Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate water soluble macro molecules like DNA, RNA, Proteins depending on their molecular weight and charge. For DNA molecules, the main factors determining the separation is molecular weight. But for protein molecules, iso-electric point and charge is also important.

The samples are placed inside wells created in the gel and electric field is applied across the gel matrix. The samples will then move in either direction or towards one direction depending on the above factors. What ever the type of electrophoresis, the mechanism and the basis is the same.

There are mainly two types of electrophoresis techniques.

Agarose Gel Electrophoresis

Most frequently used technique is Agarose Gel Electrophoresis. The Gel matrix is made using Agarose, which is a purified form of agar. Agar is extracted from seaweed or by human waste. A buffer is used when making the gel, and the gel is embedded in this buffer at the time of electrophoresis. This will enable the electric current to pass through the solution in the form of ions.

There are three commonly used buffers,

1. Tris - EDTA

2. Tris - Acetate

3. Tris - Borate

Below is a Electrophoresis machine with a casted gel. There are wells created inside the gel. In this example the wells are created near the (-) electrode, because we are using DNA as our samples. But if you are using Proteins, the wells should be created at the center of the gel, because net charge can be either (-) or (+).

Then when loading the samples, our samples have to be mixed with a dye, so that we can observe the movement of samples through the gel. The dye that is been used here moves through the gel equivalent to a nucleotide strand of 200bp.

Then as seen in the picture a voltage should be given to create an electric field, which will enable the samples to move towards the (+) electrode.

This can be used to separate nucleotide strands of 100bp to 20,000bp. It has a limited resolution. (resolution is the ability of the gel to separate nucleotide strands of different lengths clearly). To gain a higher resolution, we can increase the agarose concentration, which will increase the Resistance through the gel. But since the resistance will also increase, the time taken to run the gel will also be increased from hours to days.

After running the gel, we can see the separation under UV light.

For tasks that require higher resolution (greater separation), PAGE (poly acrylamide gel eletrophoresis) is used. PAGE can clearly separate nucleotide fragments to show one base pair (1bp) difference. It is used in tasks like DNA, RNA sequencing.

BiotechPracticals

DNA Extraction

DNA extraction is one of the basic steps in genetic engineering or modern biotechnology. Biotechnology in anyway is not a new science. Our anscesters have been using biotechnology for thousands of years. Biotechnolgy is any technology involving living organisms or biological products made by those organism. When humans manupulate biological processes within these organisms we call it biotechnology. So this is a very broad area. It has branches in Medicine, Agriculture, Industry and Environment.

Biotechnology over the years have evolved into more sophisticated science and is one of the most talked subjects in the world. No matter what field of biotechnolgy you are involved DNA extraction has same basic principles.

There can be many sources for DNA.

ex:

plant sources: grains, rice germs, plant leafs, any other suitable plant product.

if you want a bulky extract it will be nice to use some thing like strawberry, which gives a high yield.

animal sources: Blood cells (WBC), any other animal tussue.

WBC is taken because it is easy to obtain.

note: When you take a blood sample, there are RBC:WBC in the ratio of approximately 300:1. So first we will have to remove RBC from the sample. To do that RBC are lysed by a freez shock. Then only cells to be remained in the sample are WBCs. But if you are using avian blood samples, there's no need for freeze shock as the RBC of avian blood contain nuclei.

Once we obtained the sample form the source, following have to be done.

1. Remove the cell membranes of the cells and get the intra cellular constituents in to the solution.
2. Degrade the Proteins and cell debris to release DNA
3. Separate DNA from other bio molecules (proteins, AA, ex..)
4. Purify DNA sample

There are many ways to achieve these goals with varying degree of purity to our DNA sample. You can even complete these steps using some of the house hold chemicals. But the purity of the sample will be far less than when you do it in the lab under proper protocols.

Today the theory and the procedures behind DNA extraction, Visualisation of DNA, Electrophoresis of DNA and quantification of DNA sample is done. Tomorrow we will be doing the DNA extraction practical followed by the Electrophoresis of DNA.

How did people find out that DNA is the genetic material

Genetic material is the core factor that determine the character of an organism. So to prove that DNA is the genetic material, it should be proved that DNA is responsible for the changes in an organism.

In 1928 Griffith did a simple experiment with pneumococci bacteria to prove the above. He selected a smooth strain (virulent) and a rough strain (non-virulent).

First He injected smooth strain to mice and all the mice died.

