text:
they need to somehow find a way to beat Indy and Miami to go 4-4 into the bye week
then use the bye week to fix the running game. Schedule gets a little easier after that. If they can somehow get the offense going the season isn't entirely over.
If the Jets lose this game, odds are that this turd of a roster will start circling the bowl.
As such, to heal what ails this team, we are most likely as fans seeing major steps backwards prior to going forwards. In fact, one could argue that a patchwork entrance into the playoffs could be COUNTERPRODUCTIVE.
With that said, here's what I hope to see:
A) A quick hook on Sanchez if he has a bad first half. This hook hopefully ushers in the Tebow era (error) and flushes this out of the system so the GM, Owner, and Head Coach (with two of the three replaced over the next two years) can get serious about getting a decent QB no worse than Alex Smith.
B) Keep Coples and Davis on the field. They don't play enough and I don't know who in the right mind doesn't think the defense doesn't need more athleticism. How the hell does Bryan Thomas go from cut to starting in the course of one week? Isn't this alarming? It's not due to injury either.
C) Finally get out from under Pace and Scott's albatross contracts. Both are absolutely terrible.
More eventually:
Get a head coach who doesn't break out in a rash when somebody talks about Offense to him. A true head coach. We should all now know why John Harbaugh got the job over Rex in Baltimore: Rex is a defensive coordinator. Head Coaches have input on both sides of the ball, while not running either side through a proxy. You think Tom Coughlin calls plays on either side of the ball? I think he offers input on what he wants to do, but I sincerely doubt he makes the calls.
My suggestions here are John Gruden or his brother.
Get a GM with a plan that he will stick to.
To me the answer is simple: The Giants are constructed extremely well and tend to find these D-Linemen gems that seemingly appear out of thin air after a soliciting a "WHO?" on draft day. Jerry Reese's AGM is Kevin Abrams. Kevin Abrams worked for Accorsi prior to that, who worked for George Young.
This guy is a true football executive and this could truly signal, if given free reign, a new era here.
He is not a glorified accountant with no talent evaluation skills at all.
In 2-3 years, barring a rookie QB (probably pushing it to 4) we may just be competitive again by 2015.
But it's undeniable that this team is boring as all hell to watch and offers effectively nothing to be enthusiastic about with the confused and demoralized QB, whiny petulant owner about his Precious Tebow, and insistence on playing man-to-man on defense despite not ever getting pressure on the QB.
they need to somehow find a way to beat Indy and Miami to go 4-4 into the bye week
then use the bye week to fix the running game. Schedule gets a little easier after that. If they can somehow get the offense going the season isn't entirely over.
Ground and Pound works if your RB is the best weapon you have, like Ray Rice or Jamal Charles.
Keller's probably the best weapon on offense, but he can't block for crap.
Andy Luck is gong to carve our boys up. The season is already over. You think this roster is competeting for anything? Come on.
Unless we lose in which case the happy joy luck club will post the same thing next week.
I like Cro. But he did nothing monday night that every other corner this year has not done. In his last 3 games, andre johnson has had 3, 2 and 3 catch games. Hes obviously hurt or got old real fast.If Cro has a game like Monday night, Wayne will be a non-issue.
Wayne will have 5 catches, 80 yards and 1 td minimum on Sunday.
I think practicing next to the technically and mechanically flawless Revis for the past 3 years has rubbed off on Cro, who Rex Ryan has made probably the only accurate talent evaluation of during his entire tenure, Cromartie's physical attributes are nearly limitless. He has displayed a lot of amazing anticipation and technique this year through nearly every game, with the breakdowns coming 8-10 seconds in because the pass rush and d-line have been awful. Man defense inevitably breaks down after a while and Rex's insistence on playing it despite not having a pass rush at all goes to show you that he's been damned good.
The only bright spot on this skid mark isn't yesterday's carrots. It's the Jets defense being 5th against the pass, and you sure as hell can't point at a defense with 5 sacks, 2 of which are from Garret Freaking McIntyre as the reason why.
Landry, Bell, and Cromartie have been very good against the pass so far.
St. Louis due to youth and year 1 of a major rebuild with Fisher.
Seattle due to Pete Carroll being a massive loser/schmuck/ra-ra/no worries brah.
I mean signing Flynn to a huge contract then starting Wilson is laughable.
There may be nobody in professional sports who I view as a bigger joke.
And I think most Pats fans would have to be in agreement. He talks like he's some legend, but is just a massive douche.
I actually could tie this back to why Mark Sanchez hasn't panned out. He probably would blossom under Carroll simply because they are both "No worries brah" guys. Which is why Sanchez will never come out after a bad game and say "I stink." Stupid laid back West Coasters.........15 MINUTES NO WORRIES BRAH.


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Making Grand Events
It's always the little things that count. Especially when it comes to planning a good event, little details can turn out to be the ones that matter most! You gotta give props to good props. For any type of event, whether it be conference, daymeeting, banquet, team building or wedding, subtle touches to the decor add your personalized flair, and [...]Read more ›

subset: commoncrawl


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"Arise and take our stand for freedom as in the olden time." Winston Churchill
"Proclaim Liberty throughout All the land unto All the Inhabitants Thereof." Inscription on the Liberty Bell
Monthly Archives: September 2011
Hitler Learns that AttackWatch.com Is a Joke
This video has been around for a while, but I just ran across it this morning. It is part of the very funny Hitler series of which we have posted one or two in the past. In this one, Hitler gets the bad news that that the administration's AttackWatch.com site has become a standing joke on the internet: »
Springtime for Abbas
Palestinian Authority President Mahmoud Abbas turned up last week at the United Nations. He was there to petition for statehood on behalf of the PA or the PLO. There's something almost funny about Abbas's gambit. Fred Siegel reminds us that Abbas is currently in the 81st month of his 48-month term and that he is the leader of the West Bank component of the Palestinian Authority, supposedly including the Gaza »
Wherefore heartless?
A long-time reader writes about Rick Perry's position on college tuition for illegal immigrants and his claim that those who disagree with him are heartless: Rick Perry took some serious hits in Thursday's presidential debate for his defense of the view that children brought to Texas illegally by their parents should receive the same discount on their college tuition at Texas' public universities as children who reside in Texas legally. »
Another Brain Freeze at the Times
[Damn. John beat me to this story. As soon as I banged out a draft and went to post I saw that John had already worked it over. Another failure of editorial coordination here at Power Line. Should I pile on? Yeah, why not. There's one little item in this story I can add to.] Are New York Times editorial writers complete innumerate nincompoops? Okay, silly question. The answer is »
Is Solyndra A Fluke?
That's what the New York Times editorial board thinks. In an editorial yesterday, the paper denounced what it called a "panic" over the collapse of Solyndra. In the view of the Times, all is really well on the "green energy" front: Solyndra made a bad bet, investing heavily in a new type of solar array just as the price of silicon, the main ingredient in competitors' solar cells, was dropping. »
More Distraction From the Left
If the 2012 election is run on the issues, the GOP will win in a landslide. The Left's strategy for avoiding this outcome is to create as many distractions as possible. We saw it first with the Tea Party movement. The Tea Party poses a serious threat to liberalism. It is the most authentic grass-roots political movement of the last half-century, and its emphasis on limited government, fiscal responsibility and »
Why We're Reading Hayek Again
Matt Ridley, author of a splendid book, The Rational Optimist, that has won the Manhattan Institute's Hayek Prize this year has a terrific article in the Wall Street Journal over the weekend ("From Phoenecia to Hayek to the 'Cloud'") that mentions the very passage from Hayek that I've mentioned several times here on Power Line. Ridley reiterates the central Hayekian idea that I've been pounding into the heads of my »
Green Fascism at Work
Sometimes I like to point out that even if catastrophic global warming could be conclusively proven, it would not change one crucial political fact: Al Gore and the environmental left are the last people you would trust to solve the problem. This is a variation of my axiom that the environment is much too important to be left to environmentalists—they'll just screw it up further and crush our liberties. Case »
Fact Checking the "Fact Checker," Part 3
On Tuesday, I wrote a post called Who Checks the Fact-Checkers? In that post I criticized an article by Glenn Kessler of the Washington Post, who writes a regular feature called The Fact Checker. In The Fact Checker, Kessler purports to evaluate the truth or falsity of politicians' statements. In the article I criticized, which ran on Monday, Kessler accused Rick Perry of a "newbie mistake" in Perry's answer to »
A Palestinian State? Why Not?
This pretty well sums it up: »
Mrs. Warren's profession, cont'd
Given that citizens of lesser means always outnumber the rich, the classic political philosophers held that government based on majority rule was untenable. They were of the view that it would lead to organized theft from the wealthy by the democratic masses. Thus Aristotle observed in The Politics, for example: "If the majority distributes among itself the things of a minority, it is evident that it will destroy the city." »
President Solyndra
My cover story in the Weekly Standard is out this morning: "President Solyndra and His Mean Green Wealth-Wasting Machine." Here's the "nut graf," as they say in the feature magazine trade: Even if the administration eventually escapes any finding of legal wrongdoing, Solyndra threatens to haunt the green energy campaign in much the same way that the collapse of Lincoln Savings became the emblem of the savings and loan industry's »
The Empty Promise of Green Jobs
That is the title of this superb piece by the House Budget Committee. In a relatively short space and in clear prose, "The Empty Promise of Green Jobs" recounts the sorry history of the Obama administration's "green jobs" initiatives; reviews the history of failure that such efforts have universally encountered both in the U.S. and abroad; explains the reasons why government "investment" in political favored industries is a bad idea; »
Mr. Netanyahu goes to New York
I write to draw attention to Israel Prime Minister Benjamin Netanyahu's speech at the United Nations today. As Daniel Patrick Moynihan demonstrated once upon a time, there is something especially thrilling about the truth being told in this particular forum (a "theater of the absurd," in Netanyahu's reckoning). It happens rarely, but it happened today. Attention should be paid: Ladies and gentlemen, in Israel our hope for peace never wanes. »
Another Smoldering Stogie of Misinformation from the Lung Association
If you watch Fox News—and really, who doesn't watch Fox News?—you've probably seen the ads the American Lung Association is running displaying a red baby carriage with a coughing infant making its way to the U.S. capitol, urging us to urge Congress not to "weaken" the Clean Air Act. "More air pollution means more childhood asthma," the narrator says. (There isn't a direct link to the ad, but you can »
Solyndra Pleads the Fifth
This morning, two top executives of Solyndra, CEO Brian Harrison and CFO W.G. Stover, appeared before a House Energy and Commerce Committee investigative panel. They didn't testify, however. They declined to answer every question put to them on the ground that the answers might tend to incriminate them. The Fifth Amendment, which protects witnesses against self-incrimination, is pretty much an all-or-nothing thing. The witness can identify himself and perhaps answer »
A bridge too far
President Obama made an appearance yesterday at the Brent Spence Bridge in Cincinnati to promote his "jobs" (i.e., tax) bill. The bridge was intended to symbolize Republican opposition to Obama's bill, though the bill hasn't proved to be a big hit with Democrats in the Senate either. The bridge spans the Ohio River separating Ohio, home of House Speaker John Boehner, from Kentucky, home of Senate Minority Leader Mitch McConnell. »


text:
Meridian, Miss. As graduates enter the job market, what jobs are most in demand in Meridian?
