invention is a copolymer which corresponds to the following general formula (I):
in which the unit A represents a monomer of an electrically conductive polymer, the unit B represents a nucleotide, an oligonucleotide or one of the analogues thereof, x, y and z represent integers equal to or greater than 1, or y may be equal to 0, and l represents a covalent bond or a spacer arm.
FIG. 1 illustrates the preparation of Compounds No. 1, 2, 3, and 4.
FIG. 2 illustrates the preparation of Compounds No. 18, 19, and 20.
FIG. 3 illustrates the preparation of Compounds No. 5 and 6.
FIGS. 4A and 4B show a schematic representation of an electropolymerization cell (A) and cyclic voltammetry curves (intensity as a function of the potential) over 12 polymerization cycles (B).
FIG. 5 shows the kinetics of hybridization to supports of differing thickness.
FIGS. 6A and 6B show the amount of oligonucleotides remaining on the electrode (A) and the rate of denaturation as a function of the washing temperature.
FIG. 7 illustrates the preparation of Compounds No. 8, 11, 12, and 13.
FIG. 8 illustrates the preparation of Compounds No. 14 and 15.
FIG. 9 illustrates the chemical synthesis of oligonucleotides.
FIG. 10 illustrates the preparation of Compound No. 16 and 17.
FIGS. 11A and 11B show a matrix of four electrodes (A) and voltammagrarns during polymerization (B).
FIG. 12 illustrates an ultra-microelectrode according to Example 6.
FIG. 13 illustrates the preparation of Compounds No. 21 and 22.
FIG. 14 illustrates the preparation of Compounds 23 and 24.
FIG. 15 illustrates the preparation of Compounds 25, 8 and 26.
By way of non-limiting example of electrically conductive polymers for which A represents a monomer, polyacetylene, polyazine, poly(p-phenylene), poly(p-phenylene vinylene), polypyrene, polypyrrole, polythiophene, polyfuran, polyselenophene, polypyridazine, polycarbazole, polyaniline, etc. may be mentioned.
A is advantageously a pyrrole unit.
Within the context of the account of the present invention, the term nucleotide analogue is understood to mean any modified nucleotide, such as those described for example by UHLMANN [Chemical Review, 90:4, 543-584 (1990)].
When the unit B is a nucleotide, it may be not only one of those which usually form part of the composition of natural oligonucleotides, but also the analogues or derivatives thereof which are used in the laboratory.
It may be, for example:
nucleotide analogues forming part of the composition of synthetic oligonucleotides;
nucleotide derivatives bearing protected functions which are commonly used for the synthesis of nucleic acids; BZ may, in this case, constitute a synthetic intermediate of an oligonucleotide.
BZ may also be an unnatural compound which may hybridize with nucleic acids, such as those described by UHLMANN (abovementioned publication).
The units B forming part of the constitution of BZ may be identical or different, and BZ may constitute a homopolymer or a heteropolymer; in the latter case, the units B may link together in any sequence, which may or may not be predetermined.
According to a preferred embodiment of the present invention, the ratio x/y is between 1/5 and 1/100,000.
According to another preferred embodiment of the present invention, l represents a spacer arm corresponding to one of the following formulae:
in which:
n is an integer from 1 to 10;
m is equal to 0 or is an integer from 1 to 10;
p is equal to 0 or is an integer from 1 to 10;
x is equal to 0 or is an integer from 1 to 8;
y is equal to 0 or is an integer from 1 to 8;
R1, R2 and R3, which may be identical or different, represent:
where R′ represents a hydrogen atom or a C1 to C12 alkyl chain.
The subject of the present invention is the use of an electrically conductive polymer as a support for the binding, via a covalent bond, of at least one nucleotide.
Another subject of the present invention is a process f or the preparation of a copolymer of general formula (I).
According to a first variant, this process comprises at least the following steps:
a first step, during which there is prepared a copolymer of general formula (II):
in which A, x and y are as defined above, and A* represents functionalized A.
a second step, during which there is bound, to the polymer of formula (II), at least one group of general formula (III):
in which B and z are as defined above and l* is an activated arm capable of binding to A*.
In the sense of the present invention, the terms "functionalized" and "activated" are understood to mean the results of any chemical modification whose aim is to provide A and l with chemical functions capable of reacting together to form a covalent bond.
According to another variant, the process for the preparation of the copolymer of general formula (I) comprises at least the following steps:
a step a) during which there is prepared the compound of general formula (IV):
in which A, B, z and l are as defined above,
a step b), during which there is copolymerized the compound (IV) with the monomer A.
Advantageously, at least one step of one or other of the variants of the process in accordance with the invention involves at least one electrochemical reaction. This electrochemical copolymerization is advantageously carried out on an electrode surface; at the end of the reaction, an electrode whose surface consists of a copolymer in accordance with the invention is thereby obtained.
For example, in order to carry out the first variant of the process in accordance with the invention, the step for the preparation of the copolymer of general formula (II) and/or the step for binding of the group of general formula (III) may be carried out by electrochemical reaction; in the second variant, step b) is advantageously carried out by electrochemical copolymerization of the compound (IV) with the monomers A.
The electrochemical copolymerization is, for example, carried out by cyclic voltammetry, by subjecting the mixture [(IV):A] to electrical potential variations which are sufficient to bring about the polymerization by a successive oxidation and reduction; since the polymer formed is conductive, the oxidation-reduction cycle may be repeated several times.
