Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-18
2001-11-27
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S040500, C435S069100, C435S091410, C536S025400, C536S025410
Reexamination Certificate
active
06322984
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of calculus as a source of genetic information and in particular to the extraction and characterisation of microbial DNA from the dental calculus of long dead mammals.
DESCRIPTION OF THE RELATED ART
Calculus is a solid mixture of mineral salts, mainly calcium based, which builds up on the surface of teeth and certain other organs, but it may also contain phosphorus, magnesium, fluorine, cobalt, silicon and sodium (Rølla, G. et al., (1989) in Recent Advances in the Study of Dental Calculus Ed. J. M. ten Cate, IEL Press pp 123-126; Glock, G. E. and Murray, M. M. (1938) J. Dental Res. 17:257-264). Dental calculus also comprises an organic phase of bacteria and bacterial remnants, cell products and various compounds found in saliva and gingival fluid (Eggen, K. H., and Rølla, G. (1985) Scand. J. Dent. Res. 93:426-431). Dental calculus may occur supra- and sub-gingivally and there may be differences in the abundance, distribution and structure between the types.
Calculus is found in the ducts, passages, hollow organs, cysts and the surfaces of teeth in humans and other mammals. Calculus persists for many years after the death of the organism and well preserved calculus has been detected, for example, in the teeth of bodies in excess of 5,000 years old. In contrast, all the soft tissues of an organism decompose unless the body is mummified, frozen or otherwise preserved.
Other than its mere existence, most notably on teeth, little is known about the inorganic matrix which constitutes calculus. It is formed by a non-enzymic accumulation of dental plaque, calcium and other mineral salts on a surface which eventually becomes calcified. Nucleators ie. organic or inorganic structures which may serve as templates for deposition of calcium phosphates, are believed to be an important factor in calculus formation and plaque bacteria may play a role. It is widely regarded as an amorphous matrix and most scientific and clinical interest in calculus is directed towards its prevention and removal, since an accumulation of calculus on the teeth is cosmetically undesirable and can result in inflammation of the gums leading to dental problems (Mandel, I. D. and Gaffer, A. (1986) J. Clin. Periodontol, 13:249-257).
It is generally accepted that dental calculus forms to a greater or lesser extent on all mammalian teeth. A major clinical feature of calculus is that it binds tenaciously to the teeth and is difficult to remove. Its formation is spontaneous and effected by diet, chewing behaviour and the micro-environment of the mouth in particular pH. Additionally, there may be some genetic predisposition towards the greater or lesser formation of calculus and certain ethnic groups e.g. Asians, are particularly affected.
pH is a determining factor in the type of calcium salts which predominate in calculus. Animals with a salivary pH of around 8 e.g. dogs and pigs exhibit dental calculus comprising mostly calcium carbonate. Animals with a lower salivary pH of 7, for example, primates including man, exhibit dental calculus based mainly on calcium phosphate deposits (Driessens, F. C. M. & Verbeeck, R. M. H. (1989) In Recent Advances in the Study of Dental Calculus Ed. J. M. ten Cate, IRL Press pp. 7-17).
Similarly, humans with high urinary pH develop urinary calculi comprising mainly calcium carbonate; those with neutral urine develop calculi comprising calcium phosphate and individuals exhibiting low pH urine, who develp calculi, will produce stones rich in uric acid salts.
Diet can affect the formation of calculus in different ways, for example, hard, abrasive foods are believed to assist in preventing calculus formation on the teeth by mechanical agitation.
Dietary silicon can, under certain circumstances promote the formation of urinary calculi in cattle and sheep and there is some suggestion that a high silicon diet, particularly from rice, correlates with increased formation of dental calculus (Gaare, D. (1989) J. Dent. Res. 68:(Spec Issue) 1710-1711; Rølla et al., 1989).
It has recently been shown that calculus on the teeth of both living and long dead people, contains the calcified remains of microorganisms embedded in its matrix. This has not been well researched, but the number and nature of the microorganisms trapped within the calculus appears to be variable. Most bacteria occurring in dental calculus are expected to be plaque bacteria (for example Streptococci and Veillonella) or microorganisms abundant in the buccal flora of the subject. Often, only a single microorganism has been detected, embedded in the calculus matrix.
Surprisingly, not least since microorganisms are present in calculus in such low and variable numbers, it has now been shown that genetic material can be extracted from the calculus of bodies which have been dead for as long as 5,000 years. Even more surprisingly, the isolated nucleic acid, for example from calcified microorganisms embedded in dental calculus, may be isolated in a form amenable to manipulation by recombinant DNA technology and thus may be amplified, sequenced, cloned, expressed, mutated and otherwise manipulated by any molecular biology technique known in the art. Such amenability to manipulation may thus facilitate the study of gene sequences of microorganisms which lived many thousands of years ago.
Although the isolation of genetic material from cells embedded in calculus on archaeological material is a primary feature of the invention, calculus from living animals may also be used as a source of nucleic acid for genetic study.
Whereas dental calculus is believed to harbour only or primarily preserved microbial cells, calculus from other body parts, for example urinary calculi, may also harbour remnants of preserved host cells. In the case where the host has been subjected to infection or where the calculus forms in a body part normally colonised by microorganisms, the calculus may provide a record of host DNA and also DNA from any pathogen, saprophyte or commensal organisms to which the host had been exposed during its lifetime.
SUMMARY OF THE INVENTION
Thus, according to one aspect, the present invention provides the use of animal calculus as a source of nucleic acid.
In particular, the invention provides a useful source of nucleic acid for genetic study.
The calculus may be obtained from any animal body on or in which it is laid down. Conveniently however, the animal will be any mammalian animal.
The calculus may be isolated from any part of the animal, preferably mammalian body.
The nucleic acid may be of host or foreign, eg. microbial origin, and may be DNA or RNA. Preferably, however, it is DNA. More preferably if the DNA is of eukaryotic origin, it is nuclear DNA.
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Lillehaug Dag
Preus Hans R.
ADNA AS
Johannsen Diana
Myers Carla J.
Sughrue Mion Zinn Macpeak & Seas, PLLC
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