EP0298656A1 — Polynucleotide probes

Claim 1

Independent Claim 212 words

A polynucleotide in insolated or cloned form having the formula, read in the 5′ → 3′ sense H.(J.core.K) n .L wherein: (a) 'core' represents a sequence having at least 6 consecutive nucleotides, selected from the following sequences: GXGGGGYACAG and wherein: (b) (i) X is A or C or T or G and Y is A or C or T or G or nucleotide Y may be absent; (ii) J and K together represent 0 to 25 additional nucleotides within the repeating unit; and (iii) H and L each represent 0 or at least 1 additional nucleotide flanking the repeating units; (iv) the repeating unit (J.core.K) need not be an exact repeat either as to number and kind of nucleotides, provided that the polynucleotide has a recognisably repeated core sequence; (v) 'core' can also represent a variant core sequence, provided that actual core sequences in all n repeating units have on average at least 70% homology with true 'core' sequences as defined above with respect to formula (2), or a polynucleotide of complementary sequence to the above, the polynucleotide being capable of hybridising with fragments of DNA from more than one hypervariable region generated by one or more restriction enzymes which do not cleave within a sequence corresponding to the repeating unit (J.core.K).

Claim 2

Dependent Claim 212 words

A polynucleotide in insolated or cloned form having the formula, read in the 5′ → 3′ sense H.(J.core.K) n .L wherein: (a) 'core' represents a sequence having at least 6 consecutive nucleotides, selected from the following sequences: GXGGGGYACAG and wherein: (b) (i) X is A or C or T or G and Y is A or C or T or G or nucleotide Y may be absent; (ii) J and K together represent 0 to 25 additional nucleotides within the repeating unit; and (iii) H and L each represent 0 or at least 1 additional nucleotide flanking the repeating units; (iv) the repeating unit (J.core.K) need not be an exact repeat either as to number and kind of nucleotides, provided that the polynucleotide has a recognisably repeated core sequence; (v) 'core' can also represent a variant core sequence, provided that actual core sequences in all n repeating units have on average at least 70% homology with true 'core' sequences as defined above with respect to formula (2), or a polynucleotide of complementary sequence to the above, the polynucleotide being capable of hybridising with fragments of DNA from more than one hypervariable region generated by one or more restriction enzymes which do not cleave within a sequence corresponding to the repeating unit (J.core.K).

Claim 3

Dependent Claim 202 words

A polynucleotide in isolated or cloned form having the general formula, read in the 5′ → 3′ sense H.(J.core.K) n .L wherein H.J.K.L. and 'n' are as defined in claim 1 and: 'core' represents a sequence of from 6 to 16 consecutive nucleotides, read in the same sense, selected from (1) the common core region of a first animal or human hypervariable region obtained by probing animal or human genomic DNA with a probe containing a 3′ alpha-globin, or zeta-intron, or inter-zeta tandem repeat sequence (2) the common core region of a second animal or human hypervariable region obtained by probing animal or human genomic DNA with a probe containing a tandem repeat sequence comprising (1) (3) the common core region of a third animal or human hypervariable region obtained by probing animal or human genomic DNA with a probe containing a tandem repeat sequence comprising (2) each tandem repeat sequence being a repeat of at least 3 units, or a polynucleotide complementary to the aboe, the polynucleotide being capable of hybridising with fragments of DNA from more than one hypervariable region produced by one or more restriction enzymes which do not cleave within a sequence corresponding to the repeating unit (J.core.K).

Claim 4

Independent Claim 90 words

A method of preparing a polynucleotide having polymorphic hypervariable region-length-specific binding characteristics comprising (i) identifying a natural tandem repeat sequence in DNA which is capable of limiting hybridisation to other polymorphic DNA regions, (ii) identifying a natural concensus core sequence of the repeat sequence putatively responsible for such binding, and (iii) isolating or artificially building a perfect or imperfect tandem repeat sequence derived from the natural concensus core sequence having hypervariable region binding properties which exhibits lower genome locus specificity and higher polymorphic fragment acceptance than the natural repeat sequence.

Claim 5

Independent Claim 90 words

A method of preparing a polynucleotide having polymorphic hypervariable region-length-specific binding characteristics comprising (i) identifying a natural tandem repeat sequence in DNA which is capable of limiting hybridisation to other polymorphic DNA regions, (ii) identifying a natural concensus core sequence of the repeat sequence putatively responsible for such binding, and (iii) isolating or artificially building a perfect or imperfect tandem repeat sequence derived from the natural concensus core sequence having hypervariable region binding properties which exhibits lower genome locus specificity and higher polymorphic fragment acceptance than the natural repeat sequence.