Then he injected rough strain to mice and all the mice tolerated it well.

Then he heat killed smooth strain and injected it to mice. Mice lived fine. When treated with heat, proteins get denatured and they loose their three dimensional shape and function. This step proves that the factor immediately responsible for virulency of the smooth strain is proteins.

Then he mixed heat killed smooth strain and live rough strain and cultured it in a new medium.

Then he extracted a sample from the culture and injected it to the mice. mice died. This Step shows that the core factor that was responsible for the virulency of smooth strain has some how transferred to the rough strain.
Now the character of rough strain or the virulency of rough strain has changed. Griffith termed this phenomenon as TRANSFORMATION.

When the DNA is extracted from the heat killed smooth strain and injected to mice, mice died. When extracted DNA is mixed with rough strain, the virulency of rough strain increased. This results proves that DNA is the component responsible for characteristics (in this case, virulency).

Hershay and Chase helped to establish the fact. They proved that only when phage DNA enter the host bacterial cells, new phage particles are formed.

Number 42

Today one of my friends asked me to google for,

"the answer to life, the universe, and everything"

See for your self, google calculator gives "42". Isn't it great.

haha it seems that google has unlocked the mistry of universe.

i searched for this some more and found some interesting facts.

• The number 42 is The Answer to the Ultimate Question of Life, the Universe, and Everything, according to Douglas Adams' series The Hitchhiker's Guide to the Galaxy. According to the fifth Hitchhiker volume, Mostly Harmless, 42 is the location of Stavromula Beta. Thus, 42 may be the world's longest written riddle, since the riddle of the question to the answer was raised in the first volume, and not answered until the final page of the fifth, and then passes unnoticed by the story's ever-bumbling characters.

It took "deep thought" 7.5 million years to compute the answer, but google does it in 0.03 seconds!!

keep it up google!

Biometrics: Unlocking Doors With Your Eyes

Research by Ms Phang, from QUT's Faculty of Built Environment and Engineering, is helping to remove one of the final obstacles to the everyday application of iris scanning technology.

Ms Phang said the pattern of an iris was like a fingerprint in that every iris was unique. "Every individual iris is unique and even the iris pattern of the left eye is different from the right. The iris pattern is fixed throughout a person's lifetime" she said.

"By using iris recognition it is possible to confirm the identity of a person based on who the person is rather than what the person possesses, such as an ID card or password.

"It is already being used around the world and it is possible that within the next 10 to 20 years it will be part of our everyday lives."

Ms Phang said although iris recognition systems were being used in a number of civilian applications, the system was not perfect. "Changes in lighting conditions change a person's pupil size and distort the iris pattern," she said.

"If the pupil size is very different, the distortion of the iris pattern can be significant, and makes it hard for the iris recognition system to work properly."

To overcome this flaw, Ms Phang has developed the technology to estimate the effect of the change in the iris pattern as a result of changes in surrounding lighting conditions. "It is possible for a pupil to change in size from 0.8mm to 8mm, depending on lighting conditions," she said.

Ms Phang said by using a high-speed camera which could capture up to 1200 images per second it was possible to track the iris surface's movements to study how the iris pattern changed depending on the variation of pupil sizes caused by the light. "The study showed that everyone's iris surface movement is different."

She said results of tests conducted using iris images showed it was possible to estimate the change on the surface of the iris and account for the way the iris features changed due to different lighting conditions.

"Preliminary image similarity comparisons between the actual iris image and the estimated iris image based on this study suggest that this can possibly improve iris verification performance."

Adapted from materials provided by Queensland University of Technology

Genetic Engineering - Intro

Biotechnology is technology based on biology, especially when used in agriculture, food science, and medicine. The United Nations Convention on Biological Diversity has come up with one of many definitions of biotechnology.

Biotechnology is often used to refer to genetic engineering technology of the 21st century, however the term encompasses a wider range and history of procedures for modifying biological organisms according to the needs of humanity, going back to the initial modifications of native plants into improved food crops through artificial selection and hybridization. Bioengineering is the science upon which all Biotechnological applications are based. With the development of new approaches and modern techniques, traditional biotechnology industries are also acquiring new horizons enabling them to improve the quality of their products and increase the productivity of their systems.

