Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-04-16
2001-05-22
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091100, C536S024310, C536S024330
Reexamination Certificate
active
06235468
ABSTRACT:
The present invention relates to methods of genetic characterisation. In particular it relates to methods based on the analysis of telomeric repeat arrays.
The Xp/Yp pseudoautosomal region (PAR 1) has been well characterized, it is 2.6 mb in length, contains at least 6 genes and it is defined proximally by an Alu-element and telomere at the distal end (Rappold, 1993). In common with the proterminal regions of many human autosomes, the PAR1 has a high GC-content and contains many hypervariable GC-rich minisatellites. During male meiosis chromosome pairing occurs between the homologous regions of the X and Y chromosomes, and the PAR1 is the site of an obligatory recombination event (Cooke et al., 1985, Simmler et al., 1985). Recently a female recombination hotspot has been identified within the PAR1, between the loci DXYS20 (3cosPP) and DXYS78 (pMS600) and only 20-80 kb from the telomere (Henke et al., 1993). Clearly the PAR1 serves a very specialized function in the male germline but it also shares some of the physical and genetic properties that have been attributed to the proterminal regions of human autosomes.
The terminal sequences, including the telomere of the PAR1 have been cloned (Brown, 1989, Royle et al., 1992). DXYS14 (detected by 29C1, Inglehearn and Cooke, 1990) is the most distal hypervariable minisatellite, located within 20 kb of the telomere. Sequences within 1 kb of this telomere contain another PAR1 specific minisatellite which does not show any restriction fragment length variation and a truncated copy of a SINE (Royle et al., 1992). There are an estimated 20,000 copies of this family of SINEs within the genome (La Mantia et al., 1989).
Telomeres protect linear chromosomes from degradation and fusion to other chromosomes, and are thought to be a site of attachment to the nuclear matrix at times during the cell cycle. Telomeres of all human chromosomes are composed of variable length arrays of (TTAGGG) repeat units with the G-rich strand oriented 5′ to 3′ towards the telomere. Variant telomere repeat units such as (TTGGGG) and TGAGGG) have been identified but tend to be located at the proximal ends of human telomeres (Allshire et al., 1989). Telomeric repeats, in association with telomere binding proteins, are all that is required for a functional telomere and as such they protect the linear chromosome from degradation, fusion to other chromosomes, and they are thought to be a site of attachment to the nuclear matrix at times during the cell cycle (Biessmann and Mason, 1993). Telomere length decreases in somatic tissues with increasing cycles of replication and mitotic cell division (Hastie et al., 1990, Harley et al., 1990) and it is presumed that eventually functional telomeres are lost from chromosomes and the genome is destabilized. The loss of telomere repeats can be counteracted by the activity of telomerase, a specialized reverse transcriptase, which adds (TTAGGG) repeats de novo (Greider and Blackburn, 1989, Morin, 1989) but much evidence suggest that telomerase is only active in the human germline. Recently telomerase activity has been detected in ovarian carcinomas but not in the normal tissue from the patient (Counter et al., 1994) and it has been suggested that reactivation of telomerase is an essential step in tumour progression and in the immortalization of cells in culture (Greider, 1994).
Sequences composed of arrays of tandem repeats range from satellite DNAs to short arrays of dinucleotide repeats found in microsatellites. Analysis of the internal structure of alleles at minisatellite loci has been achieved by mapping the distribution of repeat unites (MVR-PCR, Jeffreys et al., 1991) which show-sequence variation from the consensus repeat. This method has revealed the real extent of allelic variation at several loci (Monckton et al., 1993, Armour et al., 1993, Neil and Jeffreys, 1993, Buard and Vergnaud, 1994) and been used to identify the processes involved in the generation of mutant alleles which arise in the germline (Jeffreys et al., 1994).
Whilst telomere repeat arrays may be expected to show variation, the level of allelic variability between individuals and hence informativeness, identified to date has been limited. We now provide a novel system called telomere variant repeat mapping by PCR (TVR-PCR) which has revealed extensive variation between unrelated alleles.
Therefore in a first aspect of the invention we provide a method of characterising a test sample of genomic DNA which method comprises contacting the test sample with type specific primer to prime selectively, within a telomere repeat array, internal repeat units of that type, and extending the type specific primers in the presence of appropriate nucleoside triphosphates and an agent for polymerisation thereof to produce a set of amplification products extending from the internal repeat units of that type to at least the end of the telomere repeat array.
Two or more specific types of telomere repeat unit may be amplified to generate corresponding sets of amplification products.
The set(s) of amplification products preferably extend to a locus flanking the telomere repeat array and acts as template for a common primer which hybridises to the flanking locus and is extended in the presence of appropriate nucleoside triphosphates and an agent for polymerisation thereof to amplify the set of amplification products. The above amplification procedures may be repeated as required, for example in a polymerase chain reaction.
The test sample of genomic DNA may be total genomic DNA or partially degraded DNA (provided that all or a relevant part of the telomere repeat array to be analysed remains unaffected).
The type specific primer is an oligonucleotide prepared either by synthetic methods or derived from a naturally occurring sequence, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, ie. in the presence of appropriate nucleoside triphosphates and an agent for polymerisation in an appropriate buffer and at a suitable temperature. In our European Patent No. 0332435, the contents of which are incorporated herein by reference, we disclose and claim a method for the selective amplification of template sequences which differ by as little as one base as well as type specific primers for use in the selective amplification method. Type specific primers for use in the present invention may therefore be designed with reference to our above mentioned European Patent, Publication No. 0332435. The selective amplification method is now commonly referred to as the Amplification Refractory Mutation System (ARMS). ARMS is a trade mark of Zeneca Limited.
It has been reported elswhere (Jeffreys et al, Nature, 1994, 354, 204-209) that amplification products may shorten progressively at each amplification cycle, due to the type specific primer priming internally on amplification products from previous cycles. Jeffreys et al unexpectedly found that this problem may be overcome by use of a tail specific primer which hybridises to the complement of the tail sequence in the extension product of the common primer and is extended in the presence of appropriate nucleoside triphosphates and an agent for polymerisation thereof to amplify the common primer amplification products. In summary the tail sequence on the type specific primer is selected so that its complement in the extension product of the common primer provides a convenient template for the tail specific primer provided that the tail sequence and complementary sequences do not hybridise to the tandemly repeated region or to an adjacent region. Examples of convenient tail sequence lengths include up to 50, up to 40, up to 30 and up to 20 nucleotides. Further details regarding the use of tailed primers are disclosed in UK patent application, publication no. 2259138 incorporated herein by reference.
Remarkably we have found that in TVR-PCR it is not essential to ‘tag’ primers with a non-co
Baird Duncan Martin
Jeffreys Alec John
Royle Nicola Jane
Cushman Darby & Cushman
Myers Carla J.
Zeneca Limited
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