A method of detecting a mutation of a specific nucleotide base in a target nucleic acid chain comprises: (a) hybridizing a labelled probe to the target to form a hybrid in which one end of the probe is positioned adjacent the specific base; (b) adding a nucleotide derivative, e.g. a thionucleotide, under conditions to cause it to join to the end of the probe if it is complementary to the specific base; (c) digesting the hybrid using an exonuclease enzyme under conditions such that the nucleotide derivative protects the probe from digestion; and observing the presence or absence of label attached to the target. The method can be used to detect mutations even when these are not present at restriction enzyme cleavage sites, and does not require the preliminary steps of restriction digestion, gel electrophoresis and DNA (or RNA) blotting.
A novel technique for determining the existence or nonexistence of a test nucleotide on a strand of DNA is provided. The determination advantageously uses an exonucleolytic agent that is capable of retaining a labeled nucleotide in a primer if there is a match between the test nucleotide on the strand of DNA and the complementary nucleotide on the primer, but not if there is a mismatch. The presence or absence of the test nucleotide then may be established by determining whether the label is preserved or lost following the reaction.
A method for the direct identification of few, preferably single nucleic acid strands of a specific target sequence (10) in a test solution comprising the following steps: a) Preparation of a reference solution with a mixture (11) of different, short primers (12, 13, 14) each with a so-called antisense-sequence (a', b', c') complementary to a section (a, b, c) of the target sequence (10), and marked with one or more dye molecules, b) mixture of the reference solution with the test solution and incubation of this mixture (15) under conditions allowing hybridization of the primers (12, 1, 14) with the nucleic acid strands to be identified, and then c) identification of the target sequence (10) in the incubated solution (15) by discriminating few, preferably one of the nucleic acid strands to be identified to which one or more primers (12, 13, 14) have hybridized against the background of the non-hybridized primers (12, 13, 14).
The invention concerns a reagent composition that employs at least two different terminators of a nucleic acid template-dependent primer extension reaction to determine the identity of a nucleotide base at a specific position in a nucleic acid of interest. The invention also concerns the method for determining such identification. The invention may be used to determine the presence or absence of a specific nucleotide sequence in a sample. It may also be employed in determination of genotype and in the identification of different alleles.
The invention concerns a reagent composition that employs at least two different terminators of a nucleic acid template-dependent primer extension reaction to determine the identity of a nucleotide base at a specific position in a nucleic acid of interest. The invention also concerns an immobilized method for determining such identification. The invention may be used to determine the presence or absence of a specific nucleotide sequence in a sample. It may also be employed in determination of genotype and in the identification of different alleles.
Synthetic nucleic acid molecules are non-covalently immobilized in the presence of a salt or cationic detergent on a hydrophilic polystyrene solid support containing an --OH, --C.dbd.O or --COOH hydrophilic group or on a glass solid support. The support is contacted with a solution having a pH of about 6 to about 8 containing the synthetic nucleic acid and the cationic detergent or salt. Preferably, the cationic detergent is 1-ethyl-3-(3'-dimethylaminopropyl)-1,3-carbodiimide hyrochloride at a concentration of about 30 mM to about 100 mM or octyldimethylamine hydrochloride at a concentration of about 50 mM to about 150 mM. The salt is preferably NaCl at a concentration of about 50 mM to about 250 mM. When the detergent is 1-ethyl-3-(3'-dimethylaminopropyl)-1,3-carbodiimide hyrochloride, the glass support or the hydrophilic polystyrene support is used. When NaCl or octyldimethylamine hydrochloride is used, the support is the hydrophilic polystyrene. After immobilization, the support containing the immobilized nucleic acid may be washed with an aqueous solution containing a non-ionic detergent. The immobilized nucleic acid may be used in nucleic acid hybridization assays, nucleic acid sequencing and in analysis of genomic polymorphisms.
