History of DNA Sequencing and Research

History of DNA Sequencing and Research DNA sequencing technology has evolved very rapidly since its inception in the 1970s, and continues to evolve and grow today. This paper will review the major innovations and developments in sequencing technology and briefly summarize their methodologies. The first group that was able to sequence DNA was the team of Allan Maxam and Walter Gilbert (Maxam and Gilbert). This was a first generation sequencing reaction, and was developed in 1976-1977. This method uses purified DNA and relies on chemical modification of DNA bases (like depurination of adenine and guanine using formic acid and methylation using hydrazine or dimethyl sulfate). The 5 end is radioactively labeled so that it can be visualized in a gel, and then fragments of modified DNA are electrophoresed. Autoradiography can then be used to visualize the sizes of each DNA fragment. The maximum read length for this technique was approximately 100 bases long. The next major innovation in DNA sequencing was the Sanger dideoxy chain termination method. This was developed in 1977 by Frederick Sanger (Sanger, Nicklen, and Coulson), and became much more popular than Maxam and Gilberts method. Sanger sequencing is a synthesis reaction and uses dideoxy nucleotides to randomly terminate synthesized strands of DNA. The DNA strands that had been terminated with ddNTPs originally were run in 4 different lanes (one for each ddNTP) and were radiolabeled so that they could be visualized with autoradiography. Later innovations made Sanger sequencing even easier when each dideoxynucleotide was labeled with different fluorescent dyes. As such, sequences could be run on a single gel in a single lane. This method was the most popular way of sequencing DNA for many years, and was prevalent until about 2004. While read length was initially about 100 base pairs long, Sanger sequencing now has a read length of about 800 to 1000 base pairs long when run in capil lary gels. With the start of the human genome project, it was necessary to find ways to sequence DNA much more quickly and more cost-effectively than had been done previously. This led to the development of so-called à ¢Ã¢â€š ¬Ã…“second generationà ¢Ã¢â€š ¬Ãƒâ€šÃ‚  DNA sequencers. It also allowed for the use of smaller samples for sequencing. One of the first major automated platforms was the Roche 454 (Margulies et al.). This utilizes pyrosequencing, which is a synthesis type sequencing reaction. This also uses emulsion PCR on beads. When a dNTP is incorporated, it releases a pyrophosphate (PPi). ATP sulfurylase is present in the reaction mix, and when PPi is released, converts it to ATP, which can activate luciferase and the emission of light. The Roche 454 can measure the amount of light given off and relate it to the number of nucleotides that have been incorporated. One problem with this type of sequencing is that it can be difficult to accurately characterize sequences of the same nucleotide in a row as the intensity of the pyrophosphate peak given off does not have a linear relationship with the number of homopolymers present. The read length for 454 is approximately 250 base pairs long, and the error mode tends to have indels. The next major second gen sequencer is the Illumina Solexa platform (Bennett). The chemistry of this platform is that it utilizes reversible terminators and sequences by synthesis. A flow cell is covered with DNA oligonucleotides that are complementary to adaptor sequences that have been ligated to the ends of fragmented genome pieces. As the genome fragments are streamed across the surface of the flow cell, they will randomly bind and go through multiple cycles of denaturation and extension, which creates clusters of clones. After these clusters have been generated, they are loaded into a sequencer which measures fluorescent signals as single nucleotides are incorporated by taking a picture and noting the location of fluorescence. Read lengths are about 26-50 bases on average, and the types of errors that are typically present tend to be SNP errors. Another important second generation sequencer is the ABI-SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequencing platform (Valouev et al.). This is another sequencing by synthesis reaction, but unlike Illumina and 454, which use polymerases, this uses ligases. After using emulsion PCR on beads to create clonal clusters, primers base pair to a known adapter sequence that has been ligated to the genomic DNA. Differently labeled probes competitively base pair to the sequencing primer, and sequencing goes through several cycles in which different primers are used each time to bind to positions offset by a single nucleotide each time. DNA bases are added in groups of two in this method. Average read lengths for this technique are on average about 35 base pairs long. The next second generation sequencing technique is Ion Torrent, which is a sequencing by synthesis technique (http://www.iontorrent.com). When nucleotides are added to a growing DNA chain, pyrophosphate and a hydrogen ion are released. Ion Torrent takes advantage of this by measuring the pH of the reaction mix after flooding a DNA strand with the four bases (one at a time) to determine sequences. One major advantage of this technique is that it doesnt require a high-cost camera set-up to measure incorporation events. However, because it indirectly measures nucleotide addition through changes in pH, it has difficulty with accuracy in calling sequences of homopolymers, resulting in indel errors (like pyrosequencing). Average read lengths using this technique are about 200 base pairs long. A more recent innovation is the Helicos-True Single Molecule Sequencing (tSMS) technique (Thompson and Steinmann). It is somewhat similar to Illumina sequencing in that it also uses fragmented DNA, adaptors, and fluorescently labeled dNTPs, but there is no amplification step. This helps eliminate issues with GC bias, which tend to affect amplification steps and can cause errors in base calling. Average read length is greater than 25 base pairs. Pacific Biosciences SMRT technology (Single Molecule Real Time sequencing) immobilizes a DNA polymerase at the bottom of a well and is a sequencing by synthesis technique (Eid et al.). Fluorescently labeled phosphate groups in dNTPs are added to the reaction mix and as the base is added to the growing DNA strand, the machine can measure the light that is given off (each base is labeled with a different fluorescent molecule). The major advantage of this technique is that it can sequence very long reads (more than 1000 bp!) which is very important in de novo sequence assembly. In addition, PacBio can also measure methylation of DNA sequences based on the kinetics of addition of base pairs (using the observation that modified base pairs tend to take longer to incorporate into a DNA strand). Furthermore, this technique can also potentially use a single molecule of DNA, which reduces any GC bias that occurs due to amplification. The final technique that will be discussed here is nanopore sequencing (Stoddart et al.). The idea behind this is that DNA may be threaded through a nanopore one base at a time. As its fed through, the sequencer can measure the change in current as it passes through (which will vary based on what base is moving through the pore). Thus, the sequence can be determined straight from the DNA without the need for modifications or reagents. In addition, because this can be done on a single molecule, there is again no need for amplification and thus no possibility of any GC bias in base calls.

