End-complementary polymerase reaction

Claim 1

A method for amplifying a target polynucleotide, comprising: contacting under conditions suitable for PCR, target polynucleotide with a bivalent primer which comprises two portions of complementarity to the target polynucleotide: (1) a first portion which is in the 5' portion of the primer and which is substantially complementary to a sequence in the 5' portion of the sequence to be amplified (target sequence) in the target polynucleotide, and (2) a second portion which is in the 3' portion of the primer and which is substantially complementary to a sequence in the 3' portion of the sequence to be amplified (target sequence) in the target polynucleotide; catalyzing under suitable reaction conditions for PCR, polynucleotide synthesis primed from the 3'-hydroxyl of the annealed bivalent primer to form a strand complementary to the target sequence, thereby forming a nascent complementary strand; denaturing the target polynucleotide and the nascent strand and allowing reannealing, under dilute conditions suitable for substantial intramolecular annealing and circle formation, the nascent strand with a complementary strand of a target polynucleotide or an amplified copy thereof to form amplification intermediates in the form of cyclized DNA as a result of the 3' terminus of an overlapped nascent strand annealing to the 3' terminus of an overlapped complementary strand which has a strand with an extendable 3'-hydroxyl which can be extended with a DNA polymerase substantially lacking exonuclease activity whereby the leading terminus of the nascent strand continually displaces the lagging portion of the nascent strand producing a concatemeric single strand emanating from the amplification intermediate; and repeating an elongation/denaturation/reannealing cycle from 1 to about 100 times as desired, resulting in formation of amplified product which comprises head-to-tail concatemers of the target sequence.

Claim 2

A method for amplifying a target polynucleotide, comprising: contacting under conditions suitable for PCR, target polynucleotide with a bivalent primer which comprises two portions of complementarity to the target polynucleotide: (1) a first portion which is in the 5' portion of the primer and which is substantially complementary to a sequence in the 5' portion of the sequence to be amplified (target sequence) in the target polynucleotide, and (2) a second portion which is in the 3' portion of the primer and which is substantially complementary to a sequence in the 3' portion of the sequence to be amplified (target sequence) in the target polynucleotide; catalyzing under suitable reaction conditions for PCR, polynucleotide synthesis primed from the 3'-hydroxyl of the annealed bivalent primer to form a strand complementary to the target sequence, thereby forming a nascent complementary strand; denaturing the target polynucleotide and the nascent strand and allowing reannealing, under dilute conditions suitable for substantial intramolecular annealing and circle formation, the nascent strand with a complementary strand of a target polynucleotide or an amplified copy thereof to form amplification intermediates in the form of cyclized DNA as a result of the 3' terminus of an overlapped nascent strand annealing to the 3' terminus of an overlapped complementary strand which has a strand with an extendable 3'-hydroxyl which can be extended with a DNA polymerase substantially lacking exonuclease activity whereby the leading terminus of the nascent strand continually displaces the lagging portion of the nascent strand producing a concatemeric single strand emanating from the amplification intermediate; and repeating an elongation/denaturation/reannealing cycle from 1 to about 100 times as desired, resulting in formation of amplified product which comprises head-to-tail concatemers of the target sequence.

Claim 8

A method of forming a polynucleotide, comprising: (1) providing two double-stranded polynucleotides overlapping at both ends, which can be denatured to single-stranded polynucleotides having complementary ends that can anneal to generate a circular form; (2) denaturing the double-stranded polynucleotides to generate the single-stranded polynucleotides; (3) incubating the single-stranded polynucleotides under conditions whereby complementary ends anneal; (4) extending the 3' ends of annealed polynucleotides, under conditions whereby each polynucleotide serves as a template for another; (5) repeating steps (2)-(4) 1-100 times, whereby the extended polynucleotides in step (4) constitute the double-stranded polynucleotides in step (2) in the next cycle.

Claim 9

A method of forming a polynucleotide, comprising: (1) providing two double-stranded polynucleotides overlapping at both ends, which can be denatured to single-stranded polynucleotides having complementary ends that can anneal to generate a circular form; (2) denaturing the double-stranded polynucleotides to generate the single-stranded polynucleotides; (3) incubating the single-stranded polynucleotides under conditions whereby complementary ends anneal; (4) extending the 3' ends of annealed polynucleotides, under conditions whereby each polynucleotide serves as a template for another; (5) repeating steps (2)-(4) 1-100 times, whereby the extended polynucleotides in step (4) constitute the double-stranded polynucleotides in step (2) in the next cycle.