End-complementary polymerase reaction
First Claim
Patent Images
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.
9 Assignments
0 Petitions
Accused Products
Abstract
The present invention is directed to a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof and for assembling large polynucleotides from component polynucleotides, each involving generating concatemers formed by PCR amplification of overlapping fragments.
-
Citations
8 Claims
-
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. - View Dependent Claims (2, 3, 4, 5, 6)
-
-
7. 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. - View Dependent Claims (8)
-
Specification