Dna Sequencing Essay, Research Paper

In 1980, Frederick Sanger won the Nobel prize in Chemistry along with his associateWalter Gilbert. They discovered a method for DNA sequencing that allows scientists torapidly determine the chemical structure of pieces of DNA. This method is known as dideoxy(or Sanger) sequencing. Sanger has said of his method to determine the sequence of DNAthat “. . . DNA is similar to a book. In a book, the information is encoded in the order of theletters 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 dideoxy method for sequencing DNA uses chemicals that slice the long moleculesof DNA at specific sites. This process is quite simple and rapid, and was an importantdiscovery that changed the field of genetic engineering. The dideoxy method uses a DNApolymerase1 to synthesize a new strand of DNA from the template; whose sequence is to bedetermined. This reaction is infected by the addition of a molecule that will cause the reactionto terminate. This is the factor that allows the sequence to be read. (Compton’s LivingEncyclopedia, “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 triphosphates (a combination of dATP, dTTP, dGTP, and dCTP)2 and thetemplate. In DNA replication, the reaction begins when a RNA primer joins the DNAtemplate. This primer has a free OH on the 3′ end that DNA polymerase can use to add morecomplimentary bases to the growing strand. As long as there is a free 3′ OH and enoughdeoxy-nucleotide triphosphates (dNTPs) around this polymerization reaction will continue. Once again, this is dependent on there being a free 3′ OH available upon which to add anotherbase. 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 isadded a new free 3′ OH is formed, the polymerase reaction proceeds. (Rosenthal, “FineStructure of a . . .”) However, in the dideoxy sequencing reaction this polymerization is terminated by theaddition of a low concentration of dideoxy-nucleotide triphosphates (ddNTPs). These arebases identical to the dNTPs, except that they lack the 3′ OH (See Figure 1). When one ofthese is incorporated into the strand, there is no longer a 3′ OH upon which to add the nextbase, thus DNA polymerase stops. This is called chain termination.Figure 1 Dideoxyribose (Wallace, The Search for the Gene) In order to determine the sequence of a strand of DNA, first the DNA must be clonedinto a plasmid, which then must be heated to seperate the two strands. Now, the primer islabelled with a radioactive tag. Finally the plasmid-primer is added to four tubes, eachcontaining DNA polymerase, all four dNTPs, and one single ddNTP, i.e. ddATP, etc3. Theconcentration of the ddNTP is kept low (approximately 1/10 of the concentration of itscorresponding dNTP) so that if the polymerase makes new strands from a large number of thesame template molecules, only occasionally will the polymerase incorporate the ddNTP andterminate 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 dATP, then all of the new strands made would terminate the firsttime a T was encountered after the primer. This would not allow us to see the sequence,because it is necessary to have the ddNTPs be incorporated only a small fraction of the time. If equal amounts of ddATP and dATP were added, then when the first T was encountered,there would be a fifty percent chance that a dATP will be incorporated, allowing thepolymerase to continue, and a fifty percent chance that a ddATP will be incorporated, causinga chain termination. The strands that added dATP would continue until they reached the nextT, when the polymerase would again either add a dATP and continue, or a ddATP andterminate. At this point, only a quarter of the templates would be left. At each new T, therewould be a statistical probability of one-half that the chain would be terminated. This showsthat the ratio of the concentrations of dATP and ddATP will determine the statisticalprobability of chain termination at each encountered T. (Wallace, The Search for the Gene)

However, at a very low concentration of ddATP, the polymerase will usuallyincorporate dATP and continue, only occasionally will it add a ddATP; with these strandsterminating. Over a large number of template molecules being used in the reaction, althoughthere is only a small chance that termination will occur at each T, there will be manytemplates that terminated at each of the T’s 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 fourtimes; once with a dideoxy base for each base in DNA (A, T, G, and C). After the reactionhas been performed using each of the different ddNTPs , they are then put throughpolyacrylamide 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 anautoradiograph is produced. (Encarta, “Nucleic Acids”) The chain terminations resulted in a staggering of fragments. It is these fragments thatare shown after PAGE. These fragments terminate at all of the different bases in thesequence. By looking at these fragments, you can see the entire sequence. For example, ifyou 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 ddATP is used and A* represents the ddATP incorporated intothe strand. These fragments are the examples of what could be expected. (The template isshown above each series of fragments, but in actuality the template is unknown. It is onlyafter PAGE that this sequence is revealed. See Figure 2’s explanation) Template –>A A T G A C T C C G T A A G G C GCompliments –>T T A* –>T T A G T C A* –>T T A G T C A G G C A* In the second tube, where ddCTP is used, the C* represents the spot where this wasincorporated. Template –>A A T G A C T C C G T A A G G C GCompliments –>T T A C* –>T T A C T G A G G C* –>T T A C T G A G G C A T T C* –>T T A C T G A G G C A T T C C* –>T T A C T G A G G C A T T C C G C* In the third tube, where ddGTP is the chain terminator, the G* represents the point ofincorporationTemplate –>A A T G A C T C C G T A A G G C GCompliments –>T T A C T G* –>T T A C T G A G* –>T T A C T G A G G* –>T T A C T G A G G C A T T C C G* In the fourth tube, ddTTP is used, and T* is used to show the point of insertion for theddTTP. Template –>A A T G A C T C C G T A A G G C GCompliments –>T* –>T T* –>T T A C T* –>T T A C T G A G G C A T* –>T T A C T G A G G C A T T* After these fragments have been separated by PAGE, they can be read from the bottomto give the sequence of the strand complimentary to the original template. By simplyreversing the sequence using the A to T, G to C, T to A, and C to G rule the original templatecan be quickly determined. So, if you had the autoradiograph shown below, whose sequenceis TTACTGAGGCATTCCGC, then you would finally know that the original DNA sequencewas 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 sequenceof genes has produced information about the promoter proximal regions and enhancers thatare used in the control of transcription and sequences important in RNA splicing, informationabout 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 bydetermining the exact sequence of nucleotides in molecules of DNA and RNA. Now that theentire process has been explained, here is a review of the entire process. (Wallace, The Searchfor the Gene) Figure 3 DNA Sequencing — Full Process (Rosenthal,”Fine Structure of a . . .”) In conclusion, the method of DNA sequencing known as dideoxy or Sangersequencing is important to the field of genetic engeeneiring and many other fields. Scientistshope 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 ofsome types of cancer and other genetically caused diseases to try and figure out why theyoccur and how they can be stopped. (Soderland, “Harvard Professor gets . . .”)