Patent ID: 7504240
Filing Date: 2009-03-17
Classification: C12P,C12Q

Abstract:
1. A method for polynucleotide amplification comprising the steps of: (a) annealing a single gene specific primer to an interior region of a desired polynucleotide, wherein the desired polynucleotide has an unknown nucleotide sequence in a region comprising its 5′-end; (b) synthesizing a daughter strand complementary to the desired polynucleotide with a polymerase enzyme by using the desired polynucleotide as a template, wherein the desired polynucleotide and the daughter strand form a linear double stranded molecule; (c) incubating the linear double stranded molecule under conditions such that terminal but not interior regions of the double stranded molecule become denatured, wherein the polymerase enzyme undergoes strand switching; (d) synthesizing a nucleotide sequence complementary to the gene specific primer at the 3′-end of the daughter strand with the strand switched polymerase enzyme by using the 5′ terminal region of the daughter strand as a template, wherein an elongated daughter strand is created; and (e) amplifying the desired polynucleotide with the polymerase enzyme by using the elongated daughter strand as a template and the single gene specific primer, wherein said amplifying is accomplished without the use of a further primer.