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US 2003/0073093 A1Published application
Methods for amplification of nucleic acids
AssigneePerlegen Sciences, Inc.
InventorsCurtis Kautzer, Nila Patil, Coleen Hacker, David P. McDonough
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Abstract
The presently claimed invention provides methods for amplifying a DNA target sequence. One embodiment of the present invention provides robust methods for amplification of target sequences. In a first aspect of the invention, a method for designing primer pairs for the amplification reaction is provided. In a further aspect of the invention, reagents and cycling parameters for the amplification reaction are provided.
Claims25
A method for amplifying a target sequence, comprising the steps of:
- mixing a reaction cocktail comprising deoxynucleotide triphosphates, 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 72° C. to about 88° C.;
- heating said reaction cocktail at a denaturing temperature of about 90° C. to about 96° C. for about 1 second to about 30 seconds;
- cooling said reaction cocktail at an annealing/extension temperature of about 50° C. to about 68° C. for about 1 minute to about 28 minutes;
- repeating said heating and cooling steps at least 10 times;
- and
- cooling said reaction cocktail to 4° C. in a final cooling step.
3 more elements
Dependencies are inferred from the claim text.