The methods of electrochemical polymerization generally used for the preparation of the ECPs, such as polymerization at set current (chronopotentiometry) or at set potential (chronoamperometry) are also applicable to the preparation of the copolymers in accordance with the invention.
The quality of the deposit may be controlled by the choice of experimental conditions: the oligonucleotide-pyrrole/pyrrole ratio, the bath temperature, the nature of the solvent, the electrochemical method used (cyclic voltammetry, chronoamperometry or chronopotentiometry). The copolymer obtained may accordingly have different qualities of porosity and of accessibility depending on the desired subsequent use, and the amount of bound oligonucleotide may be modified.
Within the context of the implementation of the process in accordance with the invention, the electrochemical reactions are advantageously carried out at the surface of an electrode. By measuring the current delivered during the reaction, the electrode effectively makes it possible to monitor the progress of the polymerization reaction (for example the thickness of the polymer formed) or the progress of subsequent reactions carried out on the copolymer.
According to a preferred embodiment of the process in accordance with the invention in one or other of its variants, it additionally comprises the elongation of the oligonucleotide Bz, in several successive steps, each of these steps consisting of the binding of one or more units B.
Elongation of the oligonucleotide Bz is carried out at the surface of the support by assembling the protected monomers, starting with at least one nucleotide or oligonucleotide bound to the surface of the electrically conductive polymer.
The standard methods for the chemical synthesis of nucleic acids may be used in the implementation of this embodiment.
The supports in accordance with the invention additionally allow the oligonucleotide to be elongated electrochemically, by using variations in the electrode potential in order to carry out the protection, deprotection and condensation reactions of the growing polymer chain.
Another subject of the present invention is an electrode, the surface of which consists of a coating comprising a copolymer of formula (I) in accordance with the invention.
Such an electrode may be obtained, for example, by depositing a layer of a copolymer of formula (I) on the surface of an electrode made of platinum, gold, chromium or titanium coated with gold, or vitreous carbon, etc.
Advantageously, several electrodes possibly bearing copolymers of different nature may be combined. A device is thus obtained which may be used for carrying out reactions for the synthesis and/or reactions for the hybridization of nucleic acids.
A particularly advantageous embodiment of a device in accordance with the invention consists in combining several electrodes, at least two of which bear a different group Bz. This may, for example, be a set of electrodes in which each bears a different nucleotide (or analogue), or a set of electrodes in which each bears an oligonucleotide of different sequence.
Insofar as it is possible to limit the electrochemical reactions resulting in the binding of the oligonucleotide to a very small surface, a device in accordance with the invention may consist of a plurality of ECP microsurfaces borne by microelectrodes distributed on a support (ECP microchip). In this way, oligonucleotides Bz which may, if so desired, all be different, may be bound in a controlled and organized manner to these microelectrodes.
The "ECP microchip" may in particular be used for sequencing nucleic acids and diagnosis.
By way of non-limiting example illustrating the above, a copolymer [polypyrrole bearing oligonucleotides/polypyrrole] in accordance with the present invention may be obtained:
1) by chemical reaction of a nucleoside, a nucleotide, an oligonucleotide or one of the analogues thereof, with a functionalized polypyrrole. For example, it is possible to carry out the condensation of aminoethylpyrrole with an oligonucleotide bearing a free phosphate or an activated carboxyl at one end.
2) by chemical or electrochemical copolymerization of the pyrrole with the product of condensation of a nucleoside, a nucleotide, an oligonucleotide or one of the analogues thereof, with pyrrole. For example, the pyrrole may be electrochemically copolymerized with an oligonucleotide having a spacer arm bearing a pyrrole at its end. The thickness of the copolymer layer obtained on a platinum surface to which it adheres strongly is from 0.1 μm to a few μm, and it may be performed on a surface of 100 μm2 for example. No interfering degradation reaction of the oligonucleotide could be demonstrated.
3) by preparation of an electrically conductive polymer bearing protected chemical functions. These functions are deprotected locally and selectively in order to allow their coupling with a nucleoside, a nucleotide or an oligonucleotide. For example, it is possible to prepare monomethoxytrityl aminoethyl pyrrole/polypyrrole, and to deprotect it locally either in acidic medium or by applying a potential. The freed amine function may then react with a nucleoside, a nucleotide or an oligonucleotide bearing, for example, an activated phosphate or an activated carboxyl.
4) by stereocontrolled simultaneous synthesis of various oligonucleotides.
The synthesis of an oligonucleotide is carried out at a point on the support by assembling protected nucleotides, starting with a nucleoside which is accessible at the surface of the copolymer. The protected nucleotides may be nucleoside phosphoramidites, nucleoside phosphonates or nucleoside phosphotriesters. Locally, the synthesis is carried out in the same way as an oligonucleotide is synthesized on a silica support in a synthesizer. However, the difference is that the synthesis of the entire set of oligonucleotides is performed simultaneously, by electrochemically performing selective deprotection or condensation operations on a very small surface, thereby making it possible to mask the oligonucleotides which should not react. This allows different oligonucleotides to be synthesized in parallel.
Obviously, the processes briefly outlined above in order to illustrate the synthesis of copolymers [pyrrole/oligonucleotides-pyrrole] are also applicable to polynucleotide analogues, for example analogues of the sugar-phosphate chain such as mono- or dithiophosphates, methylphosphonates and phosphotriesters, and nonionic non-phosphorylated analogues such as formacetals, carbamates and sulfoxides.
