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".

12/19/2012

     On this day, I learned about the various other methods of DNA seperating other then capillary electrophoresis. Of these several methods that I learned about, I was particularly interested about the method of gel electrophoresis.

     Gel electrophoresis is a method that is used to separate and then analyze DNA, RNA, and proteins on the basis of their size and charge. Particularly in the interest of my internship, it is often used to separate a mixed group of DNA and RNA fragments by length and to separate proteins by their charges. This process occurs in an agarose matrix. Agarose is a gelatinous mixture and it has a neutral charge and a lower degree of complexity--thus making it less likely to interact with biomolecules. Gels made from purified agarose have a relatively big pore size, making them useful for separating large molecules like DNA fragments. DNA is overall negatively charged and so when they are placed in a gel, an electric field is created across the very gel and so the negative DNA approaches the positive electrode while moving further away from the negative cathode. Shorter molecules move faster and travel further than the longer ones because they are able to move more easily through the pores of the gel. After the process, one will find that the different sized molecules have created distinct bands on the gel.  

    This method of DNA separation, however, is not commonly used by mentor as it is limited. For instance, the gel may melt during electrophoresis from the heat caused from passing a current across. In addition, comparing the relative quantity of the various molecules rely on the band darkness of the different spots in the gel. It's method consequently has a substantial degree of error. (That is why experiments are usually run several time to get viable results.) 

     Hopefully by the week I come back from winter vacation, my real mentor, Yolanda, will be back as well so that I may be able to start testing the various samples of DNA for myself!

http://m.youtube.com/?reload=9&rdm=mfca6b1q0#/watch?v=9f2VSyVhsGI&feature=related

<Above is a YouTube link demonstrating a slab gel separation.>

December 12

I was not able to go to my internship as my mentor Yolanda went home for the holidays.
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        Although I was not able to go to my internship on Wednesday, I decided to research more about the particular field of analytical science and about DNA separation, both in which my mentor (and thus I) is involved in.
        DNA isolation is an process that extracts DNA from other sources. There are different methods for doing this but what they all have in common is that they aim to separate DNA present in the nucleus of the cell from other cellular components. While researching online, I found that this process is crucial for genetic analysis, which is used for medical, scientific, or forensic purposes. In the field of medicine, this application is used for diagnostic purposes. To my fascination, I found that this process of DNA separation is important in the field of forensics as it allows investigators to recover DNA for identification of criminals, and the likes. While the area of my internship deals with the primary step of separating two DNA strands of similar length into separate pieces, I found it very fascinating to see how this type of research benefits the real world outside the long white coats and enclosed space called the laboratory.

reference: http://www.enotes.com/dna-isolation-methods-reference/dna-isolation-methods

December 5: Getting Started

Today, I learned all about DNA separation through the use of capillary electrophoresis.

The separation of DNA (not a double but a single strand) of the same length starts with synthesized DNA that is dyed so that the capillary machine detector can detect the separated DNA at the end of the process. Scientists are aware of how to separate DNA with different lengths, but what they do not know and what my internship is focused around is how to separate DNA of the same length. This can simply not be done by adding common separating polymers to the tube, but by adding one of the four bases (adenine, guanine, thymine, and cytosine) which helps to differentiate the speed at which the DNA sample hits the cathode end (due to the phosphate group attached to the DNA which gives it a negative charge). Why adding the bases helps separate the genes is unknown and there is currently a limit to how many parts the DNA can be separated into—as of now, it can be separated into 7 pegs.

After understanding all this, I was then explained to about the lab procedures that I would be performing. The graduate student that helped me, Yolanda, walked me through and we prepared the DNA test samples that will go in the machine for analysis. At the end of my time, I was amazed at how far I had come (and it was only the 2^nd time here) as just 2 weeks ago, I had no idea what capillary electrophoresis was and as to me, it sounded like something in another language. I am looking forward to my next meeting, as I will be able to apply what I have learned today and play an even greater role in preparing the test samples for the lab.

November 28

Was not able to go to internship due to schedule change.