Method of detecting nucleotide sequences

Claim 3

A method as claimed in claim 1 which comprises: 1) treating the sample, together or sequentially, with appropriate nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates a diagnostic primer for a diagnostic portion of a target base sequence and a corresponding amplification primer under hybridising conditions, the nucleotide sequence of the said diagnostic primer being such that it is substantially complementary to the said diagnostic portion, a terminal nucleotide of the diagnostic primer being either complementary to the suspected variant nucleotide or to the corresponding normal nucleotide, whereby an extension product of the diagnostic primer is synthesised when the said terminal nucleotide of the diagnostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesised when the said terminal nucleotide of the diagnostic primer is not complementary to the corresponding nucleotide in the target base sequence; any extension product of the diagnostic primer formed being capable of serving as a template for synthesis of an extension product of the said amplification primer after separation from its complement; 2) treating the sample under denaturing conditions to separate the primer extension product from its template where such extension product is formed; 3) contacting single strands produced in step (2), either together or sequentially, with appropriate nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates, a diagnostic primer and an amplification primer as herein defined whereby, where possible, to synthesise further extension products using the single strands produced in step (2) as templates. 4) repeating steps (2) and (3) a sufficient number of times to result in detectable amplification of the appropriate nucleotide sequence; and 5) detecting the presence or absence of the suspected variant nucleotide from the presence or absence of an amplification product obtained in step (4).

Claim 4

A method as claimed in claim 1 which comprises treating the sample, together or sequentially with either (a) a first diagnostic primer having a sequence substantially complementary to a diagnostic portion of a first nucleic acid sequence, the first diagnostic primer having a terminal nucleotide complementary to the said suspected variant nucleotide, and a second diagnostic primer having a sequence substantially complementary to a diagnostic portion of a second nucleic acid sequence, the second diagnostic primer having a terminal nucleotide complementary to the complementary suspected variant nucleotide; or (b) a first diagnostic primer having a sequence substantially complementary to a diagnostic portion of a first nucleic acid sequence, the first diagnostic primer having a terminal nucleotide complementary to the normal nucleotide which corresponds to the said suspected variant nucleotide, and a second diagnostic primer having a sequence substantially complementary to a diagnostic portion of a second nucleic acid sequence, the second diagnostic primer having a terminal nucleotide complementary to the normal nucleotide which corresponds to the said suspected variant nucleotide; the said terminal nucleotide of the first diagnostic primer and the said terminal nucleotide of the second diagnostic primer being either both at the 5′ end or both at the 3′ end of the respective primers and the first nucleic acid sequence being in the opposite sense to the second nucleic acid sequence.

Claim 5

A method as claimed in claim 1 which comprises firstly amplifying at least a part of the sample nucleic acid containing the suspected variant nucleotide and using the amplification product so obtained as the sample to be treated.

Claim 6

A method as claimed in claim 3 which comprises firstly amplifying at least a part of the sample nucleic acid containing the suspected variant nucleotide and using the amplification product so obtained as the sample to be treated.

Claim 7

A method as claimed in claim 1 which further comprises repeating the sample nucleic acid treatment at least once whereby linear amplification of an extension product is achieved using the same target base sequence as template.

Claim 9

A method as claimed in claim 6 in which the sample is treated with 1, 2 or 3 of the nucleoside triphosphates whereby an extension product to be formed can only extend as far as the presence of only the 1, 2 or 3 nucleoside triphosphates will permit.

Claim 10

A method as claimed in claim 8 in which the sample is treated with three nucleoside triphosphates.

Claim 11

A nucleotide sequence of from about 5 to 50 bp for use in the method of the present invention, a terminal nucleotide of said sequence being complementary to either a suspected variant nucleotide associated with a known genetic disorder or to the corresponding normal nucleotide, the remainder of the said sequence being substantially complementary to the corresponding target base sequence adjacent the suspected variant nucleotide or corresponding normal nucleotide the said nucleotide sequence being such that when used as a diagnostic primer in the method of the present invention an extension product of the diagnostic primer is synthesised when the said terminal nucleotide of the diagostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesised when the said terminal nucleotide of the diagnostic primer is not complementary to the corresponding nucleotide in the target base sequence.

Claim 12

A nucleotide sequence of from about 5 to 50 bp for use in the method of the present invention, a terminal nucleotide of said sequence being complementary to either a suspected variant nucleotide associated with a known genetic disorder or to the corresponding normal nucleotide, the remainder of the said sequence being substantially complementary to the corresponding target base sequence adjacent the suspected variant nucleotide or corresponding normal nucleotide the said nucleotide sequence being such that when used as a diagnostic primer in the method of the present invention an extension product of the diagnostic primer is synthesised when the said terminal nucleotide of the diagostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesised when the said terminal nucleotide of the diagnostic primer is not complementary to the corresponding nucleotide in the target base sequence.

Claim 13

A nucleotide sequence as claimed in claim 10 wherein the terminal nucleotide is at the 3′ end of the nucleotide sequence.

Claim 14

A nucleotide sequence as claimed in claim 10 or claim 11 wherein the suspected variant nucleotide results from a point mutation of the corresponding normal sequence.

Claim 15

A set of two nucleotide sequences as claimed in any one of claims 10-12 wherein a terminal nucleotide of one sequence is complementary to a suspected variant nucleotide associated with a known genetic disorder and a terminal nucleotide of the other sequence complementary to the corresponding normal nucleotide.

Claim 16

A kit for detecting the presence or absence of at least one variant nucleotide in one or more nucleic acids contained in a sample, which kit comprises:- (1) a diagnostic primer for each diagnostic portion of a target base sequence, the nucleotide sequence of each diagnostic primer being such that it is substantially complementary to the said diagnostic portion, a terminal nucleotide of the diagnostic primer being either complementary to the suspected variant nucleotide or to the corresponding normal nucleotide such that in use an extension product of the diagnostic primer is synthesised when the said terminal nucleotide of the diagnostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesised when the said terminal nucleotide of the diagnostic primer is not complementary to the corresponding nucleotide in the target base sequence; (2) each of four different nucleoside triphosphates; and (3) an agent for polymerisation of the nucleoside triphosphates in (2).

Claim 17

A kit for detecting the presence or absence of at least one variant nucleotide in one or more nucleic acids contained in a sample, which kit comprises:- (1) a diagnostic primer for each diagnostic portion of a target base sequence, the nucleotide sequence of each diagnostic primer being such that it is substantially complementary to the said diagnostic portion, a terminal nucleotide of the diagnostic primer being either complementary to the suspected variant nucleotide or to the corresponding normal nucleotide such that in use an extension product of the diagnostic primer is synthesised when the said terminal nucleotide of the diagnostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesised when the said terminal nucleotide of the diagnostic primer is not complementary to the corresponding nucleotide in the target base sequence; (2) each of four different nucleoside triphosphates; and (3) an agent for polymerisation of the nucleoside triphosphates in (2).

Claim 18

A kit as claimed in claim 14 which comprises a set of two diagnostic primers for each diagnostic portion of a target base sequence, a terminal nucleotide of one diagnostic primer being complementary to a suspected variant nucleotide associated with a known genetic disorder and a terminal nucleotide of the other diagnostic primer being complementary to the corresponding normal nucleotide.