The copolymers in accordance with the invention have good stability to mechanical stresses, to moisture, to drying, to heat and to bases, and are thus compatible with a large number of reactions, thereby allowing a wide variety of uses.
The inventors have selectively hybridized oligonucleotides to complementary oligonucleotides bound to a polypyrrole support, and have observed that the use of this support imparts the following advantages:
The copolymer in accordance with the invention is porous, thereby imparting to the oligonucleotides bound to the support a good accessibility for the hybridization with nucleic acids of complementary sequence. This accessibility is demonstrated by the observation of hybridization which is proportional to the thickness of the copolymer layer. A complementary oligonucleotide in the hybridization medium undergoes three times as much hybridization on a pyrrole/oligonucleotide-pyrrole copolymer layer which is three times thicker (and thus contains three times more oligonucleotide bound to the support). The hybridization kinetics are close to those observed with conventional hybridization supports. It should be noted that under the same conditions, a non-complementary oligonucleotide sequence does not bind to the support.
The inventors have also verified that the hybridization is reversible and that any hybridized oligonucleotide may be released by heating, or by treatment with dilute sodium hydroxide, without damaging the polypyrrole and the bound oligonucleotide.
As has been mentioned above, a controlled copolymerization may be performed on extremely small electrode surfaces. This makes it possible to produce a perfectly ordered miniaturized grid on a support, each of the points of this grid bearing an oligonucleotide of fully defined nature. The target nucleic chains bearing a sequence complementary to the chain bound to the support hybridize selectively. This results in an extremely high local density of target nucleic acids, thereby making them easier to detect, or even in certain cases eliminating the need for amplification prior to the detection. The detection of hybridization may in particular be made by means of the electrode which has been used to prepare the copolymer, and which may then be used to measure the association or dissociation phenomena which will take place at the surface thereof. The hybridization of a complementary nucleic acid may, for example, be monitored in situ by an electrical measurement on the electrode which supports the electrically conductive polymer, either by direct measurement, or by labeling the target oligonucleotide with an electroactive molecule such as a phenothiazine or a quinone, for example.
It goes without saying that the traditional methods for detecting the target sequences of nucleic acids are also applicable.
The inventors have additionally succeeded in synthesizing oligonucleotides directly on the copolymer in accordance with the invention by electrochemical deprotection in situ.
In general, the assembly of a nucleotide on a growing polynucleotide chain on a support uses a series of reactions which involve protecting groups in order to direct the reaction onto a given function and to prevent it at another. The inventors have exploited this property in order to direct the reaction for the assembly on the surfaces corresponding to the oligonucleotides chosen where it is desired to insert a nucleotide.
In accordance with the invention, the protecting group for the growing oligonucleotide chain may be removed locally by an electrochemical reaction, thereby making it possible to add a nucleotide to the chosen position.
Additional advantages follow from this possibility of carrying out, on the support in accordance with the invention, an oligonucleotide synthesis in situ. Indeed, in this case, it is possible to synthesize in situ and in parallel the set of oligonucleotides which will be arranged on the grid, instead of independently synthesizing oligonucleotides bearing a pyrrole arm and then carrying out successive copolymerizations. This makes it possible to envisage the industrial production of matrices of several thousand microsurfaces.
The present invention will be better understood with the aid of the remaining description which follows, and which refers to examples for the preparation and use of copolymers in accordance with the invention.
1st METHOD
The overall reaction scheme for this synthesis is illustrated in FIG. 1.
Preparation of Compound No. 1 (FIG. 1)
This compound may be obtained by reacting a diamine with dimethoxytrityl thiothymidine or with dimethoxytrityl thiodeoxyuridine according to the methods described by ROGET et al., [Nucleic Acids Res. 17, 7643-7651 (1989)].
Preparation of Compound No. 2 (FIG. 1)
Compound No. 1 (2 g; 3.1 mmol) is dried by coevaporation with anhydrous acetonitrile and redissolved in 20 ml of dichloromethane. 2 eq of dissuccinimidyl sebacoate (2.45 g; 6.2 mmol) are added. The reaction is left for 3 hours at room temperature. The product obtained is separated on a column of silica (gradient from 0 to 10% of methanol in chloroform) or precipitated in hexane (yield=60%). It may also be used without further purification for the synthesis of Compound No. 3.
Preparation of Compound No. 3 (FIG. 1)
This product is prepared by adding aminoethylpyrrole (1.36 g; 12.4 mmol) to the above reaction mixture or 220 mg of aminoethylpyrrole (2 mmol) to Compound No. 2 obtained after purification. The pH is brought to 8-8.5 by adding a tertiary amine (triethylamine). The reaction is left for 2 hours, and 250 ml of chloroform are added. The organic solution obtained is washed with twice 100 ml of 0.5 M NaHCO3, and 100 ml of distilled water, then dried over sodium sulfate. The product is separated on a column of silica with methanol in chloroform (0 to 10%). After evaporation of the solvent, Compound No. 3 is taken up in 10 ml of ethanol and precipitated in 400 ml of ethyl ether (Yield=60%).