Playing God in Today’s World Essay

The two fictional characters who â€Å"play God† in their own lives are two doctors, one of philosophy (Faustus) and one of medicine (Frankenstein) and, in real life, it is anyone who takes power over another’s and one’s own life and makes up his/her own rules which go against the commandments given to us by God as the athlete Lance Armstrong did during many years. First, there is Dr. Faustus, a very famous and successful scholar. He is a smart man who knows everything about almost all sciences in the world, including philosophy, theology, law, and medicine. Yet, this knowledge is not enough for him. He is greedy and obsessive about it and the more he learns, the more he desires. Soon, all that he knows makes him extremely bored; there is only one science that gives him excitement – magic. â€Å"These books by magicians- lines, circles and mysterious drawings- Yes, these are the books that I enjoy the most. Oh, what a world of power and reward they promise the hard-working student! With this knowledge, I’ll be the master of everything that moves on Earth†¦ this is your answer Faustus. Use your brain to become a god† (Marlowe 22). Faustus is willing to do anything to be the greatest in this field, so he decides to sign an agreement with the Devil and give him his soul in exchange for 24 years of magical talents and power over life itself. After this agreement is set, Faustus gets all the unimaginable power in life; learns all that he wants about this forbidden science; brings people back to life, and earns worldwide admiration and recognition for his work. Nevertheless, none of these God-like powers and tricks of magic bring him happiness. Deep in his heart he knows he is doing wrong and he will be damned forever. At last, he ends his days being lonely and is punished forever in hell. Clearly, Faustus tries to play God, but at the end, the only thing he does is to bring God’s anger upon himself. â€Å"With paper wings he flew too near the sun and heaven planned his downfall† (epilogue). Secondly, there is Victor Frankenstein, a man who has everything he needs plus a very happy family. He is a great man, who is dedicated to his studies and very clever, but he makes the same mistake as Faustus did. He plays God by trying to discover the secrets of life, and when he does, he uses them the wrong way. When his mother dies, he has to leave his sad family and go to university to study medicine. There, he becomes obsessed with the idea of knowing â€Å"the secret of life† and how to help others not to feel the sorrow he and his family did because of the loss of a loved one. Soon, after much study about death and life, he finds the answer to all the questions in his head and starts to develop a horrible project. â€Å"He takes parts from dead people and builds a new ‘man’. But this monster is so big and frightening that everyone runs away from him – even Frankenstein himself! (Comment by editor). Victor is so terrified of his own creation that the only thing he wants is to escape from it so, irresponsibly; he abandons his laboratory and the monster, which has no idea of how to speak, survive, or live. †The monster is like an enormous baby who needs love. But nobody gives him love, and soon he learns to hate. And because he is so strong, the next thing he learns is how to kill† (Comment by editor). The monster is full of resentment and decides to make Frankenstein just an unhappy and lonely as he has been his entire life. He kills all people Frankenstein loves and makes Victor look forward only to the day that he can be dead and together with his family. Clearly, Frankenstein tries to play God, but at the end, the only thing he does is to bring unhappiness and misery to his life and his loved ones’. Finally, there is Lance Armstrong, a successful and rich cyclist who has won the Tour De France seven consecutive times. He has a lot of fans around the world and is considered a strong and admirable man by many people. However, after years of arduous denials and attacks on any person who has questioned his clams of competing clean, he has been proved to use performance-enhancing drugs since 1998. Now, he is disqualified from all his titles since that year and banned from professional cycling for life. Fans, friends, and family have gone away from him and now he is left only with the money that he has made, but today this does not seem to make him happy. He did a lot of mistakes through his career and personal life and used his cancer to justify them to himself. My cocktail, so to speak, was only EPO, not a lot, [blood] transfusions and testosterone, which I almost justified because of my history. Obviously, the testicular cancer and losing [a testicle], [I] thought, surely I’m running low. †(Armstrong’s interview with Oprah). The first mistake that he makes is to forget about his spiritual values and to start violating God’s law. Victory becomes his obsession and he is wi lling to obtain it at all costs. He is not only loosing his soul, but also damaging his body and reputation. Moreover, he has given false witness in front of millions of people and fooled everyone who believes in him. Again in this case, it is clearly illustrated how â€Å"playing God† can destroy someone’s life. Considering all the above, it is evident how obsessions can lead people to self-destruction. Armstrong, Frankenstein, and Dr. Faustus have made up their own rules and ways of living, according to what they want and not caring about how they are damaging themselves and the ones around them. They are all men of successes until God decides that it’s time to pay for the mistakes they have done; and at that point, it is too late to repent. The three of them have forgotten that life is not about what we accomplish, but about the way we do it.

