Amplification of Distant Nucleic Acid Targets Using Engineered Primers
AssigneeRoche Molecular Systems, Inc.
InventorJenny A. Johnson
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Abstract
The invention is a method of amplifying nucleic acids by synthesizing an engineered amplicon containing the sequence of interest, but omitting intervening sequences present in the template molecule. The method utilizes an “amplicon bridge” incorporated into the amplification primers. The length and content of the desired amplicon can be chosen by the operator and can contain unique regions for probe binding.
Claims21
A method of amplifying a target nucleic acid in a sample, comprising:
- a) contacting the target nucleic acid in the sample with a first pair of oligonucleotide primers flanking a first portion of the target sequence;
- b) contacting the target nucleic acid in the sample with a second pair of oligonucleotide primers flanking a second portion of the target sequence, different from the first portion;
- wherein
- one oligonucleotide primer in each pair is a bridge oligonucleotide containing a bridge sequence on the 5′-terminus;
- and
- wherein the bridge sequence of the bridge oligonucleotide of the first pair is complementary to the bridge sequence of the bridge oligonucleotide of the second pair;
- c) incubating the sample under the conditions permitting annealing of the oligonucleotide primers to the complementary sequences;
- and
- d) incubating the sample under the conditions permitting extension of the oligonucleotide primers by the nucleic acid polymerase.
A set of oligonucleotides for amplifying a target nucleic acid, comprising:
- a) a first pair of oligonucleotides flanking a first portion of the target sequence,
- b) a second pair of oligonucleotides flanking a second portion of the target sequence, different from the first portion;
- wherein
- one oligonucleotide in each pair contains a bridge sequence on the 5′-terminus;
- and
- wherein the bridge sequence of the oligonucleotide of the first pair is complementary to the bridge sequence of the oligonucleotide of the second pair.
A kit for amplifying a target nucleic acid comprising:
- an amount of each of the oligonucleotides of a first pair, flanking a first portion of the target sequence;
- an amount of each of the oligonucleotides of a second pair, flanking a second portion of the target sequence, different from the first portion;
- wherein one oligonucleotide in each pair contains a bridge sequence on the 5′-terminus;
- and wherein the bridge sequence of the oligonucleotide of the first pair is complementary to the bridge sequence of the oligonucleotide of the second pair;
- and
- a nucleic acid polymerase.
A reaction mixture for amplifying a target nucleic acid comprising:
- a first pair of oligonucleotides flanking a first portion of the target sequence;
- a second pair of oligonucleotides flanking a second portion of the target sequence, different from the first portion;
- a nucleic acid polymerase;
- and nucleoside triphosphates;
- and
- wherein one oligonucleotide in each pair contains a bridge sequence on the 5′-terminus;
- and wherein the bridge sequence of the oligonucleotide of the first pair is complementary to the bridge sequence of the oligonucleotide of the second pair.
A method of generating a control molecule for an amplification reaction, the method comprising:
- a) contacting a first target nucleic acid in the sample with a first pair of oligonucleotide primers flanking a portion of the first target sequence;
- b) contacting a second target nucleic acid in the sample with a second pair of oligonucleotide primers flanking a portion of the second target sequence;
- wherein
- one oligonucleotide primer in each pair is a bridge oligonucleotide containing a bridge sequence on the 5′-terminus;
- and
- wherein the bridge sequence of the bridge oligonucleotide of the first pair is complementary to the bridge sequence of the bridge oligonucleotide of the second pair;
- c) incubating the sample under the conditions permitting annealing of the oligonucleotide primers to the complementary sequences;
- and
- d) incubating the sample under the conditions permitting extension of the oligonucleotide primers by the nucleic acid polymerase.
Dependencies are inferred from the claim text.