US Patent No. 10,920,267

ISOTHERMAL AMPLIFICATION OF OLIGONUCLEOTIDES


Patent No. 10,920,267
Issue Date February 16, 2021
Title Isothermal Amplification Of Oligonucleotides
Inventorship Steven A Benner, Gainesville, FL (US)
Ozlem Yaren, Gainesville, FL (US)
Patricia Moussatche, Gainesville, FL (US)

Claim of US Patent No. 10,920,267

1. A method for synthesizing multiple concatamers of a nucleic acid that incorporate sequences from a DNA target analyte to detect a target analyte, said process comprising:(A) providing a template that has six regions, in the following order from the 3?-end to the 5?-end, termed F3c, F2c, F1c, B1, B2, and B3,
(B) providing an external primer, termed F3, that is substantially Watson-Crick complementary to F3c,
(C) providing a first internal primer that has two regions, one F1c towards its 5?-end and the other F2 towards its 3?-end, where the two regions are joined by a linking oligonucleotide, and where F1c is substantially Watson-Crick complementary to F1 and F2 is substantially Watson-Crick complementary to F2c,
wherein polymerase-catalyzed extension of said first internal primer generates a first copy that comprises F1c, F2, F1, B1c, B2c, and B3c in the 5?- to 3? direction, wherein F1c is substantially Watson-Crick complementary to F1, F2 is substantially Watson-Crick complementary to F2c, F1 is substantially Watson-Crick complementary to F1c, B1c is substantially Watson-Crick complementary to B1, B2c is substantially Watson-Crick complementary to B2, and B3c is substantially Watson-Crick complementary to B3, and then
(D) providing a second external primer, termed B3, which is substantially Watson-Crick complementary to B3c and
(E) providing a second internal primer that has two regions, one B1c towards its 5?-end and the other B2 towards its 3?-end, where the two regions are joined by a linking oligonucleotide, and where B1c is substantially Watson-Crick complementary to B1 and B2 is substantially Watson-Crick complementary to B2c,
wherein polymerase-catalyzed extension of said second internal primer generates a second copy that comprises B1c, B2, B1, F1c, F2c, and F1 in the 5?- to 3? direction, said second copy can form a structure having two loops, and further comprising
(F) a tagged primer that is a DNA molecule comprising two regions, the first tag region carrying a fluorescence quenching moiety at or near its 5?-end and the second tag region substantially Watson-Crick complementary to a region between B1 and B2 or a region between F1 and F2, and
(G) a displaceable probe that is a DNA molecule having a fluorescent moiety at or near its 3?-end, said displaceable probe being substantially Watson-Crick complementary to the first tag region.