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Thursday 30 December 2010

Topten of Laptop (5)-Apple MacBook Pro Notebook


Technical Details

  • 2.4GHz Intel Core 2 Duo
  • 250 GB Hard Drive, 8x DVD/CD SuperDrive, 4GB DDR3 RAM
  • 13.3 inch LED-backlit display, 1280-by-800 resolution
  • NVIDIA GeForce 320M graphics processor with 256 MB of shared memory
  • Mac OS X v10.6 Snow Leopard Operating System

Product Details

Product Description

Amazon.com Product Description

The new MacBook Pro sets all-new benchmarks for Mac notebooks. This 13-inch MacBook Pro features an Intel Core 2 Duo processor and next-generation NVIDIA graphics that bring high performance to everything from 3D games to photos and videos. And the built-in battery lasts up to 10 hours.
13-inch MacBook Pro
The 13-inch unibody MacBook Pro (see larger image).
As with previous models, the new MacBook Pro features a precision unibody enclosure crafted from a single block of aluminum, creating a thin and light mobile computing workhorse (less than 1 inch thin and just 4.5 pounds) that's also highly durable. And it includes a 13.3-inch, LED-backlit glass display as well as a glass trackpad that doesn't include a button (for larger tracking area) that features Apple's Multi-Touch technology.
This version of the 13.3-inch MacBook Pro (model MC374LL/A) features a 2.4 GHz Intel Core 2 Duo processor, 250 GB hard drive, and 4 GB of installed RAM. Other features include integrated Wireless-N Wi-Fi networking, Bluetooth connectivity, an SD card slot, and two USB 2.0 ports (see full specifications below). It also comes with the Mac OS X Snow Leopard operating system as well as the iLife software suite, which includes the latest versions of iPhoto, iMovie, and GarageBand.

Key Features

13-inch MacBook Pro
The backlit keyboard--great for typing in low-light settings
(see larger image).
Intel Core 2 Duo Processing
Combining fast processing speeds with power-saving features, the Intel Core 2 Duo processor provides greater multitasking performance by combining two independent processor cores while also reducing power requirements and saving on battery life. This Intel Core 2 Duo processor has a 2.4 GHz processor speed, speedy 1066 MHz front-side bus (FSB), and 3 MB L2 cache. (An L2, or secondary, cache temporarily stores data; and a larger L2 cache can help speed up your system's performance. The FSB carries data between the CPU and RAM, and a faster front-side bus will deliver better overall performance.)
High Performance Graphics
Powered by the NVIDIA GeForce 320M graphics card, the Mac mini allows you to view flawless video in full screen HD and play the latest games with fast frame rate. It's a great match to editing video masterpieces with iMovie and managing your photos in iPhoto, and it comes with 256 MB of DDR3 SDRAM that's shared with main memory.
Buttonless Multi-Touch Trackpad
With no button on the glass trackpad, there's more room to track and click--left, right, center, and everywhere in between. Without a separate button, the trackpad gives your hands plenty of room to move on the large, silky glass surface. It also incorporates Multi-Touch gestures--including swipe, pinch, rotate, and four-finger swipe. And it also now supports inertial scrolling, an intuitive way to scroll through large photo libraries, lengthy documents and long web sites.

Key Specifications

  • 13.3-inch LED-backlit glossy widescreen display with edge-to-edge, uninterrupted glass (1280 x 800-pixel resolution).
  • 2.4 GHz Intel Core 2 Duo dual-core processor with 3 MB shared L2 cache for excellent multitasking
  • NVIDIA GeForce 320M graphics processor with 256 MB of DDR3 SDRAM shared with main memory.
  • 250 GB Serial ATA hard drive (5400 RPM)
  • 4 GB installed RAM (1066 MHz DDR3; supports up to 8 GB)
  • 8x slot-loading SuperDrive with double-layer DVD support (DVD±R DL/DVD±RW/CD-RW)

    13-inch MacBook Pro
    The left (above) and right (below) sides of the MacBook Pro (see larger image).

