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School of Natural Sciences

University Of California Merced

 

DNA Sequencing

 

DNA sequencing is the linear decoding of a stretch of DNA to determine the precise sequence of the nucleotides.

The method that is commonly used for sequencing is called the dideoxy method, also known as the Sanger method (named after its inventor Frederick Sanger).

The method involves synthesizing four deoxynucleotide triphosphates to create a DNA template which elongates as each new nucleotide is added to the 3’-OH group of the last nucleotide added. The critical role played by this method involves the synthetic nucliotides that lack the –OH at the 3’ carbon atom. Once a dideoxynucleotide triphosphate attaches to the growing DNA template, chain elongation stops due to the absence of a 3’-OH that allows attachment of another deoxynucleotide triphosphate.

The procedure involves a reaction mix of DNA to be prepared as a single strand template. The template is supplied with a mixture of all four deoxynucleotides (dATP, dGTP,dCTP, and dTTP) in ample quantities, a mixture of all four dideoxynucleotides (ddATP, ddGTP,ddCTP,and ddTTP), each in limiting amounts and labeled with a “tag” that fluoresces a different color on once scanned by the laser of the DNA sequencer, and DNA polymerase 1.

Since all four normal deoxynucleotides are present, chain elongation occurs normally until, by chance, a dideoxynucleotide is inserted by the DNA polymerase which terminates the elongation process. If there is a higher ratio of normal deoxynucleotides to dideoxy nucleotides, DNA strands will vary in length by hundreds of nucleotides before insertion of the dideoxy stops the process.

Once the incubation process has been completed, the DNA fragments are separated by length from longest to shortest as they travel through the polymer of the DNA sequencer. The resolution outcome is so good that the length of fragments can be one nucleotide apart. As the dideoxynucleotides migrate past the laser beam of the DNA sequencer, a fluorescence color is produced by each of the dideoxynecleotides. The result is a print out of the DNA sequence produced by the automatic scanner of the DNA sequencer.