Preparation of Compound No. 4 (FIG. 1)
Compound No. 3 (100 mg; 0.11 mmol) and diisopropylammonium tetrazolate (9 mg; 0.5 eq) are dried by coevaporation in a mixture of anhydrous dichloromethane (2 ml) and anhydrous acetonitrile (3 ml). The residue is taken up in 2.5 ml of dichloromethane stabilized with amylene. Bis(diisopropylaminocyanoethoxy)phosphine (39 μl; 1.2 eq) is added via a septum. After reacting for 2 hours, 20 ml of anhydrous dichloromethane are added. The solution obtained is washed twice with 25 ml of saturated NaHCO3 and then with 25 ml of distilled water. The organic phase is dried over sodium sulfate and evaporated to dryness. The phosphoramidite obtained is taken up in 2 ml of dichloromethane, precipitated in 100 ml of hexane and dried overnight in a desiccator. Compound No. 4 is obtained in a yield of 85%. It is stored under argon at −20° C. in the absence of moisture.
2nd METHOD:
The steps of this process are illustrated in FIG. 2.
Preparation of Compound No. 18 (FIG. 2)
This compound may be prepared by reacting a diamine with dimethoxytrityl thiothymidine or with dimethoxytrityl thiodesoxyuridine according to the methods described by ROGET et al. [Nucleic Acids Res.; 17, 7643-7651 (1989)].
Preparation of Compound No. 19 (FIG. 2) Compound No. 18 (4 g; 6.60 mmol) is dried by coevaporation in anhydrous pyridine and is redissolved in 4 ml of anhydrous pyridine and 40 ml of anhydrous THF (tetrahydrofuran).
1.2 eq of succinic anhydride (800 mg; 8 mmol) are added, and the mixture is left to react for 1 h. The solvents are evaporated off and, in order to remove the pyridine, the mixture is then coevaporated with toluene. 1.5 eq of aminoethylpyrrole (1.1 ml; 9.9 mmol) are added. The mixture is coevaporated with THF. 30 ml of THF and 2 eq of DCC (dicyclohexylcarbodiimide) (2.70 g; 13.2 mmol), predissolved in 30 ml of THF, are added. The reaction is left overnight. The precipitate is removed by filtration and washed with dichloromethane until it is white. The filtrate is evaporated to dryness and taken up in 250 ml of dichloromethane. The organic solution obtained is washed with 2×250 ml of saturated NaHCO3 and then with 250 ml of distilled water. The organic phase is dried over sodium sulfate and evaporated to dryness. The product (Compound No. 19) is purified by chromatography on a column of silica. It is eluted with CH2Cl2/MeOH/TEA: 97.5/2.5/1. Yield=67%.
Preparation of Compound No. 20 (FIG. 2)
Compound No. 19 (600 mg; 0.75 mmol) and diisopropylammonium tetrazolate (63 mg; 0.37 mmol) are dried by coevaporation in dichloromethane/acetonitrile: 2.5 ml/2.5 ml, and then taken up in 5 ml of dichloromethane. Bis(diisopropylaminocyanoethoxyphos-phine (280 μl; 0.9 mmol) is added via a septum. After reacting for 2 hours, 20 ml of dichloromethane are added. The organic solution obtained is washed with 2×25 ml of saturated NaHCO3 and then with 25 ml of saturated NaCl. The organic phase is dried over Na2SO4 and evaporated to dryness. The evaporation residue is taken up in 5 ml of dichloromethane. The product (Compound No. 20) is obtained by precipitation in hexane, in a yield of 78%. After drying overnight in a desiccator under vacuum, it is stored under argon at −20° C. in the absence of moisture.
Preparation of Compound No. 5
Compound No. 4 (68 mg; 0.06 mmol) is redissolved in 300 μl of anhydrous acetonitrile (0.2 M solution). This product is used to prepare an oligonucleotide (Oligo-1-pyr) of sequence Pyr-TGT ACC TGA ATC GTC CGC CAT, in which Pyr represents the nucleotide derivative corresponding to Compound No. 4. This oligonucleotide is prepared on an automatic DNA synthesizer (Applied Biosystems 381A) according to the procedures described by the manufacturer. Compound No. 4 of the invention is subjected to the same synthetic cycle as the normal phosphoramidites (A C G T). Only the concentration (0.2 M instead of 0.1 M) and the reaction time (30 seconds instead of 15 seconds) are increased for Compound No. 4.
After synthesis, the oligonucleotide-pyrrole is detritylated on the support, by the action of 3% TCA (trichloroacetic acid). It is cleaved from the support with 4×500 μl of 28% NH4OH. Heating of this solution for 16 hours at 60° C. allows the protecting groups to be removed. Compound No. 5 (represented in FIG. 3) is obtained by reverse phase chromatography using a gradient of 10 to 50% of acetonitrile in triethylammonium acetate (25 mM, pH 7).
Compound No. 20 may be used in the same way as Compound No. 4.
A—Principle of the Technique
Oxidized pyrrole rings are capable of polymerizing to form an insoluble polymer, polypyrrole. An electropolymerization cell is represented schematically in FIG. 4a: this cell comprises a working electrode (1), a counterelectrode (2) and a reference electrode (3).
If the oxidation is performed electrochemically, the polypyrrole will only be synthesized on the working electrode. This thus allows a very localized synthesis of a polymer. An oligonucleotide bearing a pyrrole ring at the end of one arm may thus be inserted into the polymer simply by copolymerizing the pyrroles. The desired polymer is thus obtained (Compound No. 6, FIG. 3).
Since the polymer formed (polypyrrole) is conductive, these reactions may be continued and several synthetic cycles may be performed (there is only a variation in the resistance of the electrode at each cycle).
