DNA Sequencing Basics
DNA sequencing, at its core, is the process of determining the order of genome bases (Adenine, Thymine, Guanine, and Cytosine) in a strand of DNA. Any process that analyzes and determines the specific order of these pairs (AT and GC) is considered DNA sequencing. Sequencing a small strand of DNA can be a fairly easy process mainly because of how quick it has become. However, sequencing an entire genome can be a long process because it requires the analyzer to break the genome up into several small strands and sequence those one by one. Thankfully, however, there are well-established methods that can be used to speed up this process.
Sanger Sequencing: Chain Termination Method
The Sanger sequencing method was introduced by Frederick Sanger in 1977 as the first real method of DNA sequencing . The method used most commonly for smaller strands of DNA (up to 900 base pairs) is known as the Sanger Sequencing (a.k.a chain termination) method. This method works through using the same process cells use in DNA replication (Polymerase Chain Reaction), but slightly modified. It uses another ingredient, dideoxy versions of the base pairs (ddATP, ddTTP, ddCTP, ddGTP) in order to terminate the chains created by the polymerase in in vitro DNA replication. The difference between these dideoxy nucleotides from the usual deoxy nucleotides is that the dideoxy nucleotides do not have a ‘hydroxyl’ group that is used to link other nucleotides to the chain, great for the separation process required in sequencing. All parts are added to a tube (DNA needing to be sequenced, polymerase, DNA nucleotides, and the dideoxy nucleotides --each separately colored-- but in a much smaller amount) and then heated to separate the strands and then cooled. The polymerase then begins synthesizing DNA starting at the primer, adding nucleotides until it adds a dideoxy nucleotide, at that point no other nucleotides can be added to the current chain so it ends. This is then repeated until every dideoxy nucleotide is in every position of the DNA in at least one reaction. After this process is over, the fragments are run through a long tube, a process called capillary gel electrophoresis. A laser is shown through the gel, illuminating the dyes used at the beginning of the sequencing. The sequence of the original DNA can be determined looking at one nucleotide at a time, so consider the DNA sequenced!
Sequencing Applications
DNA sequencing is applicable in more situations than you might expect. The CDC has been using DNA sequencing for the past several years in order to determine the different aspects of human disease (a real world example being the outbreak of ebola: see more here). A much more common application is found in cancer. The cancerous tumors can be sequenced, looking for genetic mutations to exploit through treatment. There is also the more obvious application of molecular biology, studying the proteins in the genome, giving information about possible diseases and phenotypes. Many forensic scientists also use DNA sequencing for forensic identification and paternity tests.
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Hi Will! Great post! You broke down and explained one of the most spectacular phenomena in Bio Chemistry, or in science as a whole, extremely well. I appreciate how you gave an overview of the topic in question, in this case DNA sequencing, and then broke it down into smaller more specific parts. I like how you started at the roots, Adenine, Thynine, Guanine, and Cytosyne, and then built on these roots, further explaining each and how they are involved in DNA sequencing. I appreciate your use of a visual aid, to help the reader further understand the science and order behind DNA sequencing. Although I did take Biology last year in school, I did not understand many of the concepts involving DNA very well. Your post has helped clear up any difficulties that I had understanding DNA sequencing last year. I really like how you included applications and real life examples of DNA sequencing being used to solve real world problems, furthering my understanding of the topic. Overall, I liked your post because it furthered my understanding of DNA sequencing and gave me real world examples of DNA sequencing being used.
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