Patent ID: 6251637
Filing Date: 2001-06-26
Classification: C12N,C12Q

Abstract:
In a method for cycle sequencing of DNA in the form a 1-lane technique using dideoxynucleotides labeled with a dye as a marker, wherein sequence ladders of more than 500 bases are obtained, comprising the steps of:a) mixing at least a reaction buffer, deoxynucleotides, the marker-labeled dideoxynucleotides, primers and double stranded DNA template, water and optionally further additives and adjusting the concentration thereof in relationship to each other;b) adding at least one extremely thermostable DNA polymerase,c) performing PCR sequencing cycles comprising the steps of:denaturing the template DNA at an appropriate temperature;annealing the primer at an appropriate temperature;chain elongation at an appropriate temperature; andadjusting the ramping time between the individual temperature stages in dependence on the DNA polymerase, template/primer pair and reaction buffer used;d) purifying the reaction sample; ande) separating the DNA chains and detecting to determine the DNA sequence; the improvement comprising:(i) in step c) performing the chain elongation at a temperature of 65.degree. C. to 75.degree. C.;(ii) depending on the primer/template pair and DNA polymerase used, adjusting the following conditions in relationship to each other to enable chain elongation in a temperature range of 65.degree. C. to 75.degree. C.:concentration of metal ions;concentration of nucleotides;concentration of DNA polymerase; andthe conditions for primer annealing as well as conditions for transition from primer annealing to chain elongation temperature; and(iii) adding additives for stimulation of primer annealing, chain elongation and DNA polymerase activity.