    13-inch MacBook Pro
  • Built-in iSight camera for video chatting
  • Wi-Fi wireless networking (based on 802.11n specification; 802.11a/b/g compatible)
  • Gigabit Ethernet wired networking (10/100/1000)
  • Bluetooth 2.1 + EDR (Enhanced Data Rate) for connecting with peripherals such as keyboards, mice and cell phones
  • Two USB 2.0 ports, one FireWire 800 port
  • SD card slot
  • Mini DisplayPort video output with optional adapters for DVI, VGA, dual-link DVI, and HDMI output
  • Multi-Touch trackpad for precise cursor control with support for inertial scrolling, pinch, rotate, swipe, three-finger swipe, four-finger swipe, tap, double-tap, and drag capabilities
  • Full-size backlit keyboard
  • Stereo speakers with subwoofers
  • Dimensions: 12.78 x 8.94 x 0.95 inches (WxDxH)
  • Weight: 4.5 pounds
  • Up to 10 hours of battery life
  • Meets Energy Star 5.0 requirements
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Topten of Laptop (4)-Acer Aspire AS5736Z-4790 Notebook



Acer Aspire 15.6" HD Notebook


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Item #: 22432832
Manufacturer #: AS5736Z-4790

Powered by an Intel Pentium Processor T4500, 4096MB DDR3 Dual-Channel SDRAM Memory, 320GB 5400PM SATA Hard drive, 15.6" HD Widescreen Cine Crystal LCD Display, Windows 7 Home Premium 64 Bit, 1-year Limited Warranty


Features
  • Intel Pentium Processor T4500 2.30GHz, 1MB L2 Cache, 800MHz FSB
  • 4096MB DDR3 Dual-Channel SDRAM Memory
  • 320GB 5400PM SATA Hard drive
  • 15.6" HD Widescreen Cine Crystal LCD Display
  • 802.11 b/g/n WiFi CERTIFIED
  • 1.3 MP High Definition Webcam
  • Windows 7 Home Premium 64 Bit
  • 1-year Limited Warranty
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Topten of Laptop (3)-Samsung QX410 Notebook

Display / 4GB Memory / 640GB Hard Drive - Aluminum

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Topten of Laptop (2)-Toshiba Satellite C655-S5082 Notebook

Toshiba - Satellite Laptop / Intel® Celeron® Processor / 15.6" Display / 2GB Memory / 250GB Hard Drive - Black

Model: C655-S5082 | SKU: 1261751

Special Offers:

Financing:

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Topten of Laptop (1)-Asus K52F-BBR5 Notebook

Asus - Laptop / Intel® Pentium® Processor / 15.6" Display / 3GB Memory / 320GB Hard Drive - Brown

Model: K52F-BBR5 | SKU: 1257145

Financing:

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Finali Mondiali Ferrari

Ferrari's passion and the passion for Ferrari can be best seen at the Finali Mondiali. It brings together Ferrari's personalities and motor sports programs with a rich connection to heritage.

This year Valencia hosted the finals which included the final round of the Ferrari Challenge Trofeo as well as Corse Clienti laps, a Ferrari Historic Race, parade laps, F1 demonstrations and a gala night. One impressive sight was 19 cars from the 599XX program.

2010 wasn't particularly kind to Ferrari. They missed the F1 Championship by a few points and failed to secure enough wins in the Le Mans Series, ALMS or the 24 Hours of Le Mans. Maybe 2011 might be more successful with the expected launch of the 458 GT2/GT3 contenders.
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Ferrari 458 GT2

oday the first image is being revealed of the Ferrari's new GT2 contender. Meant to replace the outgoing 430, this will be Ferrari's main hope at beating Porsche and Corvette in ALMS, FIA-sanctioned races and at Le Mans.

