Dna

Background Information

DNA sequencing enables us to perform a thorough analysis of DNA because it provides us with the most basic information of all: the sequence of nucleotides. With this knowledge, for example, we can locate regulatory and gene sequences, make comparisons between homologous genes across species and identify mutations. Scientists recognized that this could potentially be a very powerful tool, and so there was competition to create a method that would sequence DNA. Then in 1974, two methods were independently developed by an American team and an English team to do exactly this. The Americans, lead by Maxam and Gilbert, used a “chemical cleavage protocol”, while the English, lead by Sanger, designed a procedure similar to the natural process of DNA replication. Even though both teams shared the 1980 Nobel Prize, 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. Dideoxynucleotides are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH). These modified nucleotides, when integrated into a sequence, prevent the addition of further nucleotides. (Speed, 1992).This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.

The Method

Before the DNA can be sequenced, it has to be denatured into single strands using heat. Next a primer is annealed to one of the template strands. This primer is specifically constructed so that its 3' end is located next to the DNA sequence of interest. Either this primer or one of the nucleotides should be radioactively or fluorescently labeled so that the final product can be detected on a gel (Russell, 2002). Once the primer is attached to the DNA, the solution is divided into four tubes labeled "G", "A", "T" and "C". Then reagents are added to these samples as follows:

"G" tube: all four dNTP's, ddGTP and DNA polymerase

"A" tube: all four dNTP's, ddATP and DNA polymerase

"T" tube: all four dNTP's, ddTTP and DNA polymerase

"C" tube: all four dNTP's, ddCTP and DNA polymerase

As shown above, all of the tubes contain a different ddNTP present, and each at about one-hundreth the concentration of the the normal precursors (Russell, 2002). As the DNA is synthesized, nucleotides are added on to the growing chain by the DNA polymerase. However, on occasion a dideoxynucleotide is incorporated into the chain in place of a normal nucleotide, which results in a chain-terminating event. For example if we looked at only the "G" tube, we might find a mixture of the following products:

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Figure 1: An example of the potential fragments that could be produced in the "G" tube. The fragments are all different lengths due to the random integration of the ddGTP's (Metzenberg).

The key to this method, is that all the reactions start from the same nucleotide and end with a specific base. Thus in a solution where the same chain of DNA is being synthesized over and over again, the new chain will terminate at all positions where the nucleotide has the potential to be added because of the integration of the dideoxynucleotides (Russell, 2002). In this way, bands of all different lengths are produced. Once these reactions are completed, the DNA is once again denatured in preparation for electrophoresis. The contents of each of the four tubes are run in separate lanes on a #HYPERLINK "http://www.bio.davidson.edu/Courses/Molbio/SDSPAGE/SDSPAGE.html"polyacrylmide gel in order to separate the different sized bands from one another. After the contents have been run across the gel, the gel is then exposed to either UV light or X-Ray, depending on the method used for labeling the DNA.

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Figure 2: This