Mutation detection by competitive oligonucleotide priming

Claim 3

The method of claim 1, wherein said competitive oligonucleotide primers are about 8-24 mers long.

Claim 4

The method cf claim 1, wherein at least one of said competitive oligonucleotides primers is labeled and said extension product is identified by determining the presence or absence of said label in said extension product.

Claim 5

The method of claim 3, wherein said label is selected from the group consisting of radioisotopes, fluorescers, chemiluminescers, enzymes and antibodies.

Claim 6

The method of claim 1, comprising the further steps of: adding a common oligonucleotide primer prior to said identifying step; separating said extension product from its complementary strand; and repeating said preferentially hybridizing and said extending steps.

Claim 7

The method of claim 5, comprising the further step of repeating at least once, said separating, preferentially hybridizing and extending steps.

Claim 8

A competitive oligonucleotide primer method for detecting the presence or absence of a specific known nucleic acid sequence in a sample containing a mixture of separate complementary nucleic acid strands, or distinguishing between at least two different sequences in the sample, comprising the steps of: adding competitive oligonucleotide primers to a sample of nucleic acid or mixture of nucleic acids, wherein said competitive oligonucleotide primers include at least two primers, one being substantially complementary to the specific known sequence and at least one having a base mismatch with the specific known sequence; annealing said substantially complementary competitive olignucleotide primer and a common nucleotide primer to separate complementary strands under conditions in which said common primer anneals to one of the complementary strands and said substantially complementary competitive oligonucleotide primer anneals to the other complementary strand containing the specific known sequence; extending said annealed primers from their 3′ terminus to synthesize an extension product complementary to the strands annealed to said primers, said extension product, after separation from its complement, serving as a template for the synthesis of an extension product of the other of said annealed primers; separating said extension products from said templates to produce single-stranded molecules; amplifying said single-stranded molecules comprising said specific known sequence by repeating, at least once, said annealing, extending and separating steps; and identifying said amplified extension product.

Claim 9

A competitive oligonucleotide primer method for detecting the presence or absence of a specific known nucleic acid sequence in a sample containing a mixture of separate complementary nucleic acid strands, or distinguishing between at least two different sequences in the sample, comprising the steps of: adding competitive oligonucleotide primers to a sample of nucleic acid or mixture of nucleic acids, wherein said competitive oligonucleotide primers include at least two primers, one being substantially complementary to the specific known sequence and at least one having a base mismatch with the specific known sequence; annealing said substantially complementary competitive olignucleotide primer and a common nucleotide primer to separate complementary strands under conditions in which said common primer anneals to one of the complementary strands and said substantially complementary competitive oligonucleotide primer anneals to the other complementary strand containing the specific known sequence; extending said annealed primers from their 3′ terminus to synthesize an extension product complementary to the strands annealed to said primers, said extension product, after separation from its complement, serving as a template for the synthesis of an extension product of the other of said annealed primers; separating said extension products from said templates to produce single-stranded molecules; amplifying said single-stranded molecules comprising said specific known sequence by repeating, at least once, said annealing, extending and separating steps; and identifying said amplified extension product.

Claim 10

The method of claim 7, wherein at least one of said competitive oligonucleotide primers is labeled, said label being selected from the group consisting of radioisotopes, fluorescers, chemiluminescers, enzymes and antibodies.

Claim 11

The method of claim 8, wherein said identifying step includes detecting the presence or absence of said label in the amplified extension product.

Claim 12

The method of claim 8, wherein said common oligonucleotide primer is bound to a solid support, and said amplified extension product is identified by measuring the presence or absence of said label attached to said solid support.

Claim 13

The method of claim 7, wherein said specific nucleic acid sequence contains at least one mutation that causes a genetic disease.

Claim 14

A competitive oligonucleotide primer method for detecting the presence or absence of a plurality of specific known nucleic acid sequences in a sample containing a mixture of nucleic acid strands, comprising the steps of: annealing a specific oligonucleotide primer to each specific known sequence; extending each of said annealed primers from its 3′ terminus to synthesize an extension product complementary to said strand annealed to the primer, said extension product, after separation from its complement, serving as a template for synthesis of an extension product of another of said primers; separating said primer extension product from said templates on which they were synthesized to produce single stranded molecules; amplifying said single-stranded molecular comprising said specific known sequence by repeating, at least once, said annealing, extending and separating steps; adding competitive oligonucleotide primers to said sample, wherein said competitive oligonucleotide primers include at least two primers for each specific known sequence, one being substantially complementary to said specific known sequence and at least one having a base mismatch with the specific known sequence; preferentially hybridizing said substantially complementary primer to said specific known sequence under competitive conditions; extending said preferentially hybridized primer from its 3′ terminus to synthesize a competitive extension product complementary to said strand to which it is hybridized; and identifying said competitive extension product.