Offering 52 workforce programs, one of the most successful ones at Meridian Community College is the Paramedic Emergency Services Science Academy. It started last fall, and officials say most -- if not all -- students in it have received job offers since completing the one year course.
With two hospitals and many medical clinics, Meridian is a hub for health-care. However, officials with Meridian Community College's Workforce Education Program say there's also growing demand for workers in other professions here at home.
"Not only nursing and health education because they get a lot of print about that, but there are good jobs in industrial technology such as: telecommunications, electronics, business and we have a truck driving program here at Meridian Community College."
Dr. McAlister says students who complete the trucking program are poised to earn salaries ranging anywhere from $20-thousand to more than $100-thousand a year.
With 52 programs already, MCC will add to that list this fall with a Culinary Arts course that will help cater to new developments within the local hospitality industry.
"Just look around here in Meridian, out on I-20 where we have the new and evolving hospitality areas. All of the new motels and hotels that are springing up, I mean, we are uniquely situated here in Meridian at the crossroads of I-20 and I-59. So, the demand is great."
Overall, Dr. McAlister says the demand for workforce training programs is also growing drastically, with many four year college graduates now enrolling in training courses at the community college. For example, he says out of 116 applicants, 11 of the people seeking 14 spots in MCC's physical therapy program have bachelor's degrees.
"Last year there were 15 people with bachelor's degrees vying to get in the physical therapy program here. All of these nursing and health education programs and industrial business programs, we place students in jobs!"says McAlister.
Next fall Meridian Community College is adding a Medical Assisting Program to the list of programs that it offers through its Workforce Training Center.


text:
Okay...first post....nothing serious.
I've lurked JUB for a long time. Really long time, mostly in the debate, daddy, and kink forums. So, now that I'm finally 18, I finally get to join and meet you guys. Anyway, some stuff about me.
I'm an eighteen year old college freshman who wants to meet other guys, and whatever you think "meet" means, it's probably right. I want to make friends, connections, possibly something more fun because I'm dealing with a lot of sexual frustration.
I'm pretty open-minded to most things, but I really love discussion and topics like history. I'm also a massive nerd; I love DnD, Magic, Skyrim, the whole nine-yards. Body-wise, I'm a small, lithe guy with pale skin and dark hair. Basically, I'm a short, hairy elf (dwelf?) that's pretty flexible and lean, especially since I joined fencing club about a week ago. So, if I somehow come off as interesting for once, please hit me up. I really want to get to know you guys better.


text:
Patent application title: SINGLE MOLECULE SEQUENCING OF CAPTURED NUCLEIC ACIDS
Stanley Lapidus (Bedford, NH, US)
Stanley Lapidus (Bedford, NH, US)
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2010-06-10
Patent application number: 20100143932
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Patent application title: SINGLE MOLECULE SEQUENCING OF CAPTURED NUCLEIC ACIDS
Stanley Lapidus
Helicos Biosciences Corporation
Origin: BOSTON, MA US
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Publication date: 06/10/2010
Patent application number: 20100143932
The invention provides methods and devices for detecting, enumerating or
identifying target nucleic acid molecules using immobilized capture
probes and single molecule sequencing techniques.
1-25. (canceled)
26. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:introducing a sample comprising at least one target nucleic acid and other non-target molecules to at least one sequence-specific capture probe to produce target/probe duplexes;separating at least one of the duplexes from the non-target molecules;exposing the separated duplexes to at least one labeled nucleotide in the presence of a polymerase capable of catalyzing addition of the nucleotide to the duplex in a template-dependent manner, wherein the probe acts as a primer; anddetecting incorporation of the nucleotide into the probe portion, thereby obtaining sequence information from the nucleic acid.
27. The method according to claim 26, wherein prior to the exposing step, the duplexes are attached to a surface.
28. The method according to claim 27, wherein the duplexes are directly attached to a surface.
29. The method according to claim 27, wherein the duplexes are indirectly attached to a surface.
30. The method according to claim 27, wherein at least some of the attached duplexes are individually optically resolvable.
31. The method according to claim 26, wherein the probes are a plurality of different sequence-specific probes.
32. The method according to claim 26, wherein the exposing and detecting steps are repeated at least once.
33. The method according to claim 32, further comprising: removing unincorporated nucleotide and polymerase in all or some repetitions of the exposing and detecting steps.
34. The method according to claim 26, wherein the labeled nucleotide is a detectably labeled nucleotide.
35. The method according to claim 34, wherein the detectably labeled nucleotide is an optically detectably labeled nucleotide.
36. The method according to claim 35, wherein the optically detectably labeled nucleotide is a fluorescently labeled nucleotide.
37. The method according to claim 26, wherein the target nucleic acid comprises at least one mutation indicative of a disease state.
38. The method according to claim 26, wherein prior to the introducing step, the method further comprising modifying the target nucleic acid with an adaptor sequence.
39. The method according to claim 38, wherein each target nucleic acid is modified with a different adaptor sequence.
40. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:contacting a sample comprising at least one target nucleic acid and other non-target molecules to at least one sequence-specific capture probe attached to a surface to produce target/probe duplexes;separating at least one of the duplexes from the non-target molecules;exposing the separated duplexes to at least one labeled nucleotide in the presence of a polymerase capable of catalyzing addition of the nucleotide to the duplex in a template-dependent manner, wherein the probe acts as a primer; anddetecting incorporation of the nucleotide into the probe portion, thereby obtaining sequence information from the nucleic acid.
41. The method according to claim 40, wherein at least some of the attached duplexes are individually optically resolvable.
42. The method according to claim 40, wherein the exposing and detecting steps are repeated at least once.
43. The method according to claim 42, further comprising: removing unincorporated nucleotide and polymerase in all or some repetitions of the exposing and detecting steps.
44. The method according to claim 40, wherein the probes are directly attached to the surface.
45. The method according to claim 40, wherein the probes are indirectly attached to the surface.
46. The method according to claim 40, wherein the probes are a plurality of different sequence-specific probes.
47. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:performing hybrid capture on a sample comprising at least one target nucleic acid and other non-target molecules to produce target/probe duplexes;separating at least one of the duplexes from the non-target molecules; andperforming a sequencing by synthesis reaction on the separated target/probe duplexes, wherein the probe acts as a primer for the sequencing reaction.
[0001]This invention relates to methods and compositions for detecting, enumerating, and identifying nucleic acids using capture probes to capture and purify target nucleic acid in combination with single molecule sequencing of the captured or purified nucleic acid molecules.
[0002]Knowledge of the human genome has given rise to inquiry into individual differences, as well as differences within an individual, as the basis for differences in biological function and dysfunction. Differences as small as single nucleotide polymorphisms (SNPs) or combinations of SNPs can lead to phenotypic differences, and detection of combinations of SNPs can predict the likelihood that an individual will get a specific disease or how an individual will respond to treatment.
[0003]For example, most cancers develop from a series of genomic changes, some subtle and some significant, that occur in a small subpopulation of cells. Knowledge of the sequence variations that lead to cancer will lead to an understanding of the etiology of the disease, as well as ways to treat and/or prevent it. An essential first step in understanding genomic complexity is the ability to perform high-resolution sequencing. Therefore, a true understanding of the complexities in either normal or abnormal function will require specific sequence information from large numbers of target nucleic acid molecules.
[0004]Bulk sequencing techniques are often not useful for the identification of subtle or rare nucleotide changes due to the many cloning, amplification and electrophoresis steps that complicate the process of gaining useful information regarding individual nucleotides. The ability to sequence and gain information from single molecules obtained from an individual patient is the next milestone for genomic sequencing. However, effective diagnosis and management of important diseases through single molecule sequencing is impeded by lack of cost-effective tools and methods for screening individual molecules.
[0005]There have been many proposals to develop new sequencing technologies based on single-molecule measurements, generally either by observing the interaction of particular proteins with DNA or by using ultra high resolution scanned probe microscopy. See, e.g., Rigler, et al., Biotech., 86 (3):161 (2001); Goodwin, P. M., et al., Nucleosides & Nucleotides, 16 (5-6):543-550 (1997); Howorka, S., et al., Nature Biotech., 19 (7):636-639 (2001); Meller, A., et al., Proc. Natl. Acad., 97 (3):1079-1084 (2000); Driscoll, R. J., et al., Nature, 346 (6281):294-296 (1990). A recent technique employs optical detection in a sequencing-by-synthesis reaction at the single molecule level. Braslaysky, et al., PNAS, 100: 3960-3964 (2003). The present invention provides improvements in sequencing, especially single molecule sequencing.
[0006]The invention provides methods for sequencing nucleic acids. According to the invention, sequencing is facilitated by capture of target nucleic acids to be sequenced prior to sequencing. In a preferred embodiment, target nucleic acids are sequenced at the single molecule level, resulting in sample-specific, high-throughput sequence information. Thus, according to one aspect of the invention, sequence-specific capture probes are used to isolate target nucleic acids of interest. The target population may be composed of same-sequence nucleic acids or may be a population of mixed-sequence nucleic acids. The isolated target nucleic acids are then sequenced. In one embodiment, sequencing is performed directly on captured nucleic acids, using the capture probes as primers. Alternatively, target/probe duplex may be melted and the target nucleic acids may then be hybridized to primers for template-dependent sequencing.
