Patent ID: 6326143
Filing Date: 2001-12-04
Classification: C12Q

Abstract:
A method for determining an analyte in a sample, comprising(a) providing a target nucleic acid comprising a region A, a nucleobase sequence B, and a sequence I linked to the 5' terminus of the nucleobase sequence B, which nucleobase sequence B is not specific for said analyte, wherein said region A specifically binds to said analyte;(b) binding said target nucleic acid to said analyte;(c) separating the analyte bound to the target nucleic acid from the remaining part of the sample;(d) hybridizing a primer to said target nucleic acid, which primer comprises a nucleobase sequence B', wherein the nucleobase sequence B' hybridizes to said nucleobase sequence B;(e) elongating the hybridized primer to produce an elongation product E using the target nucleic acid as a template and using nucleofides, wherein at least 30% of said nucleotides contain at least one promiscuous base which is capable of base pairing with each of adenine, guanine, cytosine and thymine;(f) separating the target nucleic acid from said elongation product E;(g) hybridizing a further primer which comprises the nucleobase sequence B' to said elongation product E, wherein said elongation product E is capable of acting as a template for the elongation of said further primer;(h) elongating the hybridized further primer of step (g) to produce an elongation product E' using said elongation product E as a template and using nucleotides, wherein at least 30% of said nucleotides contain at least one said promiscuous base;(i) separating said elongation product E from said elongation product E';(j) hybridizing a further primer comprising a nucleobase sequence B' to said target nucleic acid or said elongation product E';(k) elongating said further primer of step (j) to produce another elongation product E using said target nucleic acid or elongation product E' as a template and using nucleotides, wherein at least 30% of said nucleotides contain at least one said promiscuous base;(l) separating said elongation product E of step (k) from said target nucleic acid or elongation product E';(m) optionally repeating steps (g)-(l) a sufficient number of times to generate a desired amount of double stranded nucleic acids; and thereafter(n) determining said elongation product E and/or elongation product E' as a measure of the presence or amount of said analyte,wherein the lengths of the sequence I and the nucleobase sequence B are chosen such that, when said further primer hybridizes to said elongation product E in step (g), said further primer spans a sequence formed bv elongation of said hybridized primer of step (e) and overlaps at least a part of the 3' region of said hybridized primer of step (e) by an overlap length.