Old School DNA Sequencing: Sanger Method

Before modern technology was available, DNA sequencing was much more tedious and difficult. In the 1950's Dr. Frederick Sanger determined the sequence of amino acids in protein.  This led to the understanding that DNA sequencing was collinear to the sequencing of amino acids. In the 1970's, Dr. Sanger developed a method to determine the exact sequence of nucleotides in a given gene. This method involved placing the DNA sample in  gel that was charred both negatively and positively. The longer strands of DNA would be pulled through the gel slower than the shorter strands, and the DNA separated into bands. The band positions were then "read" in order to determine DNA sequence. To stimulate this, we were given a lab packet in which DNA from four different subjects were tested. We were asked to read each sequence, compare them, and finally determine which subjects carried a disease. Pictures from the lab are posted below. 

In the step above, we determined the sequencing of each subject's DNA by evaluating the bands. This data was then placed in the chart pictured in the last image. 

After comparing the data, it became clear that "Norm" was the only healthy patient. All other subjects showed some disturbance in their DNA that caused them to be effected by the disease. Carol experienced a front shift mutation, Bob experienced a truncation mutation shift, and Abby experienced point mutation. This disease would be unrecognizable were it not for the Sanger Method.

DNA Extraction Lab

As we learn more about DNA, it is great to apply the concepts we learn so that we can gain an even deeper understanding. In this post, I am going to describe a lab we did in class in which we extracted DNA from wheat germ. In order to do this, our instructor had us work in groups so that we could collaborate our knowledge.

To begin the lab, we measured out 1 gram of raw wheat germ. This germ was then placed in a 50 ml test tube where it was mixed with 20 ml of hot tap water for 3 minutes. After being stirred, 1/4 teaspoon of detergent was added to the mixture. This was again stirred. 5 minutes after adding the detergent, we added 14 ml of alcohol. This was done very carefully so that the alcohol would not mix with the germ mixture. This was necessary in the extraction process, as DNA precipitates at the water-alcohol interface. The image below shows this step being done.

After doing this, the test tube had to sit for a few minutes so that the DNA could precipitate. After a short wait, we collected the DNA by using a wooden stick.

Overall, this was a really fun lab that expanded my knowledge of DNA.

From DNA to Protein: The Central Dogma

As we are aware, DNA is found in protein, but how does it get there? After all, DNA is located in the nucleus of all cells, and ribosome build protein. In order to cross the nucleus membrane in order for protein to be made, there has to be a step in-between that will transfer the DNA code to the ribosome, but what is that step? The answer lies in the process of the central dogma, and the material RNA. RNA, as you can predict, is very similar to DNA. Instead of having dioxyribose material, like in DNA, RNA contains ribose. As we will learn, RNA will be the key component to the central dogma, allowing protein to be successfully made. For now, we are going to define a few terms related to this process. To view the vocabulary, please click here.

DNAi Timeline

Like most things, DNA would not be as well understood as it is today without the contributions from multiple researchers. This post is going to detail the work from some of these contributing scientists that helped develop the current understanding of DNA. A great resource for information on other scientists that contributed work to the evolution of the idea of DNA can be found at this address. This is also the resource that was used for this post.

Martha Chase, Alfred Hershey, and the "Blender Experiment"
In the early 50's, a group of researchers at the Cold Spring Harbor Laboratory were researching bacteriophage genetics (also referred to as phage genetics). Phage are viruses that target, attack, and infect bacteria. At this time, it was already known that phage was constructed with an outer casing of protein that surrounded in inner core of DNA. It was also known that phage relied on bacteria to reproduce, and that phage attached themselves to bacteria by their tails. It was hypothesized that after attaching, the phage pumped genetic material into the bacterial host, causing the bacterial enzymes to be replaced by new phage particles. In 1952, Martha Chase and Alfred Hershey dedicated their work to discovering why the bacteria transformed into a phage producing organism, proving whether or not DNA was a transforming principle. Through chemical analysis, it was discovered that DNA contained high amounts of phosphorus and zero amounts of sulfur. This information was in contrast to the known information about protein, which contains sulfur but no phosphorus. This information was used in an experiment to test which component entered the bacteria for infection. This was designed in a way that radioactive phosphorus (32P) and radioactive sulfur (35P) to selectively label the phage DNA and protein. The radio labeled phage was then combined with unlabeled bacteria so that the phage could attach. The attachment was then disrupted as the culture was mixed in a Waring blender. The samples were then spun in a centrifuge in an attempt to separate the phage from the bacteria. This attempt was successful, and due to the fact that the phage is lighter than the bacteria, it remained suspended in the test tube while the bacteria collected at the bottom in the form of a pellet. After evaluating the sample 35P, it was discovered that the newly produced phage from the bacteria did not contain any radioactive sulfur, unlike the protein coat of the parent phage.  In contrast, the sample 32P contained newly produced phage from the bacteria that was contaminated with radioactive 32P. From this, it was determined that phage DNA was used inside the bacteria to produce new phage, confirming that DNA is the genetic material.

http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/Hershey_Chase_experiment.png/357px-Hershey_Chase_experiment.png