Method of characterisation of genomic DNA
AssigneeZeneca Limited
InventorsAlec John Jeffreys, Stephen Little, Richard Mark Ferrie, Jannine Brownie
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Abstract
The present invention relates generally to a method of characterizing a sample of genomic DNA and to nucleotide sequences employed in the method as well as kits comprising these. In particular the invention involves the use of primers which selectively prime specific type(s) of internal repeat unit in a tandemly repeated region. The method of the invention is particularly useful in forensic or paternity studies and provides individual sample codes suitable for computerized storage on, and retrieval from, a database.
Claims17
A method for detecting the presence or absence of at least one diagnostic base sequence in one or more nucleic acids contained in a sample, which method comprises contacting the sample with a diagnostic primer for each diagnostic base sequence, the nucleotide sequence of each diagnostic primer being such that it is substantially complementary to the corresponding diagnostic base sequence, under hybridising conditions and in the presence of appropriate nucleoside triphosphates and an agent for polymerisation thereof, such that an extension product of a diagnostic primer is synthesised when the corresponding diagnostic base sequence is present in the sample, no extension product being synthesised when the corresponding diagnostic base sequence is not present in the sample and any extension product of a diagnostic primer acts as template for extension of a further primer which hybridises to a locus at a distance from the relevant diagnostic base sequence, and wherein at least one of the diagnostic primer(s) further comprises a tail sequence which does not hybridise to a diagnostic base sequence or a region adjacent thereto, and contacting the above mixture with a tail specific primer which hybridises to the complement of the tail sequence in an extension product of the further primer and is extended in the presence of appropriate nucleoside triphosphates and an agent for polymerisation thereof to amplify the further primer amplification products whereby the presence or absence of the diagnostic base sequence(s) is detected from the presence or absence of tail specific primer extension product.
A method as claimed in claim wherein extension products of at least two diagnostic primers comprise a complementary overlap.
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:
- 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;
- at least one diagnostic primer further comprising a tail sequence for hybridisation to a tail specific primer which hybridises to the complement of the tail sequence in the extension product of the common primer and which tail sequence does not hybridise to the target base sequence or a region adjacent thereto, together with appropriate buffer, packaging and instructions for use.
Dependencies are inferred from the claim text.