[0007]In a preferred embodiment, single molecule sequencing is conducted in order to provide high-resolution, high-throughput sequence information. Template-dependent single-molecule sequencing-by-synthesis is conducted using optically-labeled nucleotides for addition to the primer or probe. Either the target (template) or the primer/probe, or both are attached to a surface that is designed to enhance optical signal detection. A particularly-preferred surface is an epoxide surface coated onto glass or fused silica. Nucleic acids are easily attached to epoxide or epoxide derivatives. A preferred method is direct amine attachment. Nucleic acids can be purchased with a 5' or 3' amine, or terminal transferase can be used to introduce a terminal amine for attachment to the epoxide ring. Alternatively, epoxide surfaces can be derivatized for nucleic acid attachment. For example, the surface can incorporate streptavidin, which binds to biotinylated nucleic acids. Either the target, primer/probe, or both can be biotinylated using known methods. Alternative surfaces include polyelectrolyte multilayers as described in Braslavasky, et al., PNAS 100: 3960-64 (2003), incorporated by reference herein. Essentially, any surface that has reduced native fluorescence and is amenable to attachment of oligonucleotides is useful for the invention.
[0008]Single molecule sequence is advantageously performed using optically-detectable labels. Especially preferred are fluorescent labels, including fluorescein, rhodamine, derivatized rhodamine dyes, such as TAMRA, phosphor, polymethadine dye, fluorescent phosphoramidite, texas red, green fluorescent protein, acridine, cyanine, cyanine 5 dye, cyanine 3 dye, 5-(2'-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY, 120 ALEXA, or a derivative or modification of any of the foregoing.
[0009]The capture step prior to sequencing may be any suitable hybrid capture method. For example, capture can occur in solution, on beads (polystyrene beads), in a column (such as a chromatography column), in a gel (such as a polyacrylamide gel), or directly on the surface to be used for sequencing. An array of support-bound capture oligos can be used to hybridize specifically to a target sequence. Additionally, chromatography-based capture techniques are useful. For example, ion exchange chromatography, HPLC, gas chromatography, and gel-based chromatography all are useful. In one embodiment, gel-based capture is used in order to achieve sequence-specific capture. Using this method, multiple different sequences are captured simultaneously using immobilized probes in the gel. The target sequences are isolated by removing portions of the gel containing them and eluting target from the gel portions for sequencing.
[0010]In an alternative embodiment, the target nucleic acid molecule either includes, or is modified to include, an adaptor sequence (such as a polyadenylation region) that is complementary to a portion of a capture probe in order to aid in the capture of the target. A preferred embodiment comprises an immobilized capture probe having a sequence that hybridizes (e.g., is complementary to) with the adaptor sequence. Methods of the invention are conducted by contacting capture probes with a sample comprising target nucleic acid molecules under conditions suitable for specific hybridization between the target nucleic acid molecule and immobilized capture probe, thereby forming target/capture probe duplex. A wash step removes debris and unhybridized nucleic acid in the sample. In one embodiment, target nucleic acid is sequenced using the capture probe as a primer for template-dependent sequencing-by-synthesis. In another embodiment, the target/capture probe duplex is melted to release target nucleic acid. The resulting purified target population is sequenced as described below.
[0011]If target nucleic acid is melted off the capture probe, the targets are either attached to a surface for sequencing or hybridized to a primer that has been attached to the surface. Surface attachment of oligonucleotides for sequencing can be direct or indirect. For example, nucleic acids are attached to an epoxide surface via a direct amine linkage as described below. Alternatively, the surface is prepared with a binding partner, the opposite of which is attached to the nucleic acid. For example, the surface can be streptavidinated and biotinylated nucleic acids can be used to form an attachment at the surface. Other binding pairs (e.g., antibody/antigen, such as digoxigenen/anti-digoxigenen and dinitorphenol/anti-dinitrophenol) can also be used.
[0012]In a preferred embodiment, the invention contemplates optical sequencing. Preferably, template-dependent sequencing-by-synthesis is conducted using optically-detectable labels. Optimal labels include fluorescent labels as described in detail below. The surface is prepared to minimize background for optical detection of incorporated nucleotides. Primer/template duplex attached to the surface is exposed to labeled nucleoside triphosphates in the presence of a suitable nucleotide polymerizing enzyme, under conditions suitable for the enzyme to add at least one nucleotide to the primer in template-dependent manner, and the added nucleotide is detected, thereby identifying at least one nucleotide in at least one attached target nucleic acid molecule.
[0013]As will be appreciated by one skilled in the art, individual features of the invention may be used separately or in any combination. A detailed description of embodiments of the invention is provided below. Other embodiments of the invention are apparent upon review of the detailed description that follows.
[0014]The present invention provides methods for sequencing nucleic acids. In a preferred embodiment, the invention comprises the use of single molecule sequencing of hybrid-captured nucleic acids. Methods of the invention are highly parallel and are amenable to multiplexing. As a result, biological samples can be rapidly analyzed over a broad dynamic range.
[0015]Methods of the invention comprise hybrid-capturing target nucleic acid and sequencing the captured targets. Captured target nucleic acids can be sequenced directly using the capture probe as a primer for template-dependent synthesis. Thus, capture probes are bound to a surface and exposed to sample nucleic acids. Complementary binding of target nucleic acid and probes occurs and the remaining nucleic acid and other sample contents are washed from the surface. Sequencing then takes place essentially as described below. In one alternative, target nucleic acids are isolated by contacting a sample containing target nucleic acid molecules with immobilized capture probes capable of hybridizing to target nucleic acid. Target nucleic acid The surface is washed to remove non-target nucleic acids and other debris, and the targets are then melted off the target/capture probe duplex. After an optional wash, the targets are bound to a surface for sequencing essentially as described below. Target nucleic acid molecules preferably are analyzed using single molecule sequencing techniques described below. In a preferred embodiment, target nucleic acid is modified by addition of a polynucleotide sequence that specifically hybridizes to a complementary portion of a primer for nucleic acid synthesis. For example, targets can be polyadenylated and captured using poly-dT probes/primers for sequencing.
Target Nucleic Acid Molecules
[0016]Target nucleic acids include deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). Target nucleic acids can be isolated or can be captured in situ. In a preferred alternative, target nucleic acid is isolated from a biological sample containing a variety of other components, such as proteins, lipids and non-target nucleic acids. Target nucleic acid molecules can be obtained from any cellular material, obtained from an animal, plant, bacterium, virus, fungus, or any other cellular organism. Target nucleic acids may be obtained directly from an organism or from a biological sample obtained from an organism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue or body fluid specimen may be used as a source for nucleic acid for use in the invention. Nucleic acid molecules may also be isolated from cultured cells, such as a primary cell culture or a cell line. The cells from which target nucleic acids are obtained can be infected with a virus or other intracellular pathogen.
[0017]A sample can also be total RNA extracted from a biological specimen, a cDNA library, or genomic DNA. Nucleic acid typically is fragmented to produce suitable fragments for capture and/or purification and analysis. In one embodiment, nucleic acid from a biological sample is fragmented by sonication. Test samples can be obtained as described in U.S. Patent Application 2002/0190663 A1, published Oct. 9, 2003, the teachings of which are incorporated herein in their entirety. Generally, nucleic acid can be extracted from a biological sample by a variety of techniques such as those described by Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). Generally, target nucleic acid molecules can be from about 5 bases to about 20 kb. Nucleic acid molecules may be single-stranded, double-stranded, or double-stranded with single-stranded regions (for example, stem- and loop-structures).
[0018]A biological sample as described herein may be homogenized or fractionated in the presence of a detergent or surfactant. The concentration of the detergent in the buffer may be about 0.05% to about 10.0%. The concentration of the detergent can be up to an amount where the detergent remains soluble in the solution. In a preferred embodiment, the concentration of the detergent is between 0.1% to about 2%. The detergent, particularly a mild one that is nondenaturing, can act to solubilize the sample. Detergents may be ionic or nonionic. Examples of nonionic detergents include triton, such as the Triton® X series (Triton® X-100 t-Oct-C6H4--(OCH2--CH2)xOH, x=9-10, Triton® X-100R, Triton® X-114 x=7-8), octyl glucoside, polyoxyethylene(9)dodecyl ether, digitonin, IGEPAL® CA630 octylphenyl polyethylene glycol, n-octyl-beta-D-glucopyranoside (betaOG), n-dodecyl-beta, Tween® 20 polyethylene glycol sorbitan monolaurate, Tween® 80 polyethylene glycol sorbitan monooleate, polidocanol, n-dodecyl beta-D-maltoside (DDM), NP-40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol n-dodecyl monoether), hexaethyleneglycol mono-n-tetradecyl ether (C14E06), octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG), Emulgen, and polyoxyethylene 10 lauryl ether (C12E10). Examples of ionic detergents (anionic or cationic) include deoxycholate, sodium dodecyl sulfate (SDS), N-lauroylsarcosine, and cetyltrimethylammoniumbromide (CTAB). A zwitterionic reagent may also be used in the purification schemes of the present invention, such as Chaps, zwitterion 3-14, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. It is contemplated also that urea may be added with or without another detergent or surfactant.
[0019]Lysis or homogenization solutions may further contain other agents, such as reducing agents. Examples of such reducing agents include dithiothreitol (DTT), β-mercaptoethanol, DTE, GSH, cysteine, cysteamine, tricarboxyethyl phosphine (TCEP), or salts of sulfurous acid.
[0020]Preferred buffer concentration is from about 5 mM to about 500 mM in solution or in solution with the sample. The buffer concentration in the lysing solution can be between about 10 mM and 300 mM.
[0021]Subsequent to exposure to a homogenization solution, samples may be further homogenized by mechanical means. Mechanical blenders, rotor-stator homogenizers, or shear-type homogenizers may be employed. Alternatively, the tissue can be homogenized in the lysis solution, and the tissue remains separated by settling, centrifugation, or filtration. These remains could then be treated with homogenization solution and extraction conditions as described above.