Before 1971, the term, biotechnology, was primarily used in the food processing and agriculture industries. Since the 1970s, it began to be used by the Western scientific establishment to refer to laboratory-based techniques being developed in biological research, such as recombinant DNA or tissue culture-based processes, or horizontal gene transfer in living plants, using vectors such as the Agrobacterium bacteria to transfer DNA into a host organism. In fact, the term should be used in a much broader sense to describe the whole range of methods, both ancient and modern, used to manipulate organic materials to reach the demands of food production. So the term could be defined as, "The application of indigenous and/or scientific knowledge to the management of (parts of) microorganisms, or of cells and tissues of higher organisms, so that these supply goods and services of use to the food industry and its consumers.

Closest Look Ever At Native Human Tissue

"This is a real breakthrough in two respects," says Achilleas Frangakis, group leader at EMBL. "Never before has it been possible to look in three dimensions at a tissue so close to its native state at such a high resolution. We can now see details at the scale of a few millionths of a millimetre. In this way we have gained a new view on the interactions of molecules that underlie cell adhesion in tissues -- a mechanism that has been disputed over decades."

So far, the only information available about a protein's position and interactions in a cell was based on either light microscopy images at poor resolution or techniques that remove proteins from their natural context. Frangakis and his group have been developing a technique called cryo-electron tomography, with which a cell or tissue is instantly frozen in its natural state and then examined with an electron micro-scope.

Electron microscopy normally requires tissue to be treated with chemicals or coated in metal, a procedure that disturbs the natural state of a sample. With cyro-electron tomography, images are taken of the untreated sample from different directions and assembled into an accurate 3D image by a computer.

The researchers applied this technique to observe proteins that are crucial for the integrity of tissues and organs like the skin and the heart, but also play an important role in cell proliferation. These proteins, called cadherins, are anchored in cell membranes and interact with each other to bring cells close together and interlink them tightly.

"We could see the interaction between two cadherins directly, and this revealed where the strength of human skin comes from," says Ashraf Al-Amoudi, who carried out the work in Frangakis' lab. "The trick is that each cadherin binds twice: once to a molecule from the juxtaposed cell, and once to its next-door neighbour. The system works a bit like specialised Velcro and establishes very tight contacts between cells."

The new insights into the cadherin system broadens the understanding of structural aspects of cell adhesion and shed light on other crucial processes such as cell proliferation. The technical advances achieved in cryo-electron tomography of frozen sections open up new possibilities to study more systems at native conditions with molecular resolution.

Adapted from materials provided by European Molecular Biology Laboratory

Global Climate Change: The Impact Of El Niño On Galápagos Marine Iguanas

According to the authors, recurring El Niño events provide an ideal system to study the impact of human-mediated climate change on ecosystems worldwide, by allowing observation of changes in populations associated with individual events.

"Since global warming is expected to cause an increase in the strength and frequency of El Niño events, it is important to evaluate the impact of El Niño on natural populations and their capacity to respond to environmental stresses," said Gisella Caccone, senior research scientist in ecology and evolutionary biology at Yale, and senior author of the paper published recently in the Public Library of Science One.

In this study, the researchers investigated the effect of sea surface warming associated with the single, intense El Niño event of 1997 to 1998 on genetic diversity in Galápagos marine iguana populations. They found that populations within the same species responded very differently.

Collaboration between German scientists who fortuitously collected and archived samples between 1991 and 1993 and Yale researchers who sampled the iguanas in 2004 enabled these unique before-and after comparisons. More than 800 samples from 11 Galápagos marine iguana populations were evaluated.

The researchers looked for changes in levels of genetic variation in nuclear microsatellite DNA and mitochondrial DNA markers of samples collected before and after this El Nino event. Changes in microsatellite frequency are sensitive enough to detect low population sizes before there is a significant loss of diversity in a population, whereas mitochondrial markers only show losses of diversity.

Caccone said that although some populations had mortality rates of up to 90%, only one population from a single island showed strong evidence of a "genetic bottleneck," suggesting that the El Niño-induced disturbance affected populations very differently even within the same species.

"Our study points out that there was a low population size on the island of Marchena during the same period in which there was serious volcanic activity as well as the El Nino," said Yale graduate student Scott Glaberman, a principal author of the study. "Since both of these forces could have acted on the population, it shows the importance of knowing the major forces influencing survival and reproduction to best interpret the results of genetic tests.

"The most striking result of our study is that although marine iguanas on some islands had a rather high mortality due to El Niño, the genetic consequences were mostly absent," said author Sebastian Steinfartz a postdoctoral fellow. "This unique study shows that natural populations may be able to balance even severe short term climatic disturbances, and that such fluctuations will not necessarily have long-term negative consequences on the population structure."