Claim 6

Dependent Claim 144 words

A polynucleotide probe comprising, with the inclusion of a labelled or marker component, a polynucleotide comprising at least three tandem repeats (not necessarily exact repeats) of sequences which are homologous with a hypervariable region of the human or animal genome to a degree enabling hybridisation of the probe to a corresponding DNA fragment obtained by fragmenting the same DNA with a restriction endonuclease, wherein: (a) the repeats each contain a core which has at least 75% homologous with a consensus core region of similar length present in a plurality of hypervariable regions from different genomic loci; (b) the core is at least 6 nucleotides long; (c) the total number of nucleotides within the repeating unit which do not contribute to the core is not more than 25; (d) the probe is capable of differentiating DNA by reference to more than one polymorphic hypervariable region.

Claim 7

Dependent Claim 144 words

A polynucleotide probe comprising, with the inclusion of a labelled or marker component, a polynucleotide comprising at least three tandem repeats (not necessarily exact repeats) of sequences which are homologous with a hypervariable region of the human or animal genome to a degree enabling hybridisation of the probe to a corresponding DNA fragment obtained by fragmenting the same DNA with a restriction endonuclease, wherein: (a) the repeats each contain a core which has at least 75% homologous with a consensus core region of similar length present in a plurality of hypervariable regions from different genomic loci; (b) the core is at least 6 nucleotides long; (c) the total number of nucleotides within the repeating unit which do not contribute to the core is not more than 25; (d) the probe is capable of differentiating DNA by reference to more than one polymorphic hypervariable region.

Claim 8

Dependent Claim 52 words

A polynucleotide probe characterised in that: (a) it consists of a labelled polynucleotide, being a polynucleotide as claimed in claim 1 or 2, wherein at least the repeat units are in a single-stranded form and (b) the probe is capable of differentiating DNA by reference to more than one polynucleotide hypervariable region.

Claim 9

Dependent Claim 42 words

A method of preparing a probe useful in genetic origin determination of human or animal DNA-containing samples which comprises introducing a label or marker into a polynucleotide according to claim 1 or 2 or as prepared by the method of claim 3.

Claim 10

Independent Claim 198 words

A method of characterising a test sample of animal or human genomic DNA, by reference to one or more controls, which comprises probing the test sample with a polynucleotide probe comprising, with the inclusion if necessary of a labelled or marker component, at least three tandem repeats, not necessarily exact repeats or sequences which are homologous with a hypervariable region of a genome to a degree enabling hybridisation of the probe therewith, detecting hybridising fragments of DNA, and comparing the hybridised fragments with a said control or controls, wherein: (a) the sample DNA is fragmented with one or more restriction enzymes which do not cleave to any relevant extent a sequence corresponding to the said tandem repeat; (b) the repeats each contain a core which is at least 75% homologous with a concensus core region of similar length present in a plurality of hypervariable regions from a different genomic loci; (c) the core is at least 6 nucleotides long; (d) the total number of nucleotides within the repeating unit which do not contribute to the core is not more than 25; (e) the hybridised fragments differentiate the sample DNA by reference to more than one polymorphic hypervariable region.

Claim 11

Independent Claim 198 words

A method of characterising a test sample of animal or human genomic DNA, by reference to one or more controls, which comprises probing the test sample with a polynucleotide probe comprising, with the inclusion if necessary of a labelled or marker component, at least three tandem repeats, not necessarily exact repeats or sequences which are homologous with a hypervariable region of a genome to a degree enabling hybridisation of the probe therewith, detecting hybridising fragments of DNA, and comparing the hybridised fragments with a said control or controls, wherein: (a) the sample DNA is fragmented with one or more restriction enzymes which do not cleave to any relevant extent a sequence corresponding to the said tandem repeat; (b) the repeats each contain a core which is at least 75% homologous with a concensus core region of similar length present in a plurality of hypervariable regions from a different genomic loci; (c) the core is at least 6 nucleotides long; (d) the total number of nucleotides within the repeating unit which do not contribute to the core is not more than 25; (e) the hybridised fragments differentiate the sample DNA by reference to more than one polymorphic hypervariable region.

Claim 12

Dependent Claim 45 words

A method, according to claim 7, of establishing the identity or otherwise of the test sample donor with one or more control sample donors, in which the control sample(s) are similarly fragmented and a comparison is made of the hybridised fragments from the respective samples.

Claim 13

Dependent Claim 44 words

A method, according to claim 7, of establishing a family connection between the test sample donor and one or more control sample donors, in which the control sample(s) are similarly fragmented and a comparison is made of the hybridised fragments from the respective samples.

Claim 14

Dependent Claim 31 words

A method, according to claim 7, wherein DNA samples from horses are compared for the purpose of equine paternity testing, equine pedigree validation, or verification of the identities of specific horses.

Polynucleotide probes

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