[0022]Target nucleic acid molecules can include or be modified to include an adaptor sequence that is complementary to the immobilized capture probe. The adaptor sequence and immobilized capture probe are chosen such that the target nucleic acid molecule comprising the adaptor sequence can hybridize to the immobilized capture probe. Typically, the adaptor sequence is a homopolymer, such as oligo(dA), and the corresponding immobilized capture probe includes an oligo(dT) sequence. The adaptor sequence may be endogenously contained within the target nucleic acid molecule, for example, the adaptor sequence can be a sequence of interest in the target nucleic acid molecule. Alternatively, the adaptor sequence can comprise vector sequence.
[0023]In one embodiment, a universal probe is used as the immobilized capture probe and the target nucleic acid molecules are modified with an adaptor comprising a sequence complementary to the universal probe, thereby allowing the modified nucleic acid molecules to hybridize to the immobilized probe.
[0024]The adaptor sequence and complementary sequence within the immobilized capture probe are of a length suitable for hybridizing the target nucleic acid molecule to the immobilized capture probe to thereby capture target nucleic acid molecules to form target probe/duplexes. The target probe/duplexes are also sufficiently stable to permit optional washing of the duplexes to remove any remaining components of the biological sample, including non-target nucleic acids. The sequence of the adaptor and the complementary sequence of the immobilized capture probe can be about 10 to about 100, and preferably 50, nucleotides in length. The adaptor sequence and complementary immobilized capture sequence can be of the same length or of different lengths. It is routine in the art to adjust probe length and/or oligonucleotide length to optimize hybridization.
[0025]The adaptor sequence can be attached to the nucleic acid molecules with an enzyme. The enzyme can be a ligase or a polymerase. The ligase can be an enzyme capable of ligating an oligonucleotide (RNA or DNA) to the nucleic acid molecules. Suitable ligases include, for example, T4 DNA ligase and T4 RNA ligase (such ligases are available commercially, from New England BioLabs (on the World Wide Web at NEB.com). In a preferred embodiment, the target nucleic acid molecules are dephoyshorylated before ligating the adaptors. Methods for using ligases are well known in the art.
[0026]The polymerase can be any enzyme capable of adding nucleotides to the target nucleic acid molecules. The polymerase can be, for example, poly(A) polymerase, including yeast poly(A) polymerase, commercially available from USB (on the World Wide Web at USBweb.com), terminal deoxyribonucleotidyl transferase (TdT), and the like. The polymerases can be used according to the manufacturer's instructions.
Purifying Target Nucleic Acid Molecules
[0027]As described herein, target nucleic acid molecules can be purified by contacting a sample containing the target molecules with one or more classes of immobilized capture probes. Where more than one target molecule is to be purified, the different target molecules can have the same or different adaptor sequences. A given class of immobilized capture probe is selected to hybridize to target nucleic acid molecules containing the corresponding adaptor sequence, under conditions suitable for the target nucleic acid molecule to hybridize to the immobilized capture probe, thereby forming target/probe duplexes. A variety of capture probes can be used to purify the target nucleic acid molecules. The capture probes typically comprise a nucleic acid with a nucleotide sequence with substantial complementarity to a region of the target nucleic acid molecule, so that the target nucleic acid molecule can hybridize to the capture probe. Complementarity between target nucleic acid molecules and the capture probes need only be sufficient to specifically bind the target nucleic acid molecule, and thus, to effectuate purification of the target nucleic acid molecule from a test sample. Probes suitable for use in the present invention include those formed from nucleic acids, such as RNA and/or DNA, nucleic acid analogues, modified nucleic acids, and chimeric probes of a mixed class comprising a nucleic acid with another organic component such as peptide nucleic acids. Capture probes can be single stranded or double stranded. Preferably, the length of the capture probe is at least 5 nucleotides, more preferably between about 5 and about 100 nucleotides, but the length can be up to several thousand nucleotides. Additional probes are described in U.S. Patent application 2002/0119480 A1, published Aug. 29, 2002, the teachings of which are incorporated herein in their entirety.
[0028]Capture probes can be coupled to agarose, dextrans, cellulose, beads, microparticles, and starch polymers using cyanogen bromide or cyanuric chloride activation. Particularly useful beads and microparticles are described in U.S. Patent Application 2003/0190663 A1, published Oct. 9, 2003, the teachings of which are incorporated herein in their entirety. Polymers containing carboxyl groups can be coupled to synthetic capture probes having primary amine groups using carbodiimide coupling. Polymers containing primary amines can be coupled to amine-containing probes with glutaraldehyde or cyanuric chloride. Polymers can also be modified with thiol-reactive groups that can be coupled to thiol-containing probes. Many other suitable methods can be found in the literature (Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, Fla. 1993). Methods for covalently attaching the capture probes to polymerizable chemical groups are also described in U.S. 2002/0119480 A1, the teachings of which are incorporated herein. In addition, as described in U.S. 2002/0119480 A1, nucleic acids can be attached to particles which themselves can be incorporated into electrophoretic media.
[0029]To purify target nucleic acid molecules, the test sample can be contacted, for example, with an electrophoretic medium that comprises at least one immobilized capture probe. An electric field is applied across the electrophoretic medium so that negatively charged molecules can migrate through the medium. The non-target components of the test sample pass into the electrophoresis buffer, while the target nucleic acid molecules are captured by hybridizing to the capture probes within the electrophoretic medium (forming target/probe duplexes). The buffer used for the electrophoresis step (containing the non-target components of the test sample) can be replaced with fresh buffer and current applied across the electrophoretic medium to denature the complex, thereby releasing the target nucleic acid molecule from the capture probe. The released target nucleic acid molecule can be eluted by applying a reversed electric field.
[0030]The method of the present invention is suitable for multiplexing. As described above, a sample can include more than one target nucleic acid molecule. If adapters are used, the different target nucleic acid molecules can have the same or different adaptor sequences. In a multiplexing reaction, where the target nucleic acid molecules contain different adaptor sequences, the test sample is contacted with two or more classes of immobilized capture probes as described above. Target nucleic acid molecules are captured by their respective immobilized capture probes. The captured target nucleic acid molecules can be released as described above. Other methods for capturing and purifying target nucleic acid molecules are described in U.S. 2002/0119480 A1, the teachings of which are incorporated herein.
Attaching Target Nucleic Acid Molecules To A Surface
[0031]In a preferred embodiment, target nucleic acid molecules are attached to a surface and subjected to analysis by single molecules sequencing. Target nucleic acid molecules are attached to the surface such that they are individually optically resolvable. Substrates for use in the invention can be two- or three-dimensional and can comprise a planar surface (e.g., a glass slide) or can be shaped. A substrate can include glass (e.g., controlled pore glass (CPG)), quartz, plastic (such as polystyrene (low cross-linked and high cross-linked polystyrene), polycarbonate, polypropylene and poly(methymethacrylate)), acrylic copolymer, polyamide, silicon, metal (e.g., alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel), polyacrolein, or composites.
[0032]Suitable three-dimensional substrates include, for example, spheres, microparticles, beads, membranes, slides, plates, micromachined chips, tubes (e.g., capillary tubes), microwells, microfluidic devices, filters, or any other structure suitable for anchoring a nucleic acid. Substrates can include planar arrays or matrices capable of having regions that include populations of target nucleic acids or primers. Examples include nucleoside-derivatized CPG and polystyrene slides; derivatized magnetic slides; polystyrene grafted with polyethylene glycol, and the like.
[0033]In one embodiment, a substrate is coated to allow optimum optical processing and nucleic acid attachment. Surfaces for use in the invention are treated to reduce background. Exemplary coatings include epoxides, and derivatized epoxides (e.g., with a binding molecule, such as streptavidin). The surface can also be treated to improve the positioning of attached nucleic acids (e.g., target nucleic acid molecules, primers, or target molecule/primer complexes) for analysis. As such, a surface according to the invention can be treated with one or more charge layers (e.g., a negative charge) to repel a charged molecule (e.g., a negatively charged labeled nucleotide). For example, a substrate according to the invention can be treated with polyallylamine followed by polyacrylic acid to form a polyelectrolyte multilayer. The carboxyl groups of the polyacrylic acid layer are negatively charged and thus repel negatively charged labeled nucleotides, improving the positioning of the label for detection. Coatings or films applied to the substrate should be able to withstand subsequent treatment steps (e.g., photoexposure, boiling, baking, soaking in warm detergent-containing liquids, and the like) without substantial degradation or disassociation from the substrate.
[0034]Examples of substrate coatings include, vapor phase coatings of 3-aminopropyltrimethoxysilane, as applied to glass slide products, for example, from Molecular Dynamics, Sunnyvale, Calif. In addition, generally, hydrophobic substrate coatings and films aid in the uniform distribution of hydrophilic molecules on the substrate surfaces. Importantly, in those embodiments of the invention that employ substrate coatings or films, the coatings or films that are substantially non-interfering with primer extension and detection steps are preferred. Additionally, it is preferable that any coatings or films applied to the substrates either increase target molecule binding to the substrate or, at least, do not substantially impair target binding.
[0035]Various methods can be used to anchor or immobilize the target nucleic acid molecule to the surface of the substrate. The immobilization can be achieved through direct or indirect bonding to the surface. The bonding can be by covalent linkage. See, Joos et al., Analytical Biochemistry 247:96-101, 1997; Oroskar et al., Clin. Chem. 42:1547-1555, 1996; and Khandjian, Mole. Bio. Rep. 11:107-115, 1986. A preferred attachment is direct amine bonding of a terminal nucleotide to an epoxide integrated on the surface. The bonding also can be through non-covalent linkage. For example, biotin-streptavidin (Taylor et al., J. Phys. D. Appl. Phys. 24:1443, 1991) and digoxigenin with anti-digoxigenin (Smith et al., Science 253:1122, 1992) are common tools for anchoring nucleic acids to surfaces and parallels. Alternatively, the attachment can be achieved by anchoring a hydrophobic chain into a lipid monolayer or bilayer. Other methods for known in the art for attaching nucleic acid molecules to supports also can be used.
[0036]In a preferred embodiment, surfaces for oligonucleotide attachment are coated with an epoxide. An epoxide may be deposited by many methods known in the art. An epoxy silane surface is preferred. Different molecules or combinations of molecules may serve to link the epoxide to a surface. Ideally, a surface will be coated with an even distribution of epoxides prior to introduction of target nucleic acid molecules. Target nucleic acid molecules can be directly or indirectly linked to an epoxide. In a direct attachment embodiment, the epoxide is introduced to a nucleic acid bearing an amine group. In a preferred embodiment, terminal transferase is used to add an amine-terminated nucleotide to a nucleic acid to be attached to the surface.