The outgoing Ferrari 430 has had an extended service with Ferrari which began in the 2006 season. Probably a high-point for the model came at the 2008 24 Hours of Le Mans when the Risi Competizione car placed first in GT2. Risi and other teams such as AF Corse and CRS Racing are probably looking at the new 458 development with a keen eye.

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Audis V6-Powered LMP1 Coupe

For the upcoming 2011 season Audi have prepared the R18: an all-new LMP1 coupe with a diesel V6 engine.

Beaten by Peugeot this year, Audi have probably spared little expense to win the 2011 24 Hours of Le Mans and beat Ferraris score of nine overall victories.

Audi failed to mention the electronics system in their 2011 contender which for the first time could include energy recovery as part of the ACO's continued focus on the environment.
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Remembering Jacques Swaters

Former racing driver, team owner of Ecurie Francorchamps and pillar of the Ferrari community, Jacques Swaters has past away at the age of 84 in Belgium.

Ecurie Francorchamps was established primarily for racing Ferraris in sports car racing and grand prix from 1952 until 1954. Later, he started Garage Francorchamps, a Ferrari dealership that featured his impressive collection including 0064M, a gorgeous two-tone 166 MM Barchetta.

No doubt, the yellow Ferraris of Jacques Swaters have shaped Supercars.net, especially the 1965 Ferrari 275 GTB Competizione Speciale. With it, Ecurie Francorchamps won their class at the 1965 24 Hours of Le Mans. Belgian photographer Dirk de Jager ran into many more of the team's old cars at the 2006 Brussels Retro Festival.
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Tom Walkinshaw Remembered

Last weekend Tom Walkinshaw passed away at the age of 63 and we lost another great figure in motor sport. Founder of TWR and a successful Touring Car driver, Walkinshaw was also an active race car manufacturer from the 1980s to the mid 1990s.

As early as 1984, TWR began their extensive partnership with Jaguar to produce Touring and Group-C cars. This resulted in two overall wins at Le Mans and three World Sportscar Championships. One of the cars that achieved this success was the 1988 Jaguar XJR-9LM.

TWR also made some of Britain's best supercars. They developed the Jaguar XJ220 and a TWR version. Furthermore they founded Jaguar Sports which produced the XJR-15. Around the same period Walkinshaw became engineering director of the Benetton F1 team and won the 1994 Formula One World Championship with Michael Schumacher behind the wheel.
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McLaren MP4-12C hits GT3

McLaren have finally announced a motor sport version of their capable MP4-12C. Built to GT3 specifications, it will face a wide-array of existing supercars on the track including the upcoming 458.

McLaren will likely use 2011 as a development season and sell a competitive package to customer teams in 2012. The new car features an updated aerodynamics package with a large rear wing and prominent fender flares.

This year we visited the opening round of the GT3 championship at Silverstone. Prospeed Competition Porsche beat Callaway's Corvette by just 10 points to take the 2010 Team's Championship. For the upcoming 2011 season, there are planned stops in both Russia and China.
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TOP 10 SUPER CARS OF 2010



There is no doubt that the Bugatti Veyron Super Sport is the 2010 supercar of the year (SCOTY). As the ultimate Veyron and the fastest, most expensive automobile on the planet, it sets the bar extremely high for the future.

Despite following a recessive year, many of the major manufacturers didn't hold back. We even saw some intriguing start-ups such as the Arash AF10, Transstar Dagger, Amoritz, Revenge Verde and the Vorax V10.

