Patent ID: 6740510
Filing Date: 2004-05-25
Classification: C12Q

Abstract:
A method for amplifying a target sequence that is between about 3 kilobases and at least about 15 kilobases in length, comprising the steps of:mixing a reaction cocktail comprising deoxynucleotide triphosphates, about 0.2 ng/&mgr;l to about 2.5 ng/&mgr;l template (target) DNA, a divalent cation, DNA polymerase enzyme, a broad spectrum solvent, a zwitterionic buffer and at least one primer pair having a length of about 28 nucleotides to about 36 nucleotides and a melting temperature of about 72Â° C. to about 88Â° C.; heating said reaction cocktail at a denaturing temperature of about 90Â° C. to about 96Â° C. for about 1 second to about 30 seconds; cooling said reaction cocktail at a single temperature within the range of about 50Â° C. to about 68Â° C. for about 1 minute to about 28 minutes, at which single temperature both annealing and extension occur; repeating said heating and cooling steps at least 10 times; and cooling said reaction cocktail to 4Â° C. in a final cooling step, thereby amplifying a target sequence that is between 3 kilobases and at least about 15 kilobases in length.