[0037]When biotin-streptavidin linkage is used to anchor the nucleic acids, the nucleic acids can be biotinylated, while one surface of the substrates can be coated with streptavidin.
[0038]Surface density of the nucleic acid molecules can be controlled by adjusting the concentration of the streptavidin applied to the surface. Reagents for biotinylating a surface can be obtained, for example, from Vector Laboratories. Alternatively, biotinylation can be performed with BLCPA: EZ-Link Biotin LC-PEO-Amine (Pierce, Cat. 21347), or any other known or convenient method. In some embodiments, labeled streptavidin of very low concentration (e.g., in the μM, nM or pM range) is used to coat the substrate surface prior to anchoring. This can facilitate immobilization of the nucleic acid with single molecule resolution.
[0039]Other examples of linkers include antigen/antibody, digoxigenin/anti-digoxigenin, dinitrophenol, fluorescein, and other haptens known in the art. Alternatively, the nucleic acid may contain other binding moieties that result in a conformational change of the epoxide ring and result in a direct attachment of the target nucleic acid molecules to the opened epoxide ring.
[0040]Alternatively, primers may be immobilized on the surface. A terminus of one or more primers may be modified to carry a linker moiety for tethering the primers to the surface or may be directly attached to the surface. Target nucleic acid molecules containing primer complementary sequence are then hybridized to the attached primer. Methods for attaching nucleic acid such as primer to the surface of a substrate are described in detail above.
[0041]In order to inhibit non-specific binding of molecules to a surface, the surface can be treated with a passivating (blocking) agent. Preferred blocking strategies include exposing the surface to a non-detectable molecule that adheres to the surface or changes the chemical properties of the surface such that non-specific binding is reduced. In methods in which optically-detectable labels are used, one way to block or passivate the surface is to expose the surface to unlabeled molecules of the same type as those that are labeled. The unlabeled molecules will out-compete labeled molecules for non-specific binding on the surface, thus reducing background due to non-specific label. Other strategies involve treating the surface with phosphate, Tris, a sulfate, or an amine that interacts with the surface to prevent non-specific binding. Non-reactive proteins are also appropriate. In a preferred embodiment, a matrix of blocking reagents is provided on the surface in order to provide a highly washable, low non-specific background surface. In some embodiments, blocking reagents are chosen to provide electrostatic repulsion of highly anionic nucleoside triphosphates.
[0042]Blocking agents may be introduced or reintroduced at any time during the analysis. Also, in some embodiments, blocking agents may be used to pre-treat the surface prior to exposing the substrate to target nucleic acid molecules or primers. In addition, blocking agents, such as a detergent (e.g., Tris) may be included in some or all wash steps in order to passivate the surface during incubation periods and/or washes.
[0043]Surface charge can be manipulated to achieve ideal conditions during both nucleic acid attachment and primer extension. For example, during the loading phase where the nucleic acid (target nucleic acid molecule or primer) is bound to the surface, the salt concentration of the solution may be increased in order to create a more positive surface charge on the substrate to facilitate reaction between the amine portion of the nucleic acid and the epoxide ring. Conversely, after the nucleic acid has been secured to the surface, the salt concentration of the solution can lowered in order to repel the nucleic acid from the surface of the substrate thereby sterically conforming the nucleic acid for annealing and sequence analysis.
[0044]In another embodiment, the substrate includes a layer of polyanions and nucleic acid molecules anchored on the layer of polyanions. Accordingly, nucleic acids are positioned to avoid being substantially parallel (e.g., is hindered from lying down on the layer of polyanions.) In some embodiments, the surface of a substrate is pretreated to create a surface chemistry that facilitates nucleic acid attachment and subsequent annealing and sequence analysis. In some of these embodiments, the substrate surface is coated with a polyelectrolyte multilayer (PEM). In some cases, biotin can be applied to the PEM, followed by application of streptavidin. The substrate can then be used to attach biotinylated target nucleic acid molecules.
[0045]In some embodiments, multiple layers of alternating positive and negative charges are used. In the case of incompletely-charged surfaces, multiple-layer deposition tends to increase surface charge to a well-defined and stable level. For example, surfaces can be coated with a PEM for attachment of primers via light-directed spatial attachment. Alternatively, target nucleic acid molecules can be attached to a PEM-coated surface chemically. PEM formation has been described in Decher et al. (Thin Solid Films, 210:831-835, 1992), the teachings of which are incorporated herein. PEM formation proceeds by the sequential addition of polycations and polyanions, which are polymers with many positive or negative charges, respectively. Upon addition of a polycation to a negatively-charged surface, the polycation deposits on the surface, forming a thin polymer layer and reversing the surface charge. Similarly, a polyanion deposited on a positively charged surface forms a thin layer of polymer and leaves a negatively charged surface. Alternating exposure to poly(+) and poly(-) generates a polyelectrolyte multilayer structure with a surface charge determined by the last polyelectrolyte added. This can produce a strongly-negatively-charged surface, repelling the negatively-charged nucleotides. Detailed procedures for coating a substrate with PEM for immobilizing nucleic acid are described in U.S. patent application Ser. No. 11/137,928, field May 25, 2005.
[0046]In another aspect of the invention, the substrate may be prepared by, for example, coating with a chemical that increases or decreases hydrophobicity or coating with a chemical that allows covalent linkage of the primers. Some chemical coatings may both alter the hydrophobicity and allow covalent linkage. Hydrophobicity on a solid substrate may readily be increased by silane treatment or other treatments known in the art. Linker molecules adhere to the surface and comprise a functional moiety that reacts with biomolecules. Many such linkers are readily available and known in the art. For example, substrates or supports are modified with photolabile-protected hydroxyl groups, alkoxy or aliphatic derivatized hydroxyl groups, or other chemicals. A preferred coating that both decreases hydrophobicity and provides linkers is poly(ethyleneimine).
[0047]Methods of the invention also optionally include a surface drying step. In some embodiments, the surface is exposed to a drying agent prior to, during and/or after a chemical reaction, such as a nucleotide incorporation step. Examples of preferred drying agents include, without limitation, phosphate buffer, an alcohol (such as, for example, EtOH), air and/or N2.
Analyzing Attached Target Nucleic Acid Molecules
[0048]As described herein, attached target nucleic acid molecules are analyzed by single molecule sequencing. At least one nucleotide is identified in at least one attached target nucleic acid molecule. Target molecules are hybridized to a primer to form nucleic acid target molecule/primer duplex on a surface. As described above, either the target nucleic acid molecule or the primer, or both, is/are attached to the surface. Thereafter, template-dependent primer extension is conducted to identify at least one nucleotide of the hybridized nucleic acid molecule using a nucleotide polymerizing enzyme and a nucleotide (e.g., dATP, dTTP, dUTP, dCTP and/or a dGTP) or a nucleotide analog. Incorporation of a nucleotide or a nucleotide analog is detected at discrete locations on the surface. Nucleic acid target molecule/primer duplexes, as well as the incorporated nucleotides, are individually resolvable in single molecule embodiments. Alternatively, bulk signal from mixed nucleic acid populations or clonal populations of nucleic acids, are obtained.
[0049]Fast reagent application and removal is another advantage of the invention. For example, concentrations of nucleotides and/or other reaction reagents can be alternated at different time points. This is a particularly useful feature in an embodiment comprising introducing one or more single species of nucleotide individually. This could lead to increased incorporation rates and sensitivity. For example, when all four types of nucleotides are simultaneously present in the reaction to monitor dynamic incorporation of nucleotides, concentrations of the each of the respective nucleotides can be alternated between a first and a second range. This leads to both better visualization of the signal when low concentrations of nucleotides are present, and increased polymerization rate when higher concentrations of nucleotides are present.
[0050]The target nucleic acid can comprise or can be modified to comprise a sequence that is sufficiently complementary to a primer to hybridize to the primer to allow template dependent addition of nucleotides to the hybridized primer. The sequence complementary to the primer can be the same as the previously described adaptor sequence. Therefore, the immobilized capture probe and the primer can comprise the same sequence. The primer sequence can be about 10 to about 1000 nucleotides in length. The primer sequence and complementary target nucleic acid molecule sequence can be of the same length or of different lengths. Conditions for hybridizing primers to nucleic acid targets are well known. The annealing reaction is performed under conditions which are stringent enough to guarantee sequence specificity, yet sufficiently permissive to allow formation of stable hybrids at an acceptable rate. The temperature and length of time required for primer annealing depend upon several factors including the base composition, length and concentration of the primer, and the nature of the solvent used, e.g., the concentration of cosolvents such as DMSO (dimethylsulfoxide), formamide, or glycerol, and counterions such as magnesium. Typically, hybridization (annealing) between primers and target nucleic acids is carried out at a temperature that is approximately 5 to 10° C. below the melting temperature of the target-primer hybrid in the annealing solvent. Typically, the annealing temperature is in the range of 55 to 75° C. and the primer concentration is approximately 0.2 μM. Under such conditions, the annealing reaction is usually complete within a few seconds.
[0051]Methods according to the invention include conducting a primer extension reaction, such as exposing the nucleic acid/primer complexes to polymerase and one or more nucleotides under conditions sufficient to extend the primer by at least one base. Sequencing, as used herein can be performed such that one or more nucleotides are identified in one or more nucleic molecules. Methods according to the invention also include the step of compiling a sequence of the molecule (nucleic acid) based upon sequential incorporation of the extension bases into the primer.
[0052]In the analyzing step, the hybridized nucleic acid molecules can be sequenced using single molecule sequencing as described, for example, in U.S. patent application Ser. No. 11/137,928, filed May 25, 2005 and/or and described in U.S. Pat. No. 6,780,591, the teachings of which are incorporated herein in their entirety. Polymerases useful in the invention include any nucleic acid polymerase capable of catalyzing a template-dependent addition of a nucleotide or nucleotide analog to a primer. Depending on the characteristics of the target nucleic acid, a DNA polymerase, an RNA polymerase, a reverse transcriptase, or a mutant or altered form of any of the foregoing can be used. According to one aspect of the invention, a thermophilic polymerase is used, such as ThermoSequenase®, 9°N®, Therminator®, Taq, Tne, Tma, Pfu, Tfl, Tth, Tli, Stoffel fragment, Vent® and Deep Vent® DNA polymerase.