As a caveat, we should mention this list is restricted to road-cars or concept cars which were launched this year and at least appeared at one show. Deciding factors included style, performance and impact on the market:



1. Bugatti 16/4 Veyron Super Sport (267 MPH, incredible CF work)

2. Ferrari 599 GTO (sold out before launch, legendary history)
3. Porsche 918 Spyder(incredible style, possible production)
4. Hennessey Venom GT (all the right hallmarks for performance)
5. Koenigsegg Agera (ultimate CCX)
6. Lamborghini Sesto Elemento (radical, ultralight CF monocoque)
7. Pagani Zonda Tricolore (Frecce Tricolori for the road)
8. Jaguar C-X75 (XJ220 successor)
9. Alfa Romeo Pandion(wildest concept this year & it drives)
10. Porsche 911 GT2 RS (it was this or the Superleggera)
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Salmonella

almonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which project in all directions (i.e. peritrichous). They are chemoorganotrophs, obtaining their energy from oxidation and reduction reactions using organic sources, and are facultative anaerobes. Most species produce hydrogen sulfide,which can readily be detected by growing them on media containing ferrous sulfate, such as TSI. Most isolates exist in two phases: a motile phase I and a nonmotile phase II. Cultures that are nonmotile upon primary culture may be switched to the motile phase using a Cragie tube.[citation needed]
Salmonella is closely related to the Escherichia genus and are found worldwide in cold- and warm-blooded animals (including humans), and in the environment. They cause illnesses like typhoid fever, paratyphoid fever, and the foodborne illness.
Salmonella is typically pronounced /ˌsælməˈnɛlə/ voicing the initial letter "L," since it is named for pathologist Daniel Elmer Salmo



Salmonella infections are zoonotic and can be transferred between humans and nonhuman animals. Many infections are due to ingestion of contaminated food. A distinction is made between enteritis Salmonella and typhoid/paratyphoid Salmonella, where the latter — because of a special virulence factor and a capsule protein (virulence antigen) — can cause serious illness, such as Salmonella enterica subsp. enterica serovar Typhi. Salmonella typhi. is adapted to humans and does not occur in animals.
ta that is often muco-purulent and bloody. In infants, dehydration can cause a state of severe toxicosis. The symptoms are usually mild. There is normally no sepsis, but it can occur exceptionally as a complication in weakened elderly patients (Hodgkin's disease, eg.). Extraintestinal localizations are possible, especially Salmonella meningitis in children, osteitis, etc. Enteritis Salmonella (e.g., Salmonella enterica subsp. enterica serovar enteritidis) can cause diarrhoea, which usually does not require antibiotic treatment. However, in people at risk such as infants, small children, the elderly, Salmonella infections can become very serious, leading to complications. If these are not treated, HIV patients and those with suppressed immunity can become seriously ill. Children with sickle cell anaemia who are infected with Salmonella may develop osteomyelitis.
In Germany, Salmonella infections must be reported .Between 1990 and 2005, the number of officially recorded cases decreased from approximately 200,000 cases to approximately 50,000. It is estimated that every fifth person in Germany is a carrier of Salmonella. In the USA, there are approximately 40,000 cases of Salmonella infection reported each year.According to the World Health Organization, over 16 million people worldwide are infected with typhoid fever each year, with 500,000 to 600,000 fatal cases.
Salmonella can survive for weeks outside a living body. They have been found in dried excrement after more than 2.5 years.[citation needed] Salmonella are not destroyed by freezing .Ultraviolet radiation and heat accelerate their demise; they perish after being heated to 55 °C (131 °F) for one hour, or to 60 °C (140 °F) for half an hour.[citation needed] To protect against Salmonella infection, it is recommended that food be heated for at least ten minutes at 75 °C (167 °F) so that the centre of the food reaches this temperature.[citation needed]
The AvrA toxin injected by the type three secretion system of Salmonella typhimurium works to inhibit the innate immune system by virtue of its serine/threonine acetyltransferase activity and requires binding to eukaryotic target cell phytic acid (IP6). This leaves the host more susceptible to infection.
The AvrA toxin injected by the type three secretion system of Salmonella typhimurium works to inhibit the innate immune system by virtue of its serine/threonine acetyltransferase activity and requires binding to eukaryotic target cell phytic acid (IP6).This leaves the host more susceptible to infection.



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Escherichia coli

Escherichia coli (commonly abbreviated E. coli; pronounced /ˌɛʃɨˈrɪkiə ˈkoʊlaɪ/, named after Theodor Escherich) is a Gram negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2,and by preventing the establishment of pathogenic bacteria within the intestine.




