End-complementary polymerase reaction
First Claim
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1. A method of multiplex amplification, comprisingcontacting at least first and second noncontiguous polynucleotide sequences with at least first, second, third and fourth bivalent primers under amplification conditions, wherein the 3′
- end of the first bivalent primer is complementary to the 3′
end of the first polynucleotide sequence, and to the 5′
end of the second bivalent primer, the 3′
end of the second bivalent primer is complementary to the 3′
end of the second polynucleotide sequence and to the 5′
end of the first bivalent primer, the 3′
end of the third bivalent primer is complementary to the 3′
end of the complement of the first polynucleotide sequence, and to the 5′
end of the fourth bivalent primer;
the 3′
end of the fourth bivalent primer is complementary to the 3′
end of the complement of the second polynucleotide sequence, and to the 5′
end of the third bivalent primer;
the first, and second bivalent primers do not form stable hybrids with each other due to lack of sequence identity in internal segments of the first and second bivalent primers;
the third and fourth bivalent primers do not form stable hybrids with each other due to lack of sequence identity in internal segments of the third and fourth bivalent primers;
conducting a multi-cyclic amplification reaction to form a contiguous amplification product comprising equimolar amounts of the first and second polynucleotide sequences and an internal segment from one of the bivalent primers.
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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.
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Citations
8 Claims
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1. A method of multiplex amplification, comprising
contacting at least first and second noncontiguous polynucleotide sequences with at least first, second, third and fourth bivalent primers under amplification conditions, wherein the 3′ - end of the first bivalent primer is complementary to the 3′
end of the first polynucleotide sequence, and to the 5′
end of the second bivalent primer,the 3′
end of the second bivalent primer is complementary to the 3′
end of the second polynucleotide sequence and to the 5′
end of the first bivalent primer,the 3′
end of the third bivalent primer is complementary to the 3′
end of the complement of the first polynucleotide sequence, and to the 5′
end of the fourth bivalent primer;
the 3′
end of the fourth bivalent primer is complementary to the 3′
end of the complement of the second polynucleotide sequence, and to the 5′
end of the third bivalent primer;
the first, and second bivalent primers do not form stable hybrids with each other due to lack of sequence identity in internal segments of the first and second bivalent primers;
the third and fourth bivalent primers do not form stable hybrids with each other due to lack of sequence identity in internal segments of the third and fourth bivalent primers;
conducting a multi-cyclic amplification reaction to form a contiguous amplification product comprising equimolar amounts of the first and second polynucleotide sequences and an internal segment from one of the bivalent primers. - View Dependent Claims (2, 3, 4)
- end of the first bivalent primer is complementary to the 3′
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5. A method of multiplex amplification, comprising
contacting at least first and second noncontiguous polynucleotide sequences with at least first and second bivalent primers and first and second flanking primers under amplification conditions, wherein the 3′ - end of the first bivalent primer is complementary to the 3′
end of the first polynucleotide sequence, and to the 5′
end of the second polynucleotide,the 3′
end of the second bivalent primer is complementary to the 3′
end of the second polynucleotide sequence and to the 5′
end of the first polynucleotide,the first flanking primer is complementary to the 3′
end of the complement of the first polynucleotide;
the second flanking primer is complementary to the 3′
end of the complement of the second polynucleotidethe first, and second bivalent primers do not form stable hybrids with each other due to lack of sequence identity in internal segments of the first and second bivalent primers;
conducting a multi-cyclic amplification reaction to form a contiguous amplification product comprising equimolar amounts of the first and second polynucleotide sequences and an internal segment from one of the bivalent primers. - View Dependent Claims (6, 7, 8)
- end of the first bivalent primer is complementary to the 3′
Specification
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Current AssigneeCodexis, Inc.
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Original AssigneeGlaxo Group Limited (GSK plc), Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
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InventorsLipshutz, Robert J., Stemmer, Willem P.C.
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Primary Examiner(s)Benzion, Gary
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Assistant Examiner(s)Tung, Joyce
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Application NumberUS09/245,802Publication NumberTime in Patent Office1,397 DaysField of Search435/91.2, 435/6US Class Current435/91.1CPC Class CodesC12N 15/10 Processes for the isolation...C12N 15/64 General methods for prepari...C12N 15/66 General methods for inserti...C12Q 1/6853 using modified primers or t...C12Q 1/686 Polymerase chain reaction [...C12Q 2525/151 repeat or repeated sequence...C12Q 2525/155 incorporating/generating a ...C12Q 2525/307 Circular oligonucleotidesC12Q 2531/125 Rolling circleC12Q 2537/143 Multiplexing, i.e. use of m...