[0053]The target nucleic acid molecule/primer complexes are contacted with dNTPs in the presence of the polymerase under conditions such that the polymerase catalyzes template-dependent addition of a dNTP to the 3' terminus of the primer. The dNTP can be detectably labeled, as described herein, and the nucleotide is identified by detecting the presence of the incorporated labeled nucleotide. As described above, unincorporated labeled dNTPs can be removed from the surface prior to detecting the incorporated labeled dNTP. The process can be repeated one or more times, wherein the template/primer complex(s) are provided with additional dNTPs, in the presence of a polymerase, followed by removing the unincorporated labeled dNTPs and detecting the incorporated labeled dNTP. The sequence of the template is determined by compiling the detected (identified) dNTPs. In this manner, the entire sequence of one or more templates can be determined. In addition, by using single molecule sequencing techniques, determining the sequence for each nucleic acid molecule attached to the surface provides the number of different or unique nucleic acid molecules in the sample. Furthermore, the number of copies of each nucleic acid sequences in a biological sample is also provided.
[0054]In order to allow for further extension and detection of subsequently added fluorophore-labeled nucleotides, the fluorophore of the incorporated nucleotide can be removed from the nucleotide incorporated into the primer. For example, the optical label (e.g., fluorescent label) can be destroyed by photochemical destruction as described in U.S. Pat. No. 6,780,591, the teachings of which are incorporated herein in their entirety. This cycle can be repeated a large number of times if sample losses are avoided. In one embodiment, such losses will be avoided by attaching the target nucleic acid molecules or primers to a surface of an array device, for example a microscope slide, and transferring the entire array device between a reaction vessel and the fluorescent reader.
[0055]The extension reactions are carried out in buffer solutions which contain the appropriate concentrations of salts, dNTP(s) and nucleotide polymerizing enzyme required for the enzyme mediated extension to proceed. For additional guidance regarding such conditions see, for example, Sambrook et al., (1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY); and Ausubel et al. (1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY).
[0056]Nucleotides particularly useful in the invention comprise detectable labels. Labeled nucleotides include any nucleotide that has been modified to include a label that is directly or indirectly detectable. Preferred labels include optically-detectable labels, including fluorescent labels or fluorophores, such as fluorescein, rhodamine, cyanine, cyanine-5 dye, cyanine-3 dye, or a derivative or modification of any of the foregoing, and also include such labeling systems as hapten labeling. Accordingly, methods of the invention further provide for exposing the primer/target nucleic acid complex to a digoxigenin, a fluorescein, an alkaline phosphatase or a peroxidase.
[0057]The sequencing can be optimized to achieve rapid and complete addition of the correct nucleotide to primers in primer/template complexes, while limiting the misincorporation of incorrect nucleotides. For example, dNTP concentrations may be lowered to reduce misincorporation of incorrect nucleotides into the primer. Km values for incorrect dNTPs can be as much as 1000-fold higher than for correct nucleotides, indicating that a reduction in dNTP concentrations can reduce the rate of misincorporation of nucleotides. Thus, in a preferred embodiment of the invention the concentration of dNTPs in the sequencing reactions are approximately 5-20 μM.
[0058]In addition, relatively short reaction times can be used to reduce the probability of misincorporation. For an incorporation rate approaching the maximum rate of about 400 nucleotides per second, a reaction time of approximately 25 milliseconds will be sufficient to ensure extension of 99.99% of primer strands.
[0059]Any detection method may be used that is suitable for the type of label employed. Thus, exemplary detection methods include radioactive detection, optical absorbance detection, e.g., UV-visible absorbance detection, optical emission detection, e.g., fluorescence or chemiluminescence. For example, extended primers can be detected on a substrate by scanning all or portions of each substrate simultaneously or serially, depending on the scanning method used. For fluorescence labeling, selected regions on a substrate may be serially scanned one-by-one or row-by-row using a fluorescence microscope apparatus, such as described in Fodor (U.S. Pat. No. 5,445,934) and Mathies et al. (U.S. Pat. No. 5,091,652). Devices capable of sensing fluorescence from a single molecule include scanning tunneling microscope (siM) and the atomic force microscope (AFM). Hybridization patterns may also be scanned using a CCD camera (e.g., Model TE/CCD512SF, Princeton Instruments, Trenton, N.J.) with suitable optics (Ploem, in Fluorescent and Luminescent Probes for Biological Activity Mason, T. G. Ed., Academic Press, Landon, pp. 1-11 (1993), such as described in Yershov et al., Proc. Natl. Aca. Sci. 93:4913 (1996), or may be imaged by TV monitoring. For radioactive signals, a phosphorimager device can be used (Johnston et al., Electrophoresis, 13:566, 1990; Drmanac et al., Electrophoresis, 13:566, 1992; 1993). Other commercial suppliers of imaging instruments include General Scanning Inc., (Watertown, Mass. on the World Wide Web at genscan.com), Genix Technologies (Waterloo, Ontario, Canada; on the World Wide Web at confocal.com), and Applied Precision Inc. Such detection methods are particularly useful to achieve simultaneous scanning of multiple attached target nucleic acids.
[0060]The present invention provides for detection of molecules from a single nucleotide to a single target nucleic acid molecule. A number of methods are available for this purpose. Methods for visualizing single molecules within nucleic acids labeled with an intercalating dye include, for example, fluorescence microscopy. For example, the fluorescent spectrum and lifetime of a single molecule excited-state can be measured. Standard detectors such as a photomultiplier tube or avalanche photodiode can be used. Full field imaging with a two-stage image intensified COD camera also can be used. Additionally, low noise cooled CCD can also be used to detect single fluorescent molecules.
[0061]The detection system for the signal may depend upon the labeling moiety used, which can be defined by the chemistry available. For optical signals, a combination of an optical fiber or charged couple device (CCD) can be used in the detection step. In those circumstances where the substrate is itself transparent to the radiation used, it is possible to have an incident light beam pass through the substrate with the detector located opposite the substrate from the target nucleic acid. For electromagnetic labeling moieties, various forms of spectroscopy systems can be used. Various physical orientations for the detection system are available and discussion of important design parameters is provided in the art.
[0062]A number of approaches can be used to detect incorporation of fluorescently-labeled nucleotides into a single nucleic acid molecule. Optical setups include near-field scanning microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, dark field microscopy, photoconversion, single and/or multiphoton excitation, spectral wavelength discrimination, fluorophore identification, evanescent wave illumination, and total internal reflection fluorescence (TIRF) microscopy. In general, certain methods involve detection of laser-activated fluorescence using a microscope equipped with a camera. Suitable photon detection systems include, but are not limited to, photodiodes and intensified CCD cameras. For example, an intensified charge couple device (ICCD) camera can be used. The use of an ICCD camera to image individual fluorescent dye molecules in a fluid near a surface provides numerous advantages. For example, with an ICCD optical setup, it is possible to acquire a sequence of images (movies) of fluorophores.
[0063]Some embodiments of the present invention use TIRF microscopy for two-dimensional imaging. TIRF microscopy uses totally internally reflected excitation light and is well known in the art. See, e g., the World Wide Web at nikon-instruments.jp/eng/page/products/tirf.aspx. In certain embodiments, detection is carried out using evanescent wave illumination and total internal reflection fluorescence microscopy. An evanescent light field can be set up at the surface, for example, to image fluorescently-labeled nucleic acid molecules. When a laser beam is totally reflected at the interface between a liquid and a solid substrate (e.g., a glass), the excitation light beam penetrates only a short distance into the liquid. The optical field does not end abruptly at the reflective interface, but its intensity falls off exponentially with distance. This surface electromagnetic field, called the "evanescent wave", can selectively excite fluorescent molecules in the liquid near the interface. The thin evanescent optical field at the interface provides low background and facilitates the detection of single molecules with high signal-to-noise ratio at visible wavelengths.
[0064]The evanescent field also can image fluorescently-labeled nucleotides upon their incorporation into the attached target nucleic acid target molecule/primer complex in the presence of a polymerase. Total internal reflectance fluorescence microscopy is then used to visualize the attached target nucleic acid target molecule/primer complex and/or the incorporated nucleotides with single molecule resolution.
[0065]Measured signals can be analyzed manually or by appropriate computer methods to tabulate results. The substrates and reaction conditions can include appropriate controls for verifying the integrity of hybridization and extension conditions, and for providing standard curves for quantification, if desired. For example, a control nucleic acid can be added to the sample. The absence of the expected extension product is an indication that there is a defect with the sample or assay components requiring correction.
[0066]Fluorescence resonance energy transfer (FRET) can be used as a detection scheme. FRET in the context of sequencing is described generally in Braslavasky, et al., Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003), incorporated by reference herein. Essentially, in one embodiment, a donor fluorophore is attached to the primer, polymerase, or template. Nucleotides added for incorporation into the primer comprise an acceptor fluorophore that is activated by the donor when the two are in proximity.
[0067]As described herein, combination articles of manufacture are provided that are suitable for performing the method of the invention. Suitable surfaces are described above. The enzyme can be a ligase or a polymerase, as described above. The adaptor sequence can optionally comprise a linker moiety at the 5' terminus of the adaptor sequence, the linker moiety being suitable for attaching a target molecule to the surface. Other suitable linker moieties are described above.
[0068]The 7249 nucleotide genome of the bacteriophage M13mp18 was sequenced using a single molecule system of the invention. Purified, single-stranded viral M13mp18 genomic DNA was obtained from New England Biolabs. Approximately 25 ug of M13 DNA was digested to an average fragment size of 40 by with 0.1 U Dnase I (New England Biolabs) for 10 minutes at 37° C. Digested DNA fragment sizes were estimated by running an aliquot of the digestion mixture on a precast denaturing (TBE-Urea) 10% polyacrylamide gel (Novagen) and staining with SYBR Gold (Invitrogen/Molecular Probes). The DNase I-digested genomic DNA was filtered through a YM10 ultrafiltration spin column (Millipore) to remove small digestion products less than about 30 nt. Approximately 20 pmol of the filtered DNase I digest was then polyadenylated with terminal transferase according to known methods (Roychoudhury, R and Wu, R. 1980, Terminal transferase-catalyzed addition of nucleotides to the 3' termini of DNA. Methods Enzymol. 65 (1):43-62.). The average dA tail length was 50+/-5 nucleotides. Terminal transferase was then used to label the fragments with Cy3-dUTP. Fragments were then terminated with dideoxyTTP (also added using terminal transferase). The resulting fragments were again filtered with a YM10 ultrafiltration spin column to remove free nucleotides and stored in ddH2O at -20° C.
