Tuesday, September 25, 2012

Historical Development of Automated Sequencing Using the Sanger Method

Autnmated sequencing has been a technique utilized since the early 1980s. Although the technology has changed dramatically since its first use, the basic chemistry is still commonly used today. It is based on four basic steps: purification of DNA, amplification using the polymerase chain reaction (PCR), separation by electrophoresis and analysis.
Sanger's Dye Terminator Chemistry
Although several methods were developed during the 1970s, the dye-terminator method invented by Fred Sanger is the accepted method used in automated sequencing. The amplification step in PCR combines a mix of raw DNA bases (dNTPs) and bases that cause termination (ddNTPs). The advantage of using ddNTPs is that a number of DNA products amplified during PCR terminate once the ddNTP is added. This creates a series of products that are different by a single base. The end product is a combined soup that contains products ranging from about 19 bases in length up to hundreds of bases, each different by a single base. On separation media, the total number of products would appear as a ladder. In addition, the ddNTPs are labeled to allow for detection.
Initially Radioactivity Was Used in the Dye-Terminator Method
Each ddNTP that represented one of the four DNA bases also contained a radioactive label. The amplified products were separated on media though electrophoresis. Then the media was removed and photographed to view the base sequence of the sample. However, the problem with this method was that there was no way to detect a difference between the ddNTP labeling a G base versus A, C or T. Therefore, it was necessary to amplify the sample in four separate reactions in which only one base ddNTP was present. One tube would terminate only when the sequence had the G base while the other three tubes labeled either A, C or T.
With this in mind, four separate reactions would be loaded separately on the media. Each base would appear as an incomplete ladder. The researcher conducting sequencing would need to draw a line across four separate lanes on the media in order to determine the sequence of all four bases.
Fluorescent Labels Replaced Radioactivity
The requirement of using four lanes to determine one sequence was soon replaced with fluorescent labels on the ddNTPs. Each ddNTP representing one of the four bases was labeled with a different fluorophore that would be detected as a different color: green for As, blue for Cs, red for Ts and yellow for Gs. The need for four lanes was eliminated. This expanded the capacity to sequence samples by four tiles.
In addition, a system capable of detecting the bases was also developed in the early 1980s, soon after Sanger developed the Dye-Terminator method. Samples were loaded on the same medium initially used for the radioactive method. Then they were fitted into a machine that would run electrophoresis. This provided a second advantage. It was no longer necessary to keep the base ladder on the gel for photographing later. Instead, as each band representing a base reached the end point on the media, the automated machine would photograph the color and send this information to a computer. Once complete, the media was simply discarded appropriately. This allowed more of the amplified products to be determined increasing capacity of the radioactive method an additional two times.
Automated Sequencing Equipment has Evolved
Separation of the radioactive products of DNA amplification was performed using a procedure called electrophoresis. Essentially a reagent called acrylamide was poured between two glass plates where it would polymerize into a gel-like matrix called polyacrylamide. The samples would be loaded into the top of the matrix and electrical current would cause the DNA to migrate through the gel. Small products migrate faster than large products when an electrical current is applied because they incur less resistance.
The same process was used when the first automated sequencers were developed. Over time this technology continued to improve so researchers could determine longer pieces of DNA and load more samples. Overall the technology changed very little until the invention of capillary sequencers. Thin glass capillaries replaced the bulky glass plates. It was no longer necessary to pour gels. Instead, a new polymer was injected automatically each time samples were to be loaded. Even better was the amount of time to run an average sample. Glass plate gel electrophoresis could take more than 12 hours to determine a sequence of 400 or 500 bases. Glass capillaries could do the same job in a little over 2 hours.
Like slab gel (glass plate) electrophoresis, capillary sequencing has continued to develop faster methods of sequencing more samples. The overall output is tremendous when compared to the original automated sequencers.
Today, science has continued to develop better methods for sequencing DNA. Next generation sequencing has the capacity to sequence an entire two megabase genome in a few days. This same job would require even the most high-tech Sanger sequencers months of preparation and processing. This does not mean automated sequencers will be replaced. There is still a great need to sequence shorter pieces of DNA at a substantially lower overall cost.

