WO2010115442A1 - Chewing mass for obtaining nucleic acids for determining sequence variants - Google Patents

Abstract

The invention relates to compact chewing masses for use in diagnostic methods while isolating nucleic acids, the use of such chewing masses, and corresponding methods. Said method allows, for example, recognizing genetic polymorphisms and selecting suitable medications and/or dosages for persons, and identifying individuals endangered by certain materials, and the like.

Description

 Gum for the extraction of nucleic acids for the determination of

sequence variants

Summary of the invention

The invention relates to gums for use in diagnostic procedures under nucleic acid isolation, the use of chewing gums and corresponding methods and methods. This allows the recognition of gene polymorphisms and the selection of appropriate forms of therapy, drugs and / or dosages for humans, as well as the identification of individuals at risk of certain substances, methods for performing a genetic analysis as well as the use of gizzards for investigations of gene polymorphisms in the context of Screening procedures and for purely informative (eg empirical) purposes.

Background of the invention

It is known from studies on drug effects, forensic studies, and other scientific studies that sequence variations of genes or other DNA segments for significant differences in degradation and efficacy of foreign substances such as drugs, environmental toxins, or the like, are responsible in the bodies of different individuals or for different susceptibility to certain diseases and the phenotypic expression of various physiological features such as eye color. Practical applications include identifying sequence changes (also known as polymorphisms) to determine whether or not certain drugs are effective in certain patients, for example, whether or not they are becoming more metabolized, or whether the dose must be increased or decreased because certain patients show increased or decreased metabolism or decreased or increased binding to target molecules such as proteins, nucleic acids or the like. The expression of various individual characteristics as well as disease risks, which do not have to be subject to environmental influences, may be dependent on genetic polymorphisms.

For example, cytochrome P450 monoxygenases in the human body often participate in the metabolism of

Medicines or other xenobiotics. So there is of CYP2D6

(Cytochrome 2 subfamily D isoenzyme 6) at least 43 alleles

(Schaffeier et al., Human Mutation 2_2, 476-485 (2003)), which disclose different metabolism rates for drug agents (e.g., antiarrhythmics, beta-antagonists, tricyclic

Antidepressants, selective serotonin reuptake inhibitors,

Neuroleptics, opiates, etc., or other xenobiotics cause, therefore, there are ultrafast, extensive, medium-fast to low metabolizers among the corresponding genetically endowed individuals - see also http: //www.pharmgkb. org / index. jsp.

For example, potent metabolizers may be suitable for therapy with drugs that are activated only by metabolism (such as codeine that is converted to morphine, for pain therapy), or they can break down drugs too quickly, so that they do not have their full effect reachable. Other cytochromes, such as CYP 2C9 or CYP 2C19, may have similar sequence variants. The same applies to greater or lesser affinity of drugs, such as inhibitors or activators, for certain receptors, enzymes, regulatory proteins, and the like.

The same applies to other metabolizing enzymes, such as glutathione S-transferases, glucuronidating, other hydroxylating, sulfating, oxidizing, ester-splitting, amide-cleaving or similar activities in the body causing enzymes or enzyme complexes.

Finally, direct targets of modulating drugs, such as certain enzymes or receptors, may also have sequence variants that sensitize or desensitize certain substances (such as drugs or substances from the environment).

It is therefore desirable to have sampling materials and methods that enable DNA to be noninvasively removed from appropriate patients or individuals, that is, without having to penetrate or otherwise injure mucous membranes or skin with needles or the like, and at the same time to enable high reliability and easy handling of the materials and reagents used. Thus, information can be obtained with the corresponding sequence variants as biomarkers for sensibilities and / or therapeutics.

It has now surprisingly been found that it is possible to use compact chewing gums, in particular chewing gums, nucleic acid samples and oral mucosal cells and / or oral mucous membrane cell fragments. noninvasively from the human oral cavity, which allows for the diagnosis of different eg metabolic rates or binding affinities, generally sequence variants, and thus allows to define more precisely human groups that therapies with certain active substances or sensitivities to certain substances are more accessible than others, which allows, the nucleic acids obtainable as biomarkers for the therapy (among other things with respect to dosage regulations, type of active substance to be administered) or the susceptibility to exposure to certain substances of representatives of certain groups of

Better estimate people. Thus, genotyping is enabled, i. the determination of certain in one

Alternatively, or as a supplement to this, it becomes possible to perform an individual gene expression analysis, for example an analysis for the frequency or activity of SNPs, or others Sequence variations to perform.

The advantages of this method are obvious: there is no cumbersome sample collection, such as with cotton swabs, and the sample collection can be done in a manner that is comfortable for the individual concerned (without pain, gagging or the like). The small traces of nucleic acids on the surface (for example in cells or cell components of the oral mucosa) of the compact chewing mass are sufficient for analysis and can be easily isolated. The compact samples are easy to store and can be kept for a long time, for example by cooling or freezing, and even stored by the subject without cooling until they can be brought to analysis. The invention therefore in one embodiment relates to a compact chewing mass for use in a method for obtaining nucleic acids from the oral cavity of a human body as part of a diagnostic procedure on a human body.

A preferred variant relates to such a compact chewing gum, wherein the diagnostic method for testing the human body for sequence variations of certain genes or DNA sections, in particular in connection with susceptibility to and / or the ability to be treated (in particular for the selection of suitable forms of therapy) of diseases or risks when exposed to certain substances. Also for other purposes, such as the determination of other physiological or pathophysiological characteristics, such as eye color, gene defects, paternity tests, genealogical examinations or the like, the corresponding diagnostic method is applicable, as well as for investigations of gene polymorphisms in the context of screening methods and