[0069]Epoxide-coated glass slides were prepared for oligo attachment. Epoxide-functionalized 40 mm diameter #1.5 glass cover slips (slides) were obtained from Erie Scientific (Salem, N.H.). The slides were preconditioned by soaking in 3×SSC for 15 minutes at 37° C. Next, a 500 pM aliquot of 5' aminated polydT(50) (polythymidine of 50 bp in length with a 5' terminal amine) was incubated with each slide for 30 minutes at room temperature in a volume of 80 ml. The resulting slides had poly(dT50) primer attached by direct amine linkage to the epoxide. The slides were then treated with phosphate (1M) for 4 hours at room temperature in order to passivate the surface. Slides were then stored in polymerase rinse buffer (20 mM Tris, 100 mM NaCl, 0.001% Triton X-100, pH 8.0) until they were used for sequencing.
[0070]For sequencing, the slides were placed in a modified FCS2 flow cell (Bioptechs, Butler, Pa.) using a 50 um thick gasket. The flow cell was placed on a movable stage that is part of a high-efficiency fluorescence imaging system built around a Nikon TE-2000 inverted microscope equipped with a total internal reflection (TIR) objective. The slide was then rinsed with HEPES buffer with 100 mM NaCl and equilibrated to a temperature of 50° C. An aliquot of the M13 template fragments described above was diluted in 3×SSC to a final concentration of 1.2 nM. A 100 ul aliquot was placed in the flow cell and incubated on the slide for 15 minutes. After incubation, the flow cell was rinsed with 1×SSC/HEPES/0.1% SDS followed by HEPES/NaCl. A passive vacuum apparatus was used to pull fluid across the flow cell. The resulting slide contained M13 template/oligo(dT) primer duplex. The temperature of the flow cell was then reduced to 37° C. for sequencing and the objective was brought into contact with the flow cell.
[0071]For sequencing, cytosine triphosphate, guanidine triphosphate, adenine triphosphate, and uracil triphosphate, each having a cyanine-5 label (at the 7-deaza position for ATP and GTP and at the C5 position for CTP and UTP (PerkinElmer)) were stored separately in buffer containing 20 mM Tris-HCl, pH 8.8, 10 mM MgSO4, 10 mM (NH4)2SO4, 10 mM HCl, and 0.1% Triton X-100, and 100 U Klenow exo.sup.- polymerase (NEN). Sequencing proceeded as follows.
[0072]First, initial imaging was used to determine the positions of duplex on the epoxide surface. The Cy3 label attached to the M13 templates was imaged by excitation using a laser tuned to 532 nm radiation (Verdi V-2 Laser, Coherent, Inc., Santa Clara, Calif.) in order to establish duplex position. For each slide only single fluorescent molecules were imaged in this step were counted. Imaging of incorporated nucleotides as described below was accomplished by excitation of a cyanine-5 dye using a 635 nm radiation laser (Coherent). 5 uM Cy5CTP was placed into the flow cell and exposed to the slide for 2 minutes. After incubation, the slide was rinsed in 1×SSC/15 mM HEPES/0.1% SDS/pH 7.0 ("SSC/HEPES/SDS") (15 times in 60 ul volumes each, followed by 150 mM HEPES/150 mM NaCl/pH 7.0 ("HEPES/NaCl") (10 times at 60 ul volumes). An oxygen scavenger containing 30% acetonitrile and scavenger buffer (134 ul HEPES/NaCl, 24 ul 100 mM Trolox in MES, pH 6.1, 10 ul DABCO in MES, pH 6.1, 8 ul 2M glucose, 20 ul NaI (50 mM stock in water), and 4 ul glucose oxidase) was next added. The slide was then imaged (500 frames) for 0.2 seconds using an Inova301K laser (Coherent) at 647 nm, followed by green imaging with a Verdi V-2 laser (Coherent) at 532 nm for 2 seconds to confirm duplex position. The positions having detectable fluorescence were recorded. After imaging, the flow cell was rinsed 5 times each with SSC/HEPES/SDS (60 ul) and HEPES/NaCl (60 ul). Next, the cyanine-5 label was cleaved off incorporated CTP by introduction into the flow cell of 50 mM TCEP for 5 minutes, after which the flow cell was rinsed 5 times each with SSC/HEPES/SDS (60 ul) and HEPES/NaCl (60 ul). The remaining nucleotide was capped with 50 mM iodoacetamide for 5 minutes followed by rinsing 5 times each with SSC/HEPES/SDS (60 ul) and HEPES/NaCl (60 ul). The scavenger was applied again in the manner described above, and the slide was again imaged to determine the effectiveness of the cleave/cap steps and to identify non-incorporated fluorescent objects.
[0073]The procedure described above was then conducted 100 nM Cy5dATP, followed by 100 nM Cy5dGTP, and finally 500 nM Cy5dUTP. The procedure (expose to nucleotide, polymerase, rinse, scavenger, image, rinse, cleave, rinse, cap, rinse, scavenger, final image) was repeated exactly as described for ATP, GTP, and UTP except that Cy5dUTP was incubated for 5 minutes instead of 2 minutes. Uridine was used instead of thymidine due to the fact that the Cy5 label was incorporated at the position normally occupied by the methyl group in Thymidine triphosphate, thus turning the dTTP into dUTP. In all 64 cycles (C, A, G, U) were conducted as described in this and the preceding paragraph.
[0074]Once 64 cycles were completed, the image stack data (i.e., the single molecule sequences obtained from the various surface-bound duplex) were aligned to the M13 reference sequence. The image data obtained was compressed to collapse homopolymeric regions. Thus, the sequence "TCAAAGC" would be represented as "TCAGC" in the data tags used for alignment. Similarly, homopolymeric regions in the reference sequence were collapsed for alignment. The sequencing protocol described above resulted in an aligned M13 sequence with an accuracy of between 98.8% and 99.96% (depending on depth of coverage). The individual single molecule sequence read lengths obtained ranged from 2 to 33 consecutive nucleotides with about 12.6 consecutive nucleotides being the average length.
[0075]The alignment algorithm matched sequences obtained as described above with the actual M13 linear sequence. Placement of obtained sequence on M13 was based upon the best match between the obtained sequence and a portion of M13 of the same length, taking into consideration 0, 1, or 2 possible errors. All obtained 9-mers with 0 errors (meaning that they exactly matched a 9-mer in the M13 reference sequence) were first aligned with M13. Then 10-, 11-, and 12-mers with 0 or 1 error were aligned. Finally, all 13-mers or greater with 0, 1, or 2 errors were aligned. At a coverage depth of greater than or equal to 1, 5,001 bases of the 5,066 base M13 collapsed genome were covered at an accuracy of 98.8%. Similarly, at a coverage depth of greater than or equal to 5, 83.6% of the genome was covered at an accuracy of 99.3%, and at a depth of greater than or equal to 10, 51.9% of the genome was covered at an accuracy of 99.96%. The average coverage depth was 12.6 nucleotides.
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Patent applications by Stanley Lapidus, Bedford, NH US
Patent applications by HELICOS BIOSCIENCES CORPORATION
Patent applications in class Involving nucleic acid
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The eleventh day of both dark (KrishnaKrishnaIt is described that there are three aspects of the Supreme that must...) and bright (Shukla) lunar fortnight, according to Hindu calendar is known as Ekadashi. This day is considered sacred as it is dedicated to Lord Vishnu. There can be twenty four or twenty five Ekadashi in a year but the most auspicious one is Nirjala Ekadashi. The day falls on the eleventh day of the Shukla paksha (waxing fortnight) in the month of Jyestha (May-June), according to Hindu calendar. Thus, it is also known as Jyeshtha Shukla Ekadashi.
Nirjala is a combination of two words i.e., 'nir' meaning without and 'jala' meaning water. Thus this day devotees observe a strict fast without consuming even water. This is the most austere and sacred Ekadashi. It also is most rewarding ones. People who cannot keep fasts on other Ekadashi can keep only this one and all their desires will be fulfilled as Nirjala Ekadashi Vrat Vrat is the Hindi term of fast . It is a process in which devotees do not consume food or/and water on the days dedicated to the Gods they believe in, to show their dedication and to gain health and prosperity(fast) is the most fruitful one.
Legend of Nirjala Ekadashi Vrat
Bhima, the strongest of all Pandavas, was a lover of food and was unable to abstain himself from eating. When he saw his brothers and his wife, observing fasts on all the Ekadashis, Bhima felt bad and asked sage Vyasa (the author of the Hindu epic, Mahabharata) for a solution. Vyasa told him to observe only one fast i.e., Nirjala Ekadashi fast. If he is able to observe this fast religiously then he would attain the virtues equivalent to all twenty four Ekadashis. Thus this day is also known as PandavaBhima Ekadashi or PandavaNirjala Ekadashi.
Only staunch devotees of VishnuVishnuVishnu is one of the Supreme Gods of Hindus and is a part of Hindu Trinity,.. should undertake the fast as it falls in extreme summer and in the heat staying without water becomes unbearable. The fast is observed for twenty four hours, from sunrise of Ekadashi to the sunrise of next day. The day preceding Ekadashi, people consume only one meal and that too exclusive of rice.
The morning of Nirjala Ekadashi starts with devotees taking an early bath and chanting Vishnu's names. They visit temples to offer prayers to him. At home, people bathe Vishnu's idol with Panchamrit, i.e., a mixture of milk, curd, ghee (clarified butter), sugar and honey.Then clean the idol with clean water and dress him in new and clean clothes. Tulsi (Holy basil), fruits and sweets are also offered to him. In the evening, he is worshipped with Durva grass It has many medicinal values and people who perform the rituals associated with the day, are benefitted by its positive effects on skin disorders, high sugar level, obesity, insomnia, fatigue, anemia and digestion. Hence it symbolizes purity of body. One can also purify his house by sprinkling water in all the corners using durva grass. According to some beliefs, the tip of the grass represents Shiva, the middle represents Vishnu and the roots represent Brahma. Thus it as pure as the holy trinity(CynodonDactylon). People sing hymns and prayers throughout the night and stay vigil and complete their fast the next morning after sunrise and consume water and food, again without rice.
