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.

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".

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.

Mitosis - Cell Division

Mitosis

We all are aware of reproduction process in humans and in the animal kingdom. Have you ever thought about how reproduction takes place in the plant kingdom and in other eukaryotic organisms?

Plants and other eukaryotic cells do reproduce by separating the duplicated chromosomes and this process is known as mitosis. In this topic let's learn about mitosis and its various stages.

The basic mechanism of mitosis was described in early 1880s. The term mitosis was first coined by the scientist named "Fleming" in the year 1882. He studied and observed mitosis both in vivo and in stained preparation to study the process of cell division in unicellular animals.

Mitosis is generally defined as a type of cell division which occurs to form daughter cells, with same number of chromosomes as that of parental cells. This process is seen in all vegetative cells. In case of plants, mitosis is observed in all roots and shoot tip. In case of eukaryotic animals, mitosis is observed in organs related to the production of blood cells, skin cells, wound healing regions, etc.

STAGES OF MITOSIS:

Mitosis is divided in to six stages.

· INTERPHASE:

A cell during this stage of mitosis shows a clearly defined nuclear envelope, nucleolus and chromatin. We can also see the increased size of nucleolus. This phase is called as a resting period as division of cytoplasm and nucleolus does not take place. It is the longest phase of cell cycle which requires one to two days for completion.

· PROPHASE: (In Greek pro-first phase stage)

In this stage, the chromatin material of nucleus gradually condenses into distinct chromatin threads by using water. They gradually become thicker and shorter forming chromosomes. Nucleus and nuclear membrane gradually disappears at the end of prophase. It is the longest phase of cell cycle.

· METAPHASE:

In this stage of mitosis the chromosomes move towards the equator of the cell, by attaching themselves to the spindle fibers with the help of their centromere. At the end of metaphase the centromere of each chromosomes divides to reach each chromatids to have its own centromere. These chromosomes further become shortened and thickened. The spindle fibers formed are of two types, namely chromosomal and polar spindle.

· ANAPHASE:

By the separation of chromatids, daughter chromosomes are formed towards the opposite pole of the cell along with the spindle fibers. At the end of anaphase each set of daughter chromosomes reach their respective pole. It is the shortest phase of mitosis as it lasts for few minutes.

· TELOPHASE: (In Greek telo-end phase stage)

In this stage of mitosis, two groups of chromosomes are formed, one at each pole. These chromosomes undergo uncoiling and become thread like structures. At the end of telophase nucleolus and nuclear membrane reappears and cytokinesis takes place by giving rise to new two cells.

· CYTOKINESIS:

In this stage of mitosis the cytoplasm of a single cell is divided in to two separate daughter cells.

SIGNIFICANCE OF MITOSIS:

Mitosis cell division is the process by which single cells reproduce themselves and multicellular organisms grow. This process is replication or multiplication. Mitosis is also called as an equatorial cell division and it takes place only in unicellular animals.

How To Use A Test Cross

A test cross was defined in a recent article as a way to detect a real nature or ability (known to vary within limits) of a living organism by the way of a cross between that organism and a tester genetically neutral in its contribution to the common offspring. Here the question is to know in which case a test cross can be the best choice for a genetic analysis. And the answer is we rather use this cross when we want:

1. To discover the genotype of an individual with a dominant phenotype. With a single locus for example such an individual may be dominant homozygous or heterozygous and you cannot tell which one is true just by looking at its phenotype.


2. To discover how many types of gametes a double heterozygote produces and then see if genes are linked or not. The expectations are: two types of gametes, both parental, with the same frequency (this means both loci are linked but no cross over occurred during meiosis), four types of gametes with the same frequency (when both genes have an independent behaviour but you do not know if it is because of a real physical independence or because of a linkage on a wide distance between them so that every single meiosis is made with a cross over), or four types of gametes in two groups having different frequencies (which means both loci are linked on a short distance and only some meiosis are associated with a cross over, so that at the end we have parental gametes over numbering recombined gametes).


3. To discover how many types of gametes a triple heterozygote produces and then see if genes are all linked, and if so see the order in which they are linked, or if only two loci out of three are linked and then identify them and compute the genetic distance between them, or if the three genes behave independently. Here we expect the target individual to produce height types of gametes in the same amount each (which shows an independent behaviour, true or faked), height types of gametes in four groups with different frequencies (this is the genetically known sign of a linkage between three loci, with a high frequency for both parental gametes, lower frequencies for the two kinds of simply recombined gametes and a very low frequency for both double recombined gametes), or any other number of gamete types in between, including just two types of parental gametes 50% each (in the case the three genes are linked but some how there is no single cross over during meiosis).

Saving the Tuart Forest

The tall Tuart (Eucalyptus Gomphocephala) forest located in between Busselton and Capel in Western Australia's picturesque south west, is one of the rarest forest ecosystems in the world. The trees are named after the local Wardandi aboriginal people's name Too-art, and the forest is a diverse ecosystem and home to over 80 species of birds, reptiles, frogs, bats and many animals including number of endangered species, such as the Western Ringtail and Western Brushtail Possums, Chuditch or Native Cat and the Quenda or Southern Brown Bandicoot.