Thursday, September 13, 2012

How to Study Biology to Get an A-Plus

Biology is the study of life in its entirety. The growth of biology as a natural science is interesting from many points of view. One feature of this growth is changing emphasis. Initially it was description of life forms, identification, nomenclature, classification of all recorded life forms. In recent years, Physics and Chemistry have been applied to biology and the new science of Biochemistry and Biotechnology have become the dominant faces of Biology. Medicinal practice, green revolution and the advancement in biotechnology has made the presence of biology felt by the common man.
The key to success in Biology is hard work. It means management of time and energy. Biology is a vast subject which requires a great and clear-cut understanding of each topic. Trying to cram notes will never lead to success unless rigorous effort is made in comprehending them. Being a very vast subject, developing interest in it is very important for success. One must have a passion for nature, to understand how things have developed and marvel at the beauty of nature.
To do well at biology, both the quantity and quality of time spent on it are important. A substantial time commitment is required to understand the subject in its entirety. Many students are discouraged when they get unsatisfactory even after spending hours studying for tests,. This may happen when the studies done by them do not lead to comprehension but only cramming effort. The test requires one to integrate concepts from different lectures, and to apply these principles of biology covered in class to evaluate new situations during the test. High-quality work entails preparing for such questions. Preparing entails organizing the mass of new information in such a way that it helps you understand the way the concepts are related to each other
To be successful, a student must carry out lecture follow-up activity i.e. rewriting their lecture notes. This could be done by re-organizing the information studied during the session in a way that conforms to your mental "landscape." Better than rewriting your notes, it helps you to discern the patterns and relationships between concepts leaving no doubts.
The lecture follow-up activity would involve the following:
1. Make a list of the important concepts from the lecture.
2. Rank the concepts from most general to most specific.
3. Circle the concepts that are linked with a solid line.
4. Label the line with a linking phrase.
5. Work down the page, adding increasingly specific concepts and looking for cross-links, which should be drawn with dashed lines.
6. Do a second version for all the concepts with the goal to add formerly unnoticed cross-links and to organize the map so that it flows as logically and as clearly as possible.
Often students are not able to organize themselves in the correct manner and are not able to do well despite their best efforts. At this time, it is best to get expert help which can help the student put things in order.
To sum up all, the mantra to excel in biology is hard work and practice. The combination of these two can do wonders if done properly. The emphasis should always be on the deep understanding of the concepts. Further practice will enable development of right kind of approach required to answer questions during the exam. Amalgamation of a good understanding with a good approach to answer questions would lead to the path of - "SUCCESS".

Friday, September 7, 2012

The Fossil Record, A History of Life

When we think of keeping records, what comes to mind is a filing cabinet filled with a bunch of file folders with tax information or business records from years gone by. The fossil record is life's evolutionary filing cabinet, and it's full of genetic history that tells the tale of life and death on this planet in accordance with natural selection.
If you have ever been to the Grand Canyon or the badlands of Drumheller Alberta or even just the mountains or foothills, you will have seen some of the filing cabinet of the fossil record in the cliffs and hill sides. Some of what is seen there, may have been either an ancient lake or the very bottom of the ocean millions of years ago. When people climb or ski in the Rocky Mountains, they are actually standing on an almost 2 billion year old ocean floor that got pushed high up into the air as the Continental Divide plates shifted and squeezed together. When the tectonic processes of subduction, causes a continent to ride forcefully over an oceanic plate, or the convergence of two or more continents, a new formation of a mountain ranges occurs.
These events have pushed up huge mountain ranges, on the land, and in the seas, and many of these mountains, like the Mountains in the Appalachians, over long periods of time, have been almost completely worn away. In plate tectonics, one plate gets pushed upward from the ocean bottom, becoming the mountain tops and the other plates get pushed under, and back down into the earth's molten core. The event that formed the Rockies, took place somewhere around 85 million years ago, back in a time when life was just beginning to blossom on the planet, and there wasn't a vertebrate to be found anywhere.
Some of the earliest fossils ever found, have been found on mountain tops. It was a time before the mammals, and long before the dinosaurs thundered across the earth. The Rocky Mountains, or the Rockies, as we call them here in Canada, are the major mountain range in western North America. The Rocky Mountains stretch more than 3,000 miles from the northern British Columbia, in western Canada, to New Mexico. 
In a sense the fossil record is all around us, but until it gets "dug up" and filed, it officially won't count. This record of life is the accumulation of artifacts by the diligent hands of those amateurs and professionals in the field who put the "files in the folders" and record what they have found. Without human hands and minds, there would be just a jumble of old bones and shells, ancient rocks lying in a heap or packed away in crates in a museum's basement. It's only due to the people who sort and catalogue each piece that we finally get an image of how life evolved over the millennia!

