Method of detecting nucleotide sequences
First Claim
1. A method for detecting the presence or absence of at least one variant nucleotide in one or more nucleic acids contained in a test sample from an individual, with reference to a control or controls, which method comprises the following steps:
- treating the sample, together or sequentially, with appropriate nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, a diagnostic primer for each diagnostic portion of a target base sequence and a corresponding amplification primer under hybridizing conditions,the nucleotide sequence of said diagnostic primer being such that it is substantially complementary to said diagnostic portion, a 31 terminal nucleotide of the diagnostic primer being either complementary to a suspected variant nucleotide or to the corresponding normal nucleotide, whereby an extension product of the diagnostic primer is synthesized when said terminal nucleotide of the diagnostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesized 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 said amplification primer after separation from its complement;
amplifying any extension product; and
detecting the presence or absence of the suspected variant nucleotide from the presence or absence of amplification product obtained as above, with reference to the control or controls.
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Abstract
The present invention relates to a method for detecting the presence or absence of one or more variant nucleotide sequences. The method comprises (i) contacting a nucleic acid sample with a diagnostic primer which is substantially complementary to a diagnostic portion of a target base sequence, whereby extension of the diagnostic primer on a target template under appropriate conditions is only achieved where a terminal nucleotide of the diagnostic primer is complementary to either a suspected variant nucleotide or a corresponding normal nucleotide of the target base sequence, and (ii) detecting the presence or absence of an extension product. Kits for performing diagnostic tests are also disclosed.
157 Citations
11 Claims
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1. A method for detecting the presence or absence of at least one variant nucleotide in one or more nucleic acids contained in a test sample from an individual, with reference to a control or controls, which method comprises the following steps:
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treating the sample, together or sequentially, with appropriate nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, a diagnostic primer for each diagnostic portion of a target base sequence and a corresponding amplification primer under hybridizing conditions, the nucleotide sequence of said diagnostic primer being such that it is substantially complementary to said diagnostic portion, a 31 terminal nucleotide of the diagnostic primer being either complementary to a suspected variant nucleotide or to the corresponding normal nucleotide, whereby an extension product of the diagnostic primer is synthesized when said terminal nucleotide of the diagnostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesized 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 said amplification primer after separation from its complement; amplifying any extension product; and detecting the presence or absence of the suspected variant nucleotide from the presence or absence of amplification product obtained as above, with reference to the control or controls. - View Dependent Claims (2, 3, 4, 5, 9, 10, 11)
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6. A kit for detecting the presence or absence of at least one or more nucleic acids contained in a sample, which kit comprises:
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two diagnostic primers for each diagnostic portion of a target base sequence, a terminal nucleotide at the 3'"'"' of one diagnostic primer being complementary to a suspected variant nucleotide associated with a known genetic disorder and a terminal nucleotide at the 3'"'"' of the other diagnostic primer being complementary to the corresponding normal nucleotide, such that in use an extension product of the diagnostic primer is synthesized when said terminal nucleotide of the diagnostic primer is complementary to the corresponding nucleotide in the target base sequence, no extension product being synthesized when said terminal nucleotide of the diagnostic primer is not complementary to the corresponding nucleotide in the target base sequence; and (ii) a corresponding amplification primer for the two diagnostic primers. - View Dependent Claims (7, 8)
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Specification