The Effects Urbanisation Is Having on Australias...

Today, this briefing note is put to you the National Taskforce on Environmental Science and Sustainable Development in response to Urbanisation and its effects on Australias’ waterways. This is fast becoming a critical environmental issue affecting Australia. It has long been known that plants act as a natural filter of water, removing all the properties that would harm the creatures that ingest it. A key issue facing developers today is an expanding population. With expanding urban sprawl vastly affecting the green space urban development, strategies continue to focus on removing the encroachment of land as a development strategy and going ‘up’. This essay will focus on the eutrophication of waterways within Australia and in†¦show more content†¦Ammonium is the form of nitrogen taken up most readily by phytoplankton because nitrate must first be reduced to ammonia before it is assimilated as amino acids in organisms (Geoscience Australia, 2013). The excessive level of certain nutrients in waterways is often linked to human activities. A comprehensive investigation on how nutrients in waterways were increasing, was undertaken by a body of individual scientists and their findings were as follows; - Run-off from agricultural areas; - Storm water and wastewater; - Turbidity and nutrient levels occur within Australia’s river systems, generally coincide in Australias river systems. A large proportion of the suspended sediment in Australian rivers results from vegetation removal, leading to gully and stream bank erosion and sheetwash. Phosphorus is overwhelmingly associated with such particulate loads (The National Land and Water Resources Audit, 2002). - Dentrification is a major sink for inorganic nitrogen in estuaries. Nitrogen loss due to denitrification can exceed 50% of the total nitrogen input, and is probably the main reason why nitrogen can sometimes be the limiting nutrient in estuaries (Berelson, W.M., Heggie, D., Longmore, A., Kilgore, T., Nickolson, G., Skyring, G. 1998). - Water column phosphorus concentrations have also been shown to increase under anoxic conditions (Webb, K.L. and DElia, C.F. 1980). This is because some of the iron oxyhydroxides that

How Are Proteins Transported Out Of The Nucleus - 805 Words

How are proteins transported out of the nucleus? Eukaryotic cells rely majorly on the aqueous pores of the nuclear envelope, the double membrane system surrounds the nucleoplasm, as a pathway for transportation of proteins between the nucleus and the cytosol. The process is commonly known as a kind of gated transportation as to the fact that the nuclear pore complexes (NPC) that are embedded in the aqueous pores serve as gates that only select certain molecules to pass. Proteins, as macromolecules, cannot directly diffuse through the pathway of NPCs due to the presence of the disordered region of channel nucleoporins; the bundles of the channel nucleoporins are compactly aligned in disarray in the central pore, and certain phenylalanine-glycine (FG) repeats, which are present on the bundles, are believed to associate via low-affinity, cohesive interactions to form a permeability barrier of the pore (Xu Powers, 2013) and stop macromolecules to pass through freely, thus it requires energy input and aids from other molecules to traffic proteins through NPCs. Proteins that are needed to move out of the nucleus are often referred as cargos; these proteins have a specific part of their amino acid sequences, nuclear export signals, that contains information to lead the protein to their desired destination, which is the cytosol. Proteins with their corresponding signals can be recognized by and bonded to a soluble protein, nuclear export receptors (Nakielny et al. 1999); theseShow MoreRelatedThe Role Of Proteins Of A Cell Range From Structural, Enzymatic And Hormonal Functions1345 Words   |  6 Pagesit can carry out the next process. Translation of the information collected from RNA is the next step. The information collected is transported to the protein. Certain proteins play specific roles so the body synthesizes the right protein for the specific job at certain time. Production of proteins is one of the most significant processes taking place in the cell. The role of proteins in a cell range from structural, enzymatic and hormonal functions. DNA is responsible for protein synthesis in allRead MoreThe Role Of Biology And Macromolecules1464 Words   |  6 Pagesdefining feature that separates eukaryotes from prokaryotes. 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