The word "fossil" comes from the Latin word "fossus", which literally means, "having been dug up". The remains of animals and plants are also known as "zeolites". The study of fossils that have formed periodically over "geological time", which is the time, as looked at through the records of the planetary evolution itself, makes the study of Paleontology possible. The earth's crust shifts and slides constantly on a mantle of molten rock. The thin part of solid earth that we walk on and that oceans and all the life in them exist, has been changing for millions of years.
If it were possible to flip the pages of the fossil record like a motion picture, people would see the movement and surge of life across four billion years of geological time passing. As scientists record and piece more and more of the record together the "big picture" becomes that much clearer! But first human hands have to put every piece of stone, every imprint of every bit of life from the past, into some sort of order, from the chronological "heap" where the bones are now.
The fossils of the oldest forms of life are not actually in their original form. But instead they are merely a stone photocopy of what they were originally. If you can imagine, after the animal dies, in order for it to become a fossil, it must first fall in an area where water and or volcanic ash or sand can cover it. This is why not every animal or plant gets fossilized. Once the animal is covered completely the process of decay begins, but because it happens without the presence of air, it takes a very long time. As each minute bit of organic matter is lost it gets filled in with some of the sand or ash above it. Over time the organic matter is pretty much completely replaced with sand or ash and when it hardens, a fossil is formed!
The youngest fossils start appearing around the beginning of the Holocene Epoch, and the oldest ones on record are estimated during the Archaen Eon, approximately 3.4 billion years ago. Throughout human history people have tried to explain what fossils were and how they came to be where they were found. Most of the explanations were embedded in myth and folklore, and so have very little importance in modern times.
In China fossils bones were thought to have belonged to dragons, and were often ceremonially worshiped and put into medicines. The ancient Greeks were the first of people in history to realize that perhaps these belonged to creatures that lived in the sea. Later in human history, Leonardo Da Vinci would also proclaim that the fossils found had in the distant past been the remains of living creatures. Early Naturalists, the people who studied the nature of things around them, began to contemplate and develop a way to define and catalogue these discovered fossils. It wasn't until Darwin and his peers that links were drawn, and the tree of life began to become more visible. Charles Darwin described the "process of descent" with modifications caused by evolution. He described evolution as the adaptation of all living things to the natural pressures put upon them by their environment.
Since Darwin's time, discoveries have been made which pushes back the fossil record to between 2.3 billion and 3.5 billion years. Almost since the formation of the earth and its geological record! Today archaeologists have discovered that the fossil record shows how some species have remained essentially unchanged for millions of years. A species will only undergo major change, if its environment changes in a way which leaves it significantly less well adapted to survive. Without the fossil record people would all still be asking the age old questions regarding how humans got here.
Most of the fossils found during the Precambrian Period were microscopic bacteria or microfossils. However, macroscopic fossils are now known to have mostly originated during the late Proterozoic. This gives us a glimpse into the time when no mammals or fish lived on the planet. A record that shows that after the evolution of microscopic life, there was a surge of plant life that flourished in its ocean and perhaps even land environments for millennia before anything walked or swam on earth.
Perhaps not all things are disclosed by the fossil records science currently has, but then again, perhaps someday soon there will be revelations which will complete humanity's understanding of how life began in the first place. Perhaps it will help people to avoid the annihilations and extinctions of those long dead creatures that no human would truly understand, without the backdrop story revealed by the current fossil record. Today the fossil record is life's evolutionary epic that unfolded over four billion years on this planet and it will continue to unfold long after humans are gone.