The Need For Automated Sequencing, Refinements In Automation
The process of determining the order of nucleotides (A, C, G, and T) along a DNA strand is called DNA sequencing. Knowing the nucleotide sequence of a gene or region of DNA is important in studying relatedness between species and between individuals and for a better understanding of how genes function. Several techniques have been developed for "reading" the sequence of any particular DNA segment. One of these techniques was developed by Fred Sanger in 1977 and is called the chain termination method (or Sanger method). The essence of the technique is the creation of a set of DNA fragments that match the chain to be sequenced. Each fragment is one nucleotide longer than the last. By determining the identity of the final nucleotide in each fragment, the sequence of the whole chain can be determined.
The chain termination method makes use of special forms of the four nucleotides that, when incorporated at the end of a growing chain during DNA synthesis, stop (terminate) further chain growth. In four separate reactions, each containing a different terminator base (called a dideoxynucleotide), a collection of single-stranded fragments is made. These fragments all differ in length and all end in the dideoxynucleotide added to the particular reaction. Gel electrophoresis is then used to separate the fragments according to their length. By knowing which terminator base is associated with which fragment on the gel, the base sequence can be constructed.
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