4. DNA Sequencing

The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA olignucleotide. The advent of DNA sequencing has significantly accelerated biological research and discovery. Several methods for rapid DNA sequencing have been devised in the past few years. One of them have gained a reputation of being particularly reliable and simple: Sanger’s Method. Sanger’s method became the standard because of its practicality (Speed, 1992). Sanger’s method, which is also referred to as dideoxy sequencing or chain termination, is based on the use of dideoxynucleotides (ddNTP’s) in addition to the normal nucleotides (NTP’s) found in DNA. With the many advancements in technology that we have achieved since 1974, it is no surprise that the Sanger method has become outdated. However, the new technology that has emerged to replace this method is based on the same principles of Sanger\\'s method. Automated sequencing has been developed so that more DNA can be sequenced in a shorter period of time. With the automated procedures the reactions are performed in a single tube containing all four ddNTP\\'s, each labeled with a different color dye. Cycle sequence is similar to PCR. It uses most of the same ingredients, follows the same basic procedure, and is done in a thermocycler as well. One key difference is that only one primer is used in each cycle sequencing reaction so that the amplification of product is linear, not exponetial. Another key difference is that dideoxynucleotides are used which interrupts the extention of the DNA strand when incorporated. There are three major steps in a sequencing reaction (like in PCR), which are repeated for 30 or 40 cycles. Denaturation at 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle). Annealing at 50°C : The primer is jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore. Extension at 60°C : This is the ideal working temperature for the polymerase (normally it is 72 °C, but because it has to incorporate ddNTP\\'s which are chemically modified with a fluorescent label, the temperature is lowered so it has time to incorporate the \\'strange\\' molecules. The bases (complementary to the template) are coupled to the primer on the 3\\'side (adding dNTP\\'s or ddNTP\\'s from 5\\' to 3\\', reading from the template from 3\\' to 5\\' side, bases are added complementary to the template). When a ddNTP is incorporated, the extension reaction stops because a ddNTP contains a H-atom on the 3rd carbon atom (dNTP\\'s contain a OH-atom on that position). Since the ddNTP\\'s are fluorescently labeled, it is possible to detect the color of the last base of this fragment on an automated sequencer. The oligo- and polynucleotide population is separated according to chain length by polyacrylamide gel electrophoresis. Such electrophoresis can take place in slab gels or in capillaries. Variations of this method have been developed for automated sequencing machines. In one method, called cycle sequencing, the dideoxynucleotides—not the primers--are tagged with different colored fluorescent dyes, thus all four reactions occur in the same tube and are separated in the same lane on the gel. As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded and the sequence is reconstructed from the pattern of colors representing each nucleotide in the sequence.


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