Charity and donations are also a part of the rituals. Some people offer food to Brahmins It is one of the caste of Hindus. They are known for their knowledge of ancient, sacred scriptures(the learned class) and some offer items like clothes, umbrellas, hand-fans and pitchers filled with water to needy.
Observing this fast is considered equivalent to performing a pilgrimage. It is believed that Vishnu himself took the form of the day, Ekadashi to absolve humans from their sins. Devotees who offer their dedication to Vishnu on this day receive happiness, prosperity, salvation and forgiveness. As the fast takes place before monsoon, it is also believed to help in cleansing the mind, body and soul, just like rain cleanses the earth of its impurities.
It is also believed that after death, the soul is taken by YamaYamaAlso known as Yamaraja, Yama is the controller of the hell (Naraka).... He judges the soul by giving an account of all the deeds performed and then he decides whether the soul goes to hell or heaven. But one who keeps Nirjala Ekadashi fast, does not go through any judgment. Their souls are taken by the messengers of Vishnu and brought to his abode.
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Thaipusam 2017
Kartik Purnima (Dev Diwali) 2016
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A Tale of Two Away Games
The Phillies final two games in Seattle were a stark contrast to each other. Saturday night's 5-1 win was against 2010 Cy Young winner Felix Hernandez. But on Sunday, the Phillies lost to Jason Vargas, an average pitcher who entered the game with a 4.13 ERA.
With Vance Worley starting Saturday, the Phillies managed to score some runs. Worley held the Mariners to just one run over five innings and then Shane Victorino provided most of the offense.
Victorino ended the game just a single short of the cycle and had four RBI on three hits, including a 2-run homer. Jimmy Rollins had three hits as well. Both Ryan Howard and Raul Ibanez added two hits apiece.
But Sunday, the Phillies seemingly could not hit their way out of a barn. Howard had two hits, Ben Francisco had one and that was it for the offense. Vargas tossed a complete game shut-out as the Phils sat and watched.
Cole Hamels pitched a great game, allowing only two runs on a few very cheap, bloop hits. Hamels also collected his 1,000 career strike out in the fourth inning.
If he had won the game, Hamels would have been the first pitcher in the majors to 10 wins. He and six other pitchers, including Roy Halladay, are stuck at nine wins. But it was not meant to be. Wasting such a great performance was a real shame.
But welcome to Interleague play with the Phillies. Every year, it is a struggle for them. After losing two out of three to the Mariners, the Phillies are off tomorrow as they travel to St. Louis for three games with the Cardinals starting on Tuesday.
Photo by Jenn Zambri Photography
*Read more about the Phillies at my other home page, Phightin' Phils Phorum in the MTR Media network*


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There's a reason coaches and players unanimously want to get off to a good start. Not only does it give your team an early advantage on the scoreboard, but it sets the tone for the rest of the contest.
Thompson Valley and Mountain View both had plenty of traffic on the basepaths in the first inning, despite each only coming up with one base hit.
The difference was, the Mountain Lions scored three times, aided by a couple of walks and some poor defense.
The Eagles, meanwhile, left the bases loaded without a single run to their credit.
That 3-0 lead was more than enough for MVHS starting pitcher Hunter Porterfield, who put forth an effort on the mound that his team has been seeking all season.
Porterfield scattered five hits, struck out five and allowed just one run to cool off a red-hot TVHS offense, as the Lions made good use of their strong start for an 11-1 win Thursday at Brock Field.
The win kept the Lions' faint playoff hopes alive in snapping a two-game skid to improve to 7-8 overall and 4-6 in the Northern Conference with four games left and still plenty of work to do to reach districts.
"I think Hunter really set the tone for the day. He was in command from the beginning," said MVHS coach Brian Smela, whose team still needs to win out and get some help to earn one of the league's three automatic playoff bids. "We knew how good of a hitting team they were, and we knew he was going to have to be at his best.
"He was great today. I think it loosened the lineup up, and it made us play more comfortable throughout the game just knowing how well he was throwing throughout the game."
After getting Drew Meintzer to pop up to end the bases-loaded threat in the first, Porterfield retired nine of the next 12 batters before a seeing-eye single by Jesse Shaw scored Nate Giefer to put the Eagles on the board in the fifth.
But that lone run never stood a chance against a balanced Lions' order that had all nine starters come up with at least one hit or one run.
Ozzie Pearcy and Adam Baumann did the most damage. Baumann was 2-for-3 with a pair of extra-base hits, including a run-scoring triple in the first. Pearcy was 2-for-4 with three RBI, the final two of which ended the game in the fifth on a hard-hit single to right field to enact the 10-run mercy rule.
Pearcy said the difference Thursday, the first of a two-game set between the two teams, was matching the intensity the Eagles tend to bring to the crosstown rivalry.
"They're ahead of us in the league right now, and they always come out and try to play us like it's the biggest game of the year. They're always looking to pound the ball," said Pearcy, who also had a run-scoring triple in the fourth. "Going in, we decided to up our ante, more intense, really bring our game. We kind of just hit a roll, kept the intensity up, and it just steamrolled from there."
While Mountain View needs to win out to return to the postseason after winning the 4A title a year ago, Thompson Valley remains still very much in the hunt after entering the day having won seven of its past eight.
The one constant during that run for the Eagles was their offense, having scored at least eight runs in seven straight until running into Porterfield, who didn't allow a single extra-base hit and was very much stress-free after wiggling out of the first-inning jam.
"We had an opportunity in the first that we didn't capitalize on. Then in the bottom of the first defensively, we didn't take care of our pitcher," said TVHS coach Jay Denning, whose team fell to 9-6 overall and 6-5 in league play. "Momentum swing right there at the top of the game. We could never get it back from them."
Contact Reporter-Herald Sports Writer Sean Star at 970-669-5050 ext. 512, email@example.com or twitter.com/rhpreps
Mountain Lions 11, Eagles 1
At Brock Field
Thompson Valley
000 01 — 1 5 2
Mountain View
302 24 — 11 9 0
W — Hunter Porterfield. L — Nate Hewson.
Thompson Valley hitting (ab-r-h-rbi) — Garrett Smith 2-0-1-0, Brad Soucek 2-0-0-0, Nate Giefer 2-1-0-0, Jason Cox 3-0-2-0, Jesse Shaw 2-0-1-1, Drew Meintzer 3-0-0-0, Brandon Freed 2-0-1-0, Jordan Parsons 2-0-0-0, Dillon Feuerstein 2-0-0-0. Totals 20-1-5-1.
Mountain View hitting (ab-r-h-rbi) — Aaron Morrison 2-1-0-1, Ozzie Pearcy 4-0-2-3, Adam Baumann 3-2-2-1, Dakotah Shea-Shelley 2-2-1-1, Justin Clinton 2-1-1-1, Hunter Porterfield 3-0-1-10, Jerrod Klug 3-1-0-0, Cody Howell 2-2-0-1, Max Moree 3-2-2-1. Totals 24-11-9-9 . 2B — Baumann. 3B — Baumann, Pearcy.
Thompson Valley pitching (ip-h-r-er-bb-so) — Hewson 3-4-6-5-3-3, DJ DeWaard 1.1-4-5-5-2-0, Feuerstein 0-10-0-0-0.
Mountain View pitching (ip-h-r-er-bb-so) — Porterfield 5-5-1-1-3-5.


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sprunger pow
Kyle Sprunger awarded Bluffton's first-ever men's tennis HCAC player-of-the-week honorMarch 25, 2002
Bluffton College freshman Kyle Sprunger (Berne, Ind./South Adams H.S.) fashioned a perfect 3-0 record in both singles and doubles last week and has been named the men's tennis player-of-the-week in the Heartland Collegiate Athletic Conference.
Sprunger is Bluffton's first-ever men's tennis player-of-the-week recipient in the four years in which the Beavers have competed in the HCAC.
Sprunger won 8-3 at No. 4 singles on Thursday at Tiffin University before moving up to play at No. 3 singles on Saturday against Baldwin-Wallace College and HCAC foe Transylvania University. He defeated his B-W opponent in three sets (2-6, 7-5, 6-3) before notching a win over his Transy opponent in two sets (6-1, 6-0).
In No. 2 doubles action Sprunger and his teammate were winners over Tiffin 8-1 and Baldwin-Wallace 9-7 and claimed a forfeit win over Transylvania.
Bluffton defeated Tiffin 9-0 and Transylvania 7-2, but fell to B-W 5-4. The Beavers are now 2-1 overall on the season and 1-0 in the HCAC.


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|7 years ago :: Apr 28, 2011 - 4:15PM #1
I am a 23 year old single mother of a 3 year old girl. I am hard working (although not employed at this moment) physically and mentally, I am still going to College and I take pride in my motherly instincts. In the past few months I have broken away from my old relationship (not my daughters father) and now I am single.
I am not taking it too well, in fact I have little hope that I will find a decent guy to partner with. By decent I mean a man with a real job and always looking to improve himself.
Wondering if anyone else feels the same way or has some hope to shed on my view?
Quick Reply
|7 years ago :: Apr 28, 2011 - 9:04PM #2
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|7 years ago :: May 10, 2011 - 4:00PM #3
You seem keen to get a move on to someone new. I wouldn't recommend you move too quickly, from what you're saying.
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|7 years ago :: May 28, 2011 - 7:05PM #4
I was single for about 10 years before I met my partner. I dated quite extensively during that time so believe me I understand a bit about relationships.
When you feel disheartened about men and relationships and feel you may never meet anyone it is the time to be by yourself and nuture yourself NOT to rush into a new relationship.
That whole desperation thing you bring into the relationship when you are in that place usually means the relationship is doomed anyways.
Much better to get along with your life and start doing some of the things that make you and your child happy. That energy tends to attract the right sort of partner naturally anyway.
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King Kong
Many HU's i look at say they have a rear aux-in port and i was wondering how exactly you plug in an ipod/mp3 player if its in the rear of the HU. Do they have to run a cord to the glovebox or something? and if not the glovebox where else? do they have to cut a hole in the dash if you want it elsewhere?