Claim 15

A competitive oligonucleotide primer method for detecting the presence or absence of a plurality of specific known nucleic acid sequences in a sample containing a mixture of nucleic acid strands, comprising the steps of: annealing a specific oligonucleotide primer to each specific known sequence; extending each of said annealed primers from its 3′ terminus to synthesize an extension product complementary to said strand annealed to the primer, said extension product, after separation from its complement, serving as a template for synthesis of an extension product of another of said primers; separating said primer extension product from said templates on which they were synthesized to produce single stranded molecules; amplifying said single-stranded molecular comprising said specific known sequence by repeating, at least once, said annealing, extending and separating steps; adding competitive oligonucleotide primers to said sample, wherein said competitive oligonucleotide primers include at least two primers for each specific known sequence, one being substantially complementary to said specific known sequence and at least one having a base mismatch with the specific known sequence; preferentially hybridizing said substantially complementary primer to said specific known sequence under competitive conditions; extending said preferentially hybridized primer from its 3′ terminus to synthesize a competitive extension product complementary to said strand to which it is hybridized; and identifying said competitive extension product.

Claim 16

The method of claim 12, wherein said competitive oligonucleotide primers are about 8-24 mers long.

Claim 17

The method of Claim 12, wherein at least one of said competitive oligonucleotide primers is labeled and said competitive extension product is identified by determining the presence or absence of said label in said competitive extension product.

Claim 18

The method of claim 14, wherein, the label is selected from the group consisting of radioisotopes, fluorescers, chemiluminescers, enzymes and antibodies.

Claim 19

The method of claim 12, comprising the further steps of: adding a common oligonucleotide primer prior to said identifying step; separating said competitive extension product from its complementary strand; and repeating said preferentially hybridizing and said extending steps.

Claim 20

The method of claim 16, comprising the further step of repeating at least once said separating, preferentially hybridizing and extending steps.

Claim 21

The method of claim 14, wherein, said common oligonucleotide primer is bound to a solid support and said amplified competitive extension product is identified by measuring the presence or absence of label on said solid support.

Claim 22

A competitive oligonucleotide primer method of detecting a genetic disease which results from at least one mutation in a specific known nucleic acid sequence, comprising the steps of: adding competitive oligonucleotide primers to a sample of nucleic acid or a mixture of nucleic acids, wherein said competitive oligonucleotide primers include at least two primers, one being substantially complementary to the normal genetic sequence and at least one being substantially complementary to the mutated genetic sequence; preferentially hybridizing said substantially complementary primers to the appropriate strands containing said specific known sequence under competitive conditions; extending said preferentially hybridized primers from their 3′ terminus to synthesize an extension product complementary to said strands to which the primers are hybridized; identifying said extension products.

Claim 23

A competitive oligonucleotide primer method of detecting a genetic disease which results from at least one mutation in a specific known nucleic acid sequence, comprising the steps of: adding competitive oligonucleotide primers to a sample of nucleic acid or a mixture of nucleic acids, wherein said competitive oligonucleotide primers include at least two primers, one being substantially complementary to the normal genetic sequence and at least one being substantially complementary to the mutated genetic sequence; preferentially hybridizing said substantially complementary primers to the appropriate strands containing said specific known sequence under competitive conditions; extending said preferentially hybridized primers from their 3′ terminus to synthesize an extension product complementary to said strands to which the primers are hybridized; identifying said extension products.

Claim 24

The method of claim 19, wherein at least one of said competitive oligonucleotide primers is labeled, said label being selected from the group consisting of radiosotopes, fluorescers, chemiluminescers, enzymes and antibodies.

Claim 25

The method of claim 20, wherein each competitive oligonucleotide primer is differentially labelled.

Claim 26

The method of claim 20, comprising the further steps of: dividing said sample into a plurality of portions prior to adding said competitive oligonucleotide primers; adding a different, labeled competitive oligonucleotide primer to each of said portions; and measuring the presence or absence of said label in said extension products in each of said portion.