Fluorescence-based dideoxynucleotide

Several modes of detecting a single base extension have been investigated, including measuring the incorporation of fluorescent, haptenated, or radioactive ddNTPs [6, 7] or gel electrophoresis based-detection of fluorescent primers extended by non-fluorescent nucleotides [8]. Recently, the Applied Biosystems SNapshot lYimer Extension Kit was introduced. In this assay, a primer is extended by one or more fluorescent- labelled dideoxynucleotides with subsequent detection in a fluorescent-based DNA sequencer. Several primers can be analysed within one lane of the DNA sequencer. 

Alternatively, oligonucleotides are arrayed onto the glass slide and a one-base minisequencing reaction is carried out on the slide with the arrayed oligonuleotides as the primer and sample PCR products as the template. SNP is typed by scanning the array to determine which fluorescent dideoxynucleotide has been incorporated. Again many SNPs can be typed at once by minisequencing from a multiplex of PCR products. 

A modification of Sanger s method has resulted in the commercial availability of automated DNA sequenators based on Sanger s use of dideoxy analogs of nucleotides. Instead, however, of tagging a primer with P, the purine and pyrimidine base portions of the dideoxynucleotides are each modified to contain a side chain that bears a different fluorescent dye, and all the dideoxy analogs are present in the same reaction. After electrophoretic separation of the products in a single lane, the gel is... 

Each terminating dideoxynucleotide is labeled with a fluorescent dye that gives a specific color depending on the base carried by that terminating nucleotide. (An alternate method is to label the primer, a short oligonucleotide sequence used to initiate replication of the specific DNA, with specific fluorescent dyes, instead of the dideoxynucleotide terminators, but the general method is the same.) One of the dye systems in use (patented by ABl) consists of a donor chromophore that is initially excited by the laser and which then transfers its energy to an acceptor moiety which produces the observed fluorescence. The donor is tethered to the dideoxynucleotide by a short linker. 

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