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...
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