E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily manipulated or duplicated through a process of metagenics, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology and microbiology.
E. coli was discovered by German paediatrician and bacteriologist Theodor Escherich in 1885, and is now classified as part of the Enterobacteriaceae family of gamma-proteobacteria.



A strain of E. coli is a sub-group within the species that has unique characteristics that distinguish it from other E. coli strains. These differences are often detectable only at the molecular level; however, they may result in changes to the physiology or lifecycle of the bacterium. For example, a strain may gain pathogenic capacity, the ability to use a unique carbon source, the ability to take upon a particular ecological niche or the ability to resist antimicrobial agents. Different strains of E. coli are often host-specific, making it possible to determine the source of faecal contamination in environmental samples. For example, knowing which E. coli strains are present in a water sample allows to make assumptions about whether the contamination originated from a human, another mammal or a bird.
New strains of E. coli evolve through the natural biological process of mutation and through horizontal gene transfer. Some strains develop traits that can be harmful to a host animal. These virulent strains typically cause a bout of diarrhoea that is unpleasant in healthy adults and is often lethal to children in the developing world. More virulent strains, such as O157:H7 cause serious illness or death in the elderly, the very young or the immunocompromised.

E. coli is Gram-negative, facultative anaerobic and non-sporulating. Cells are typically rod-shaped and are about 2 micrometres (μm) long and 0.5 μm in diameter, with a cell volume of 0.6 - 0.7 μm3. It can live on a wide variety of substrates. E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide. Since many pathways in mixed-acid fermentation produce hydrogen gas, these pathways require the levels of hydrogen to be low, as is the case when E. coli lives together with hydrogen-consuming organisms such as methanogens or sulphate-reducing bacteria.
Optimal growth of E. coli occurs at 37°C (98.6°F) but some laboratory strains can multiply at temperatures of up to 49°C (120.2°F). Growth can be driven by aerobic or anaerobic respiration, using a large variety of redox pairs, including the oxidation of pyruvic acid, formic acid, hydrogen and amino acids, and the reduction of substrates such as oxygen, nitrate, dimethyl sulfoxide and trimethylamine N-oxide.
Strains that possess flagella can swim and are motile. The flagella have a peritrichous arrangement.
E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population. This process led to the spread of the gene encoding shiga toxin from Shigella to E. coli O157:H7, carried by a bacteriophage.

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Wednesday 15 December 2010

TYPE OF PYROSEQUENCINGS

In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.

 

DNA sequencing

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Thus far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as Pyrosequencing are gaining an increasing share of the sequencing market. More genome data is now being produced by pyrosequencing than Sanger DNA sequencing. Pyrosequencing has enabled rapid genome sequencing. Bacterial genomes can be sequenced in a single run with several X coverage with this technique. This technique was also used to sequence the genome of James Watson recently.
The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in fundamental research into why and how organisms live, as well as in applied subjects. Because of the key nature of DNA to living things, knowledge of DNA sequence may come in useful in practically any biological research. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for genetic diseases. Similarly, research into pathogens may lead to treatments for contagious diseases. Biotechnology is a burgeoning discipline, with the potential for many useful products and services.

 

 

Sanger sequencing

Part of a radioactively labelled sequencing gel
In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.

View of the start of an example dye-terminator read (click to expand)
An alternative to the labelling of the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the incorporation of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability.
This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. The major reason for this is that the primers do not have to be separately labelled (which can be a significant expense for a single-use custom primer), although this is less of a concern with frequently used 'universal' primers. [This is changing rapidly due to the increasing cost-effectiveness of 2nd and 3rd generation systems from Illumina, 454, ABI, Helicos & Dover.]