Before white settlement of the south west, Tuart woodland stretched 400km from north of present-day Perth to Dunsborough near Cape Naturaliste. Now after 175 years of white settlement, less than 30,000 hectares remain and in that, less than 10% of the original understorey.

The reasons for the decline are many, but 3 in particular seem to dominate:

Clearing. In a settlement of poor soils and only seasonal rainfall, the Tuart forest was at once one of the most fertile and so was cleared for farming and town sites and

Logging. Tuart was highly prized for its hard timber which was widely used for ship building, railway trucks, bridges, cog wheels for mills, flooring, stair treads and so on. By 1904 only 40,000Ha remained

Grazing. Begun as early as the 1830's most of the Tuart forest was leased and fenced by the early 1900's. Clovers and grasses were introduced whilst native plants thought to be poisonous to stock such as Zamias were cleared. Even after the Tuart forest was protected in 1918 in what became State Forest No 1, the grazing of cattle was still carried on under the tall trees. Although cattle have now been removed since the declaration of the 2049Ha National Park in 1987, Western Grey Kangaroos have taken their place multiplying to large numbers. High on their list of menu favourites are young Tuart trees!

Early in the 2000's there was a lot of protesting about a proposed mineral sands mine within the Tuart forest, with the predictable bumper stickers "Save the Tuart forest" and sit-ins, etc. Eventually the mine went ahead, but under strict guidelines and conditions (including re-forestation), one of which was that the miner undertake a comprehensive study to establish a list of, and to re-plant native understorey, as well as replacement Tuart trees. Mining has now been completed and rehabilitation has begun.

An observant visitor to the forest today might notice the absence of young Tuarts and the proliferation of weeds such as Arum Lily, and be tempted to draw the conclusion that once the old trees eventually die off, the Tuart forest would cease to exist.

So both the mining company on its old minesite, and the Department of Conservation (DEC) have begun programmes of replanting not only young Tuart trees, but also the range of understorey plants which the research indicated were endemic to the forest. The programmes would be a waste of resources if that was all there was to it, but the areas of rehabilitation have been fenced off with high fences to keep kangaroos out until the young trees and the other vegetation reach sufficient size to be able to survive the kangaroos appetite.

A new section of this can be seen on the site of what was an old Forests Department pine plantation near Inn the Tuarts Guest Lodge, the only accommodation in the Tuart forest, at the end of Rushleigh Rd, just off the Tuart Tourist Drive, about 7km north-east of Busselton city centre. Another visible rehab site is near the bird hide on the Vasse-Wonnerup estuary, accessed from the Spotlight Possum Walk near historic Wonnerup House, off Layman Rd.The old minesite is visible from Tuart Drive around 12 km from Busselton, but is not accessible to the public at this stage, although inspections may be possible by prior arrangement.

So by removing a pine plantation, clearing and sand mining and excluding the native kangaroos, the future of the Tuart forest is a little more assured.

Why Can't We Charge Up Human Cells With Energy Bypassing the Organic Process?

Back in 2000 there were some NASA scientists screwing around with lasers and they build a tiny little model aircraft out of balsa wood and Cellophane with receptors to allow for power absorption using a small laser. Once the laser powered up the batteries of the model airplane it could keep flying until it ran out of juice then of course you could decide if you really wanted to re-charge it again the same way.

Basically, the little model could in essence or in theory fly around forever, never landing to recharge. Interesting yes? Well, I have some other ideas I'd like to run by you as well.

You see, the other day, I was perusing MIT's top 35 superstar science graduates and ran across a gentleman named Ryan Bailey who has done some rather brilliant work with nano-particles used internally to help medical equipment see what's going on inside. The strategies are brilliant, but the applications are unbelievably amazing. How amazing?

Well, I asked myself the same question and came up with some new questions of my own. The first of many is actually the title of this article; "Why Can't We Charge Up Human Cells with Energy Bypassing the Organic Process?"

Now then, it's not my desire to fully explore the whole Scientology Theory or religion, rather, just borrow a few of L. Ron Hubbard's Science Fiction ideas and couple them with the cordless charging technology of for personal tech devices - then use vibrational frequencies to super charge the human cells at various levels and frequencies for optimal performance.

What if we used such a scheme to power up humans at a space colony? They would require far less food, although you'd still have to give them nutrients and be very careful not to over boost them with frequency bombardment melting the nerve endings for instance.

Yes, this idea is still on the back burner. It's just a potential future concept, which may or may not ever be feasible. Still, in theory, it should be possible right? Therefore we must quince this curiosity and fun research to see. We could experiment on cell tissue, mice, or 3-D scaffold printed tissue to try it out. If things worked we could progress the experiments from there.

Is this a potential component that would allow long-term space flight or space colonies to survive with drastically reduced food supplies? It may very well be, we don't know, but I speculate the answer is yes. Please consider all this and think on it.