The polymerization is carried out on a platinum electrode 60 mm2 in a solution containing 10−2 M pyrrole, 5.10−7 M substituted pyrrole, oligonucleotide bearing a 5′ pyrrole group (Oligo-1-pyr) and 0.1 M LiClO4 (doping agent).
The oligonucleotide bearing the 5′ pyrrole (Compound No. 5, Oligo-1-pyr) was synthesized according to the method described above in Example 1, and purified by reverse phase HPLC. An oligonucleotide of the same sequence (Oligo-1) not bearing pyrrole served as a negative control.
These two products were 5′-labeled with 32P in order for the copolymerization reactions to be monitored more easily.
The reactions for the oxidation of the monomer and for the reduction of the polymer are provided for by a cyclic variation of the potential between −0.4 and +0.9 V/ECS. FIG. 4b represents the cyclic voltammetry curves (intensity as a function of the potential) over 12 polymerization cycles.
Integration of the current with respect to time (amount of electrons consumed) allows an evaluation of the mass of polymer formed on the surface of the electrode and thus the thickness of the film (of the order of 0.2 μm for 5.10−2 C).
Stability of an oligonucleotide under the electropolymerization conditions
Checking by HPLC of the oligonucleotide in solution subjected to the electropolymerization shows no degradation of the latter.
Actual migration of an oligonucleotide subjected to a potential
A nucleic acid is a polyanionic molecule capable of migrating in an electric field; however, owing to the presence of perchlorate ions in the medium, no migration is observed. Moreover, no adsorption of the oligonucleotides onto a preformed polypyrrole can be measured.
Specificity and level of incorporation of oligonucleotides during the copolymerization
1—Polymerization of the pyrrole is carried out in the presence of the unmodified oligonucleotide 1 oligo-1 (TGT ACC TGA ATC GTC CGC CAT).
Oligo-1: 10−9 M in the reaction medium
Oligo-1 on support: 4.10−12 mol, i.e. 0.4% of nonspecific incorporation.
2—The polymerization is carried out in the presence of the modified oligonucleotide Oligo-1-pyr (P TGT ACC TGA ATC GTC CGC CAT)
Oligo-1-pyr: 10−9 M in the reaction medium
Oligo-1-pyr on support: 7.2·10−12 mol, i.e. 0.72% incorporation.
44% of the oligonucleotides-pyrroles detected on the support are efffectively bound by the pyrrole group. However, by adding 0.2 M thymidine 5′phosphate to the electropolymerization solution, the specificity of anchoring then increases to 80%, by decreasing the binding of the non-modified oligonucleotide.
Electrochemical reactivity of the oligonucleotide-pyrrole
The starting solution contains 1 oligonucleotide-pyrrole per 20,000 pyrrole monomers. By calculating the mass of the polymer formed and the amount of oligonucleotide bound, it is estimated that the polymer comprises 1 oligonucleotide-pyrrole per 60,000 pyrrole chain units.
Oligonucleotide-pyrrole is thus incorporated 3 times less than a free pyrrole, thereby constituting an entirely satisfactory level of incorporation.
Density of binding
Under the experimental conditions outlined above, 5.3 pmol/cm2 of oligonucleotides are bound.
The proportion of oligonucleotide integrated into the polymer (1/60,000) may readily be improved by increasing the [oligonucleotide-pyrrole/pyrrole monomer] ratio in the reaction medium. This may be achieved in three different ways:
increasing the amount of oligonucleotide;
decreasing the concentration of free pyrrole;
decreasing the reaction volume.
A polypyrrole support bearing the oligonucleotide Oligo-1 was synthesized according to the method described in Example 2. The electropolymerization was carried out up to charges of 5×10−2 C in order to obtain a support 0.2 μm in thickness, and of 15×10−2 C in order to obtain a support 0.6 μm in thickness. The hybridization reactions are carried out in a 20 mM phosphate buffer pH 7.4, 300 mM NaCl, 0.5% SDS. The washes are performed in the same buffer but which is diluted 4-fold. All these reactions are carried out at room temperature.
The accessibility of the grafted oligonucleotides was verified by their capacity to hybridize with a 32P-labeled complementary oligonucleotide in the surrounding liquid medium.
a) Hybridization
The kinetics for the hybridization of the supports of various thicknesses is comparable, and the total hybridization capacity is proportional to the thickness of the support, as shown in FIG. 5, which represents on the x axis the hybridization time (in minutes) and on the y axis the amount of 32P-labeled complementary oligonucleotide bound to the support (in cpm), for two different thicknesses: ()=support 0.2 μm in thickness; (▴)=support 0.6 μμm in thickness.
b) Denaturation
It is possible to monitor the duplex denaturation continuously, thereby revealing the reversibility of the hybridization phenomenon. FIGS. 6a) and 6 b) respectively illustrate the amount of oligonucleotides remaining on the electrode and the rate of denaturation as a function of the washing temperature (for a temperature variation of 1° C. per minute) on the supports of different thickness: (): 0.2 μm; (▴)=0.6 μm.
Moreover, it has been verified that the oligonucleotide-polypyrrole support is not affected by denaturation/renaturation cycles.
Under the experimental conditions used, the maximum rate of denaturation is achieved at about 60° C., which corresponds to the theoretical melting point of the oligonucleotide (61.5° C.).