Pyrosequencing

Pyrosequencing, which was developed by Pål Nyhren and Mostafa Ronaghi, has been commercialized by Biotage (for low throughput sequencing) and 454 Life Sciences (for high-throughput sequencing). The latter platform sequences roughly 100 megabases [now up to 400 megabases] in a 7-hour run with a single machine. In the array-based method (commercialized by 454 Life Sciences), single-stranded DNA is annealed to beads and amplified via EmPCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pairs. Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow. [1]

RNA sequencing

RNA is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by transcription from DNA, the information is already present in the cell's DNA. However, it is sometimes desirable to sequence RNA molecules. In particular, in Eukaryotes RNA molecules are not necessarily co-linear with their DNA template, as introns are excised. To sequence RNA, the usual method is first to reverse transcribe the sample to generate DNA fragments. This can then be sequenced as described above.
For more information on the capabilities of next-generation sequencing applied to whole transcriptomes see: RNA-Seq.

Protein sequencing

If the gene encoding the protein can be identified it is currently much easier to sequence the DNA and infer the protein sequence. Determining part of a protein's amino-acid sequence (often one end) by one of the above methods may be sufficient to enable the identification of a clone carrying the gene.

Polysaccharide sequencing

Though polysaccharides are also biopolymers, it is not so common to talk of 'sequencing' a polysaccharide, for several reasons. Although many polysaccharides are linear, many have branches. Many different units (individual monosaccharides) can be used, and bonded in different ways. However, the main theoretical reason is that whereas the other polymers listed here are primarily generated in a 'template-dependent' manner by one processive enzyme, each individual join in a polysaccharide may be formed by a different enzyme. In many cases the assembly is not uniquely specified; depending on which enzyme acts, one of several different units may be incorporated. This can lead to a family of similar molecules being formed. This is particularly true for plant polysaccharides. Methods for the structure determination of oligosaccharides and polysaccharides include NMR spectroscopy and methylation analysis.



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Principle of Pyrosequencing Technology

Step 1
A sequencing primer is hybridized to a single-stranded PCR amplicon that serves as a template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase, and apyrase as well as the substrates, adenosine 5' phosphosulfate (APS), and luciferin.

Step 2
The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxyribo-nucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.




 
Step 3
ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) chip and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated.

Step 4
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.

Step 5
Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfa-thio triphosphate (dATP·S) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace. 


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PYROSEQUENCING TECHNIQUE

Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, relying on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.[1] The technique was developed by Mostafa Ronaghi at the Royal Institute of Technology in Stockholm in 1996.[2][3][4]

 

Procedure

"Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The Pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemiluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are added and removed after the reaction, sequentially. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.
ssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.
1.     The addition of one of the four deoxynucleotide triphosphates (dNTPs)(in the case of dATP we add dATPαS which is not a substrate for a luciferase) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.
2.     ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program.
3.     Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.
Currently, a limitation of the method is that the lengths of individual reads of DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods (e.g. Sanger sequencing). This can make the process of genome assembly more difficult, particularly for sequences containing a large amount of repetitive DNA. As of 2007, pyrosequencing is most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available.
The templates for pyrosequencing can be made both by solid phase template preparation (streptavidin-coated magnetic beads) and enzymatic template preparation (apyrase+exonuclease).

Commercialization

The company Pyrosequencing AB in Uppsala, Sweden commercialized machinery and reagents for sequencing short stretches of DNA using the pyrosequencing technique. Pyrosequencing AB was renamed to Biotage in 2003 which was acquired by Qiagen in 2008[5]. Pyrosequencing technology was further licensed to 454 Life Sciences. 454 developed an array-based pyrosequencing technology which has emerged as a platform for large-scale DNA sequencing. Most notable are the applications for genome sequencing and metagenomics. GS FLX, the latest pyrosequencing platform by 454 Life Sciences (now owned by Roche Diagnostics), can generate 400 million nucleotide data in a 10 hour run with a single machine. Each run would cost about 5,000-7,000 USD, pushing de novo sequencing of mammalian genomes into the million dollar range.

"Collection"


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