"Basic surveys and ecology of organisms are not the most glamorous aspects of research, but if used together with the exciting genetic approach in our study, they make for a very powerful approach," said Glaberman.

"This underscores the importance of having baseline studies for any population of interest, and the value of archiving samples for the use of future research," Caccone said. "It sets the basis for future research to determine which environmental and biological factors make specific populations more vulnerable than others."

Other authors on the study are Deborah Lanterbecq at Yale, Cruz Marquez at the Charles Darwin Research Station, Santa Cruz, Galápagos, Ecuador and Kornelia Rassmann in Germany. Granting from the National Geographic Society, the German Research Community, the Belgian American Educational Foundation, and the Yale Institute for Biospheric Studies supported the research.

Citation: Public Library of Science (PLoS): December 12, 2007.

Adapted from materials provided by Yale University

Using Carbon Nanotubes To Seek And Destroy Anthrax Toxin And Other Harmful Proteins

Scientists have long been interested in wrapping proteins around carbon nanotubes, and the process is used for various applications in imaging, biosensing, and cellular delivery. But this new study at Rensselaer is the first to remotely control the activity of these conjugated nanotubes.

A team of Rensselaer researchers led by Ravi S. Kane, professor of chemical and biological engineering, has worked for nearly a year to develop a means to remotely deactivate protein-wrapped carbon nanotubes by exposing them to invisible and near-infrared light. The group demonstrated this method by successfully deactivating anthrax toxin and other proteins.

"By attaching peptides to carbon nanotubes, we gave them the ability to selectively recognize a protein of interest -- in this case anthrax toxin -- from a mixture of different proteins," Kane said. "Then, by exposing the mixture to light, we could selectively deactivate this protein without disturbing the other proteins in the mixture."

By conjugating carbon nanotubes with different peptides, this process can be easily tailored to work on other harmful proteins, Kane said. Also, employing different wavelengths of light that can pass harmlessly through the human body, the remote control process will also be able to target and deactivate specific proteins or toxins in the human body. Shining light on the conjugated carbon nanotubes creates free radicals, called reactive oxygen species. It was the presence of radicals, Kane said, that deactivated the proteins.

Kane's new method for selective nanotube-assisted protein deactivation could be used in defense, homeland security, and laboratory settings to destroy harmful toxins and pathogens. The method could also offer a new method for the targeted destruction of tumor cells. By conjugating carbon nanotubes with peptides engineered to seek out specific cancer cells, and then releasing those nanotubes into a patient, doctors may be able to use this remote protein deactivation technology as a powerful tool to prevent the spread of cancer.

Kane's team also developed a thin, clear film made of carbon nanotubes that employs this technology. This self-cleaning film may be fashioned into a coating that -- at the flip of a light switch -- could help prevent the spread of harmful bacteria, toxins, and microbes.

"The ability of these coatings to generate reactive oxygen species upon exposure to light might allow these coatings to kill any bacteria that have attached to them," Kane said. "You could use these transparent coatings on countertops, doorknobs, in hospitals or airplanes -- essentially any surface, inside or outside, that might be exposed to harmful contaminants."

Kane said he and his team will continue to hone this new technology and further explore its potential applications.

Details of the project are outlined in the article "Nanotube-Assisted Protein Deactivation" in the December issue of Nature Nanotechnology.

Co-authors of the paper include Department of Chemical and Biological Engineering graduate students Amit Joshi and Shyam Sundhar Bale; postdoctoral researcher Supriya Punyani; Rensselaer Nanotechnology Center Laboratory Manager Hoichang Yang; and professor Theodorian Borca-Tasciuc of the Department of Mechanical, Aerospace, and Nuclear Engineering.

The group has filed a patent disclosure for their new selective nanotube-assisted protein deactivation technology. The research project was funded by the U.S. National Institutes of Health and the National Science Foundation.

Adapted from materials provided by Rensselaer Polytechnic Institute.

Bio Technology

I selected to do Bio Technology for my electives, So i'll have to learn something on biotech. Ok, let's start with some definitions.

According to Oxford medical dictionary it is,

"the development of techniques for the application of biological processes to the production of materials of use in medicine and industry"

NIH gives following definition,

"A set of biological techniques developed through basic research and now applied to research and product development."



So it's all about finding new solutions to old problems. that's cool isn't it.