Method for determining polynucleotide sequence variations

Abstract

A method of determining the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide.

What is claimed is:  
     
         1 . A method of determining the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide, comprising: 
 a) providing a sample of the first polynucleotide;    b) selecting a region of the first polynucleotide potentially containing the variation;    c) subjecting the selected region to a template producing amplification reaction to produce a first plurality of double stranded polynucleotide templates which include the selected region;    d) selecting a region of the templates potentially containing the variation;    e) producing a first family of labeled, linear polynucleotide fragments from both strands of the templates simultaneously by a fragment producing reaction including, 
 i) a set of at least two primers comprising a first primer and a second primer,  
 ii) at least four types of nucleotide triphosphates, comprising at least two different sets of two Watson-Crick-pairing bases, and  
 iii) two types of fragment terminators, comprising a first terminator and a second terminator;  
   where the first primer and the second primer flank the selected region of the template strands;    where the first primer has a first primer label and the second primer has a second primer label;    where at least a portion of one of the types of nucleotide triphosphates is labeled with a first nucleotide label;    where the first terminator and the second terminator are non-Watson-Crick-pairing;    where the first terminator is labeled with a first terminator label and the second terminator is labeled with a second terminator label;    where each of the first primer label, the second primer label, the first nucleotide label, the first terminator label and the second terminator label are all distinguishable from each other;    where each of the first family of fragments are terminated by either the first terminator or the second terminator at the 3′ end of the fragment; and    where the first family of fragments include at least one fragment terminating at each possible base, represented by the either the first terminator or the second terminator, of that portion of the selected region of both template strands flanked by a primer; and    f) determining the location and identity of the bases in the selected region of the first polynucleotide by detecting the first primer label, the second primer label, the first nucleotide label, the first terminator label and the second terminator label present in the fragments.