01/23/13

I could not go this Wednesday because of MLK day.
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 What is Electrophoresis in general?

Electrophoresis, according the Wikipedia is “the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.” Expressed in layman’s terms, this can be translated to the definition: the movement of electrically charged particles in a fluid under the influence of an electric field. This phenomenon was observed first by Ferdinand Frederic Reuss in 1807. He noticed that the application of a constant electric field resulted in clay particles mixed in water to migrate.

The electrophoresis of positively charged particles is called cataphoresis. In a similar sense, the electrophoresis of negatively charged particles is called anaphoresis. Electrophoresis is used anytime a separation of molecules is needed. For example, DNA electrophoresis is used to study the genetic makeup of plants, animals, and humans. This is an analytical method used frequently in molecular biology and medicine as it is applied for the separation and characterization of nucleic acids, proteins, viruses, small organelles, and the likes.

Electrophoresis. Retrieved from http://en.wikipedia.org/wiki/Electrophoresis.           

01/16/13

My ride to RPI was cancelled because of the snow.

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Capillary Zone Electrophoresis

Due to the fact that I could not go to my internship again, this week, I did my research on CZE. 

(While researching this topic, I realized over and over again how hard and challenging science is. Almost all of the scientific terms I had rarely heard of, and understanding what I was reading required a lot of research.)

The simplest form of CE is capillary zone electrophoresis (CZE), also known as free solution capillary (FSCE). This form is based on the differences in the charge-to-mass ratio. For this method of electrophoresis, all that is needed is a “well-chosen buffer”. Separation is able to take place because of the relatively simple interaction of the analytes with the pH of the buffer.

The CZE samples being analyzed move in the EOF (electroosmotic flow: “the movement of ions through a solute under the control of an applied potential") but then separate into different bands as a result in the differences in their electrophoretic mobilities, µ. (Electric mobility is the speed at which macromolecules pass through a matrix “in the presence of the electric field”)

The differences in µ make each analyte’s overall velocity slightly different. This difference in velocity is also known as separation.  

The steps required for CZE are very straightforward. First, one washes the capillary with buffer. Then, the sample—already dissolved in the same buffer—is injected and the EOF is established.  

Capillary zone electrophoresis is extremely useful for the separation of peptides and proteins since “complete resolution can often be obtained for analyses differing by

only one amino acid substituent.” While researching, I found it particularly interesting that this is very important in tryptic mapping where mutations and post-translational modifications can be found.

Beckman Coulter. Introduction to Capillary Electrophoresis. Retrieved from

https://www.beckmancoulter.com/wsrportal/bibliography?docname=360643-CEPrimer1.pdf.

Chasteen, T. F. Modes of Electrophoresis. Retrieved from http://www.shsu.edu/-chm_tgc/primers/pdf/CES.pdf.

01/09/13

I was not able to go to my internship because Yolanda [my mentor] was busy.
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        Although I could not go to my internship last Wednesday, I was able to learn about the history of capillary electrophoresis. Historically, DNA sequencing products were separated using gels (like those I have talked about in my previous post) that had to be manually poured between the two glass plates. Capillary electrophoresis was introduced as an novel and automated alternative to slab gel electrophoresis 30 years ago by Jorgensen. By 1989, Bob Brownlee and his coworkers introduced the first commercial instrument for quick and high resolution capillary electrophoresis separation. With the introduction of capillary electrophoresis, work has been greatly facilitated. Due to the reason that you don't need to pour the gels with this method, DNA sequence analysis has been made a lot easier. In addition, this method is far more time efficient as one is able to process more samples at once. The currently used routine Sanger based methods in conjunction with the capillary electrophoresis can directly sequence up to 1000 nucleotides in length in a single reaction. 
          Although there are a lot of positive aspects of the current technology, the cost of sequencing using capillary electrophoresis is quite expensive, costing around $0.02 per base. Just by itself, this number to some, might seem incredibly small and thus contradictory to my previous assertion. However, when calculated, the expense comes out to be $60 million dollars to sequence a human genome. (We are able to calculate this cost because it is known that one human genome contains 3 billion bases.) With the increasing importance of new sequencing and re-sequencing efforts, novel technologies have been arising to replace the currently used methods, promising orders of less expensive sequencing costs per genome--with the goal of $1,000 by the year 2014. According to writers of the article "DNA Sequencing by Capillary Electrophoresis"Barry L. Karger and Andras Guttman, "For the future of personalized medicine, especially for multifactorial diseases, everyone's genome will be sequenced...with a likely cost of several hundred dollars per genome".