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Coupled polymerase chain reaction-restriction-endonuclease digestion-ligase detection reaction process

  • US 7,014,994 B1
  • Filed: 03/17/2000
  • Issued: 03/21/2006
  • Est. Priority Date: 03/19/1999
  • Status: Expired due to Term
First Claim
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1. A method for identifying one or more low abundance sequences differing by one or more single-base changes, insertions, or deletions from a high abundance sequence, in a sample containing a plurality of target nucleotide sequences comprising:

  • providing a sample potentially containing one or more low abundance target nucleotide sequences with at least one sequence difference each from the high abundance target sequences;

    providing a primary oligonucleotide primer set characterized by (a) a first oligonucleotide primer containing a target-specific portion, and (b) a second oligonucleotide primer containing a target-specific portion, wherein the primary oligonucleotide primers hybridize to complementary strands of high and low abundance target nucleotide sequences to permit formation of a polymerase chain reaction product, but have a mismatch which interferes with formation of such a polymerase chain reaction product when hybridized to any other nucleotide sequence present in the sample;

    providing a polymerase;

    blending the sample, the primary oligonucleotide primers, and the polymerase to form a primary polymerase chain reaction mixture;

    subjecting the primary polymerase chain reaction mixture to two or more polymerase chain reaction cycles;

    providing a secondary oligonucleotide primer set characterized by (a) a first oligonucleotide primer, having a target-specific portion and a 5′

    upstream secondary primer-specific portion, and (b) a second oligonucleotide primer, having a target-specific portion and a 5′

    upstream secondary primer-specific portion, wherein the secondary oligonucleotide primers in a particular set hybridize to complementary strands of the primary extension products to permit formation of a secondary polymerase chain reaction product which contains or creates a restriction endonuclease recognition site when amplifying the high abundance target, but does not contain or create a restriction endonuclease recognition site when amplifying the one or more low abundance targets;

    providing a polymerase;

    blending the primary extension products, the secondary oligonucleotide primers, and the polymerase to form a secondary polymerase chain reaction mixture;

    subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles, wherein high abundance secondary extension products contain a restriction site but low abundance secondary extension products do not;

    providing a restriction endonuclease;

    blending the secondary extension product and the restriction endonuclease to form an endonuclease digestion reaction mixture;

    subjecting the endonuclease digestion reaction mixture to an endonuclease digestion reaction such that the restriction endonuclease recognizes and cleaves the restriction endonuclease recognition site contained within or created when amplifying the high abundance target but not the low abundance target in the secondary extension products, thus selectively destroying the high abundance secondary extension products;

    providing a tertiary oligonucleotide primer set characterized by (a) a first tertiary primer containing the same sequence as the 5′

    upstream portion of the first oligonucleotide primer of the secondary oligonucleotide primer set, and (b) a second tertiary primer containing the same sequence as the 5′

    upstream portion of a second oligonucleotide primer of the secondary oligonucleotide primer set, wherein the set of tertiary oligonucleotide primers are amplification primers for amplification of all the secondary extension products;

    blending the secondary extension products, the tertiary oligonucleotide primer set, and the polymerase to form a tertiary polymerase chain reaction mixture;

    subjecting the tertiary polymerase chain reaction mixture to two or more polymerase chain reaction cycles;

    providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a tertiary extension product-specific portion and a detectable reporter label, and (b) a second oligonucleotide probe, having a tertiary extension product-specific portion, wherein the oligonucleotide probes in a particular set ligate together when hybridized adjacent to one another on a complementary tertiary extension product-specific portion, but have a mismatch which interferes with said ligation when hybridized to any other nucleotide sequence present in the sample;

    providing a ligase;

    blending the tertiary extension product, the plurality of oligonucleotide probe sets, and the ligase to form a ligase detection reaction mixture;

    subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the tertiary extension products, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective tertiary extension products, if present, and ligate to one another to form a ligation product sequence containing (a) the detectable reporter label and (b) the tertiary extension product-specific portions connected together, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences other than their respective complementary tertiary extension products but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment; and

    detecting the reporter labels of the ligation product sequences, thereby identifying the presence of one or more low abundance target nucleotide sequences in the sample.

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