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In 1980, Frederick Sanger won the Nobel prize in Chemistry along with his associate Walter Gilbert. They discovered a method for DNA sequencing that allows scientists to rapidly determine the chemical structure of pieces of DNA. This method is known as di deoxy (or Sanger) sequencing. Sanger has said of his method to determine the sequence of DNA that...
DNA is similar to a book. In a book, the information is encoded in the order of the letters of the alphabet. If you can read this information, you can understand the book... We[Frederick Sanger and Walter Gilbert] have worked out a method for reading DNA... It slike saying you can learn to read a book. (Soderland, Harvard Professor gets... ) The di deoxy method for sequencing DNA uses chemicals that slice the long molecules of DNA at specific sites. This process is quite simple and rapid, and was an important discovery that changed the field of genetic engineering.
The di deoxy method uses a DNApolymerase 1 to synthesize a new strand of DNA from the template; whose sequence is to be determined. This reaction is infected by the addition of a molecule that will cause the reaction to terminate. This is the factor that allows the sequence to be read. (Compton's Living Encyclopedia, DNA) To understand this reaction, we must first recall the basic mechanisms of DNAreplication. It is a polymerase reaction that makes a new strand of DNA from deoxy-nucleotide triphosphate's (a combination of d ATP, d TTP, d GTP, and d CTP) 2 and the template.
In DNA replication, the reaction begins when a RNA primer joins the DNA template. This primer has a free OH on the 3 end that DNA polymerase can use to add more complimentary bases to the growing strand. As long as there is a free 3 OH and enough deoxy-nucleotide triphosphate's (dates) around this polymerization reaction will continue. Once again, this is dependent on there being a free 3 OH available upon which to add another base.
These bases added are the compliments to the template strand sequence. That is, an Ain the template will join with a T, a G with a C, and vice versa. Since every time a base is added a new free 3 OH is formed, the polymerase reaction proceeds. (Rosenthal, Fine Structure of a... ) However, in the di deoxy sequencing reaction this polymerization is terminated by the addition of a low concentration of di deoxy-nucleotide triphosphate's (ddntp's). These arrases identical to the dates, except that they lack the 3 OH (See Figure 1). When one of these is incorporated into the strand, there is no longer a 3 OH upon which to add the next base, thus DNA polymerase stops. This is called chain termination.
Figure 1 Di deoxyribose (Wallace, The Search for the Gene) In order to determine the sequence of a strand of DNA, first the DNA must be cloned into a plasmid, which then must be heated to seperate the two strands. Now, the primer is labelled with a radioactive tag. Finally the plasmid-primer is added to four tubes, each containing DNA polymerase, all four dates, and one single dd NTP, i. e. dd ATP, etc 3. The concentration of the dd NTP is kept low (approximately 1 / 10 of the concentration of its corresponding d NTP) so that if the polymerase makes new strands from a large number of these template molecules, only occasionally will the polymerase incorporate the dd NTP and terminate in the sequence of the template. (Rosenthal, Fine Structure of a... ) It is very important that this concentration be kept low because if, for example, onlyddATP was added, with no d ATP, then all of the new strands made would terminate the first time a T was encountered after the primer.
This would not allow us to see the sequence, because it is necessary to have the ddntp's be incorporated only a small fraction of the time. If equal amounts of dd ATP and d ATP were added, then when the first T was encountered, there would be a fifty percent chance that a d ATP will be incorporated, allowing the polymerase to continue, and a fifty percent chance that a dd ATP will be incorporated, causing chain termination. The strands that added d ATP would continue until they reached the next T, when the polymerase would again either add a d ATP and continue, or a dd ATP and terminate. At this point, only a quarter of the templates would be left. At each new T, there would be a statistical probability of one-half that the chain would be terminated. This shows that the ratio of the concentrations of d ATP and dd ATP will determine the statistical probability of chain termination at each encountered T. (Wallace, The Search for the Gene However, at a very low concentration of dd ATP, the polymerase will usually incorporate d ATP and continue, only occasionally will it add a dd ATP; with these strands terminating.
Over a large number of template molecules being used in the reaction, although there is only a small chance that termination will occur at each T, there will be many templates that terminated at each of the Ts represented in the sequence. When this concludes, the result is a number of DNAs of varying lengths; dependent on where the Ts met a ddATPwhich caused a chain termination in the sequence. (Wallace, The Search for the Gene) In order to sequence an entire piece of DNA, this reaction must be performed four times; once with a di deoxy base for each base in DNA (A, T, G, and C). After the reaction has been performed using each of the different ddntp's, they are then put through polyacrylamide gel electrophoresis (or PAGE) which separates the DNA strands by length. This is then exposed to X-ray film (in a technique called autoradiography), and an autoradiograph is produced. (Encarta, Nucleic Acids) The chain terminations resulted in a staggering of fragments. It is these fragments theatre shown after PAGE. These fragments terminate at all of the different bases in the sequence.
By looking at these fragments, you can see the entire sequence. For example, iou wanted to find out the sequence of the piece of DNA that went AATGACTCCGTAAGGCG, you would get the these result frome each of the tubes. (Rosenthal, Fine Structure of a... ) In the first tube, where dd ATP is used and A represents the dd ATP incorporated intothe strand. These fragments are the examples of what could be expected. (The template is shown above each series of fragments, but in actuality the template is unknown. It is only after PAGE that this sequence is revealed.
See Figure 2 s explanation) Template >Gcompliments >T T A >T T A G T C A >A In the second tube, where dd CTP is used, the C represents the spot where this was incorporated. Template >Gcompliments >T T A C >T T A C T G A G G C >C >C >C In the third tube, where dd GTP is the chain terminator, the G represents the point ofincorporationTemplate >Gcompliments >T T A C T G >T T A C T G A G >T T A C T G A G G >G In the fourth tube, dd TTP is used, and T is used to show the point of insertion for theddTTP. Template >Gcompliments >T >T T >T T A C T >T >T After these fragments have been separated by PAGE, they can be read from the bottom give the sequence of the strand complimentary to the original template. By simply reversing the sequence using the A to T, G to C, T to A, and C to G rule the original template can be quickly determined. So, if you had the autoradiograph shown below, whose sequences TTACTGAGGCATTCCGC, then you would finally know that the original DNA sequence was AATGACTCCGTAAGGCG. (Wallace, The Search for the Gene) Figure 2 Sample Autoradiograph (Wallace, The Search for the Gene) The development of this method of determining the sequence of DNA has resulted inthe discovery of a tremendous amount of information. Specifically, determining the sequence genes has produced information about the promoter proximal regions and enhancers theatre used in the control of transcription and sequences important in RNA splicing, information about the amino acid sequences of proteins, and the discovery of introns.
Introns are pieces ofthe DNA that do not get used in determining the code of RNA. They were discovered by determining the exact sequence of nucleotides in molecules of DNA and RNA. Now that the entire process has been explained, here is a review of the entire process. (Wallace, The Search for the Gene) Figure 3 DNA Sequencing Full Process (Rosenthal, Fine Structure of a... ) In conclusion, the method of DNA sequencing known as di deoxy or Sangersequencing is important to the field of genetic engeeneiring and many other fields. Scientists hope to use this process to fully map out the genes of certain organisms, including humans. Scientists also hope to use methods similar to the one shown above to find out the genes of some types of cancer and other genetically caused diseases to try and figure out why they occur and how they can be stopped. (Soderland, Harvard Professor gets... )
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