I.—BINDING OF THE 1st NUCLEOTIDE
1st METHOD
Preparation of Compound No. 13
The reaction scheme is illustrated by FIG. 7.
The support (Compound No. 8, FIG. 7) is prepared by electropolymerization of a solution of pyrrole and aminoethylpyrrole (10−2 M/10−3 M) in the presence of 0.1 M LiClO4 in acetonitrile. The electropolymerization takes place by sweeping from −0.3 V to +0.85 V relative to 10−2 M Ag/Ag+on a 60 mm2 platinum electrode.
Preparation of Compound No. 11 (FIG. 7)
Compound No. 8 is washed with anhydrous acetonitrile (2×5 ml) and then with triethylamine in acetonitrile (500 μl/5 ml).
10 mg of activated nucleoside (Compound No. 2) are dried by coevaporation in anhydrous acetonitrile, taken up in 500 μl of anhydrous acetonitrile and added to the support in a hermetically stoppered flask. The mixture is placed under gentle mechanical agitation for 24 hours. The support is removed and washed with acetonitrile and then with dichloromethane until the color of the trityl has disappeared in the washing solvents.
The amine functions of the support which have not reacted with the nucleoside have to be blocked. This has been carried out by "capping" with an acetic anhydride/N-methylimidazole mixture in pyridine. The reaction is left for 6 hours. The functionalized support (Compound No. 11) is then washed thoroughly with 3×10 ml of pyridine, 3×10 ml of acetonitrile and 3×10 ml of dichloromethane successively.
2nd METHOD
The reaction scheme is illustrated in FIG. 8.
Preparation of Compound No. 14 (FIG. 8)
The amino polypyrrole (Compound No. 8) is washed with anhydrous acetonitrile (2×5 ml) and then with triethylamine in acetonitrile (500 μl/5 ml). The activated nucleoside (Compound No. 2) (20 mg) is dried by coevaporation in anhydrous acetonitrile, then taken up in 1 ml of anhydrous acetonitrile and added to the support (Compound No. 8) in a hermetically stoppered flask. The mixture is placed under gentle mechanical agitation for 24 hours. The grafted support (Compound No. 14) is washed with acetonitrile and then with dichloromethane until the color of the trityl has disappeared in the washing solvents during their acidification.
Preparation of Compound No. 15 (FIG. 8)
The secondary alcohol functions provided by the nucleoside and the amine functions of the support which have not reacted must be masked. For this, blocking is performed with an acetic anhydride/N-methylimidazole mixture in pyridine (1 ml) for 6 hours. Washing with pyridine (2×5 ml), acetonitrile (2×5 ml) and dichloromethane (2×5 ml) allows Compound No. 15 to be obtained.
Preparation of Compound No. 12 (FIG. 7)
The trimer d(CCT) was prepared on a platinum electrode coated with polypyrrole, by two methods:
synthesis with chemical deprotection, according to the usual cycle of the phosphoramidite synthesis,
synthesis with electrochemical detritylation.
a) Chemical synthesis
The following steps are carried out, as many times as necessary, each step corresponding to the binding of a nucleotide; these steps are represented in FIG. 9:
detritylation of the support with 4×500 μl of 2% trichloroacetic acid in dichloromethane;
rinsing with acetonitrile in order to remove the reagent (5×1 ml);
washing with anhydrous acetonitrile for DNA synthesis (3×1 ml);
addition of 250 μl of 0.1 M phosphoramidite and 250 μl of 0.5 M tetrazole;
coupling (2 min) and removal of the nucleoside solution;
rinsing with acetonitrile (5×1 ml);
acetic anhydride/methylimidazole capping (500 μl, 1 min);
rinsing with acetonitrile (2×1 ml);
oxidation with iodine/lutidine 1 min (500 μl, 1 min);
rinsing with 5×1 ml of acetonitrile;
detritylation, and start of a new cycle, etc.
Measurement of the trityls after each cycle respectively gives: 0.090 OD/2 ml (dT), 0.095 OD/2 ml (dCT) and 0.087 OD/2 ml (dCCT).
b) Synthesis with electrochemical deprotection
The synthesis steps are the same as for the chemical synthesis above, but the detritylation is performed by application of a potential of 1.2 V for 5 min.
The detritylation cannot be quantified since the trityl cation formed is captured by the anode, which subtracts it from the measurement. The coupling cycle was, however, performed.
Preparation of Compound No. 13 (FIG. 7)
Cleavage of the support and removal of the protecting groups are done with 2 ml of aqueous ammonia in a glass tube closed with a screw-thread stopper, and the reaction is carried out for 48 hours at room temperature.
The controls prepared on a silica column are deprotected with 4×250 μl of aqueous ammonia in order to detach them from the support (t=4×˝ h). The ammonia solution is then left for 48 hours at room temperature. The solutions are evaporated and analyzed by reverse phase chromatography on a 5 μm by 25 cm C4 column. A gradient of 0 to 30% of B (25 mM triethylammonium acetate, pH 7 and 50% acetonitrile) in A (25 mM triethylammonium acetate, pH 7) is applied over 30 min.
Preparation of Compound No. 16 (FIG. 10)
Compound No. 16 is synthesized according to the same procedure as Compound No. 12, with similar results for the detritylation. This shows that the nature of the spacer arm has little influence on the chemical synthesis.
Preparation of Compound No. 17 (FIG. 10)
Compound No. 16 is deprotected for 48 hours at room temperature in 28% aqueous ammonia in a hermetically stoppered flask. The dimethoxytrityl group is then cleaved with 3% trichloroacetic acid (3×3 ml) and measured in order to verify that the oligonucleotide is still on the support.
A matrix of four electrodes represented in FIG. 11a is made by insertion of four platinum wires (1) (diameter 0.6 mm) into a glass cylinder (2) (diameter 5 mm×height 10 mm). One of the electrodes is used as counter-electrode (3). This matrix system makes it possible to bind various oligonucleotides to each point of the matrix.
The electrode matrix is placed inside a container (4) in which the reaction is carried out, and in which a reference electrode (5) is also immersed.
The 3 working electrodes are successively electrochemically coated with a copolymer composed of pyrrole and of oligonucleotides capable of detecting, by hybridization, a mutation in codon 61 of the human ras H gene. These 3 oligonucleotides bearing a 5′ pyrrole group are as follows:
normal oligo: 5′ Pyr TCCTCCTGGCCGG 3′ (SEQ ID NO: 2)
A-mutated oligo: 5′ Pyr TCCTCCAGGCCGG 3′ (SEQ ID NO:3)
C-mutated oligo: 5′ Pyr TCCTCCCGGCCGG 3′ (SEQ ID NO:4)
Each oligonucleotide is successively copolymerized on each electrode under the conditions described in Example 2, but in a reaction volume of 300 μl instead of 3 ml.
The voltammagrams obtained are represented in FIG. 11b: ((1) polymerization on the first electrode; (2) polymerization on the second electrode; (3) polymerization on the third electrode). These voltammagrams are very uniform and very reproducible both at reduced charge (2 to 4×10−4 C, top curves) and at high charge (1 to 1.3×10−3 C, bottom curves). Under these conditions, 6×10−14 mol of oligonucleotide are bound on 0.3 mm2 (i.e. 18 pmol/cm2) for a film thickness of 0.1 μm (charge of 10−4 C).
Detection of a bridging mutation of a nucleic acid by hybridization on a 3-point matrix
Three nucleic acid fragments 51 nucleotides in length are used in order to simulate the desired natural ras H mutations.
These three nucleic acids have the sequence:
normal ras H (SEQ ID NO:5):
T-mutated ras H (SEQ ID NO:6):
G-mutated ras H (SEQ ID NO:7):
They are specifically recognized by hybridization with the matrix-bound probes; normal oligo, A-mutated oligo and C-mutated oligo respectively.
The hybridization reaction is performed at 25° C. for 1 hour, in 20 mM phosphate buffer, pH 7.4, 300 mM NaCl, 0.5% SDS containing 0.1 pmol of nucleic acid to be detected, 5′-labeled with 32P. The matrix is then washed in the same buffer at 35° C. The detection is performed by autoradiography of the matrix on a photographic film. Under these conditions, hybridization of the target nucleic acid takes place only on the electrode bearing the oligonucleotide of strictly complementary sequence; no cross-hvbridization can be detected.
The specific detection of a bridging mutation is thus possible by virtue of this matrix.
A system represented in FIG. 12 is composed of 10 gold electrodes arranged on a glass plate; the width of the electrodes may vary from 10 to 100 μm and the length of the active zone (zone immersed in the solution) is of the order of 2 mm. Another system was manufactured by selective deposition of gold onto a silicon oxide insulating substrate, followed by insulation of the connections. Matrices consisting of square electrodes with a side length of 25 to 200 μm are thus obtained.
In both cases, the copolymerization of pyrrole and of oligonucleotide-pyrrole may be carried out on each electrode, according to the process described in Example 5, and the polypyrrole films obtained are of good quality and their thickness can be fully controlled, as described in Example 2.
The synthesis was carried out on an amino polypyrrole support (Compound No. 8), bearing an arm which is cleavable for the needs of subsequent analysis of the oligonucleotide formed.
Thymidine amidite, introduced in position (5) from the 3′ end of the oligonucleotide, is deprotected by application of a potential of +1.1 V for 15 min when the protecting group is thiopixyl, or of −1.3 V for 15 min when the protecting group is the p-nitrobenzoyl group. The other nucleosides are introduced in the form of tritylated amidites, and are deprotected chemically, by detritylation with trichloroacetic acid.
1) Protection with a p-nitrobenzoyl Group:
The steps for the preparation of the protected nucleoside are illustrated by FIG. 13.
Synthesis of p-nitrobenzoylthymidine (Compound No. 21: FIG. 13)
Thymidine (2.42 g; 10 mmol) is dried by coevaporation in pyridine, then taken up in 200 ml of anhydrous pyridine and cooled to 4° C. p-Nitrobenzoyl chloride (2.04 g; 11 mmol) is added. The temperature is allowed to rise and the reaction is left overnight at room temperature. The reaction is stopped with 5 ml of saturated sodium bicarbonate. The reaction mixture is concentrated and then taken up in 500 ml of chloroform. The organic solution obtained is washed with 2×500 ml of 0.5 M NaHCO3 and then with 250 ml of saturated NaCl. The aqueous phases are counter-extracted with 100 ml of CHCl3. The organic phases are evaporated. The pure product is obtained by column chromatography on silica. It is eluted with 5% of methanol in chloroform. Yield=58%.
Synthesis of p-nitrobenzoylthymidine amidite (Compound No 22: FIG. 13)
Compound No. 21 (1.96 g; 5 mmol) and diisopropylammonium tetrazolate (428 mg; 2.5 mmol) are dried by coevaporation using an anhydrous dichloromethane/acetonitrile solvent. These reagents are taken up in 25 ml of anhydrous dichloromethane and bis(diisopropylaminocyanoethoxy)phosphine (1.8 g; 6 mmol) is added. After reacting for 2 h (in the absence of oxygen and moisture) the reaction mixture is diluted with 250 ml of dichloromethane and washed successively with 2×250 ml of 0.5 M sodium bicarbonate and with 250 ml of saturated sodium chloride. The organic phase is evaporated. The residue is taken up in 10 ml of dichloromethane. The product (Compound No. 22) is obtained by precipitation in hexane, then dried under vacuum and stored under argon. Yield=84%.
2) Protection with a Thiopixyl Group
The steps for the preparation of the protected nucleoside are illustrated by FIG. 14.
Synthesis of tbiopixylthymidine (Compound No. 23: FIG. 14)
Thymidine (2.42 g; 10 mmol) is dried by coevaporation in pyridine, taken up in 100 ml of anhydrous pyridine, cooled to 4° C. and reacted with thiopixyl chloride (3.4 g; 11 mmol). After gradual rise of the temperature to room temperature, the reaction is left to continue overnight (8 to 12 h approximately). The reaction is stopped with 10 ml of NaHCO3. The solvent is evaporated off and the residue is taken up in 250 ml of dichloromethane. The organic solution obtained is extracted with 2×250 ml of saturated NaHCO3 and then with 250 ml of distilled water. The aqueous phases are counter-extracted with 100 ml of chloroform. The organic phase is evaporated. The product is purified on a column of silica with 0.5% TEA (triethylamine) in the solvents. It is finally eluted with 5% of methanol in dichloromethane (+0.5% TEA). Yield=52%.
Synthesis of thiopixylthymidine amidite (Compound No. 24, FIG. 14)
Compound No. 24 is prepared from Compound No. 23 (5 mmol) according to the same procedure as for Compound No. 22. Yield=78%.
3) Binding of the First Nucleoside
The steps for this binding are illustrated in FIG. 15.
This oligonucleotide is synthesized on a strip of platinum (3×10 mm) on which a mixture of pyrrole and aminoethylpyrrole (9:1) has been copolymerized.
The first nucleoside (position 1-3′ end) is coupled with aminoethylpyrrole (Compound No. 8) according to the methods described for the functionalization of the silica supports [K. MIYOSHI et al., Nucleic Acids Res.; 8, (22), 5473-5489 (1989)] starting with an activated ester, N-isobutyryl 2′deoxyguanosine (Compound No. 25: FIG. 13a).
Compound No. 26 (10 mg) is dissolved in 500 μl of acetonitrile. The functionalized-polypyrrole-coated platinum electrode and 1 μl of triethylamine are added. The reaction is agitated mechanically for 20 hours at room temperature. The grafted electrode (Compound No. 26) is removed and washed thoroughly with acetonitrile and then with dichloromethane.
The unreacted amine functions are blocked with acetic anhydride (10% in 500 μl of pyridine) for 6 hours. The grafted electrode is washed with pyridine and with methanol, and then dried.
4) Synthesis of the Oligonucleotide dTTCTGAGG
The grafted electrode is placed in an emptied OPC® column (APPLIED BIOSYSTEMS). Filling is completed with teflon chips so as to minimize the residual volume. The nucleosides in positions 2, 3 and 4 are added according to the instructions of the manufacturer (APPLIED BIOSYSTEMS) for the "1 μmol cycle" on a 381A synthesizer. Chemical deprotection of the dimethoxytrityl group between each step is performed with TCA in dichloromethane under the conditions recommended by the manufacturer.
The amidite in position 5 (Compound No. 22 or Compound No. 24) is coupled according to the same method as for the normal amidites, with a coupling time of 1 min. Oxidation of the phosphite triester bond created and capping are performed according to the standard procedure. The electrode is removed from the column and electrochemical deprotection is performed.
If Compound No. 22 is used, the p-nitrobenzoyl group is cleaved by immersing the electrode in the following electrolyte: 0.1 M tetrabutylammonium perchlorate in methanol, and by applying a potential of −1.3 V for 15 min.
If Compound No. 24 is used, the thiopixyl is cleaved by applying a potential of +1.1 V for 15 min, the electrolyte being 0.1 M tetrabutylammonium perchlorate in acetonitrile.
In both cases, after cleavage of the protecting group of the T nucleoside in position 5′, the electrode is replaced in the column with the teflon chips, and the synthesis is continued by successively adding the amidites C (position 6), T (position 7) and T (position 8).
When the synthesis is complete, the oligonucleotide is cleaved from the support: the electrode is treated with 4×500 μl of 28% aqueous ammonia in a stoppered flask for 4×˝ h. The 4 fractions are combined in a stoppered 4 ml Wheaton flask, and are left for 16 h at 55° C. in order to deprotect the oligonucleotide.
After coevaporation in the presence of TEA, an aliquot ({fraction (1/100)}th) of the oligonucleotide obtained is 5′-labeled with 32P in the presence of polynucleotide kinase and then analyzed by polyacrylamide gel electrophoresis. The acrylamide gel electrophoresis analysis shows the presence of the desired product (octamer) and the absence of the oligonucleotide (pentamer), the presence of which would indicate poor electrochemical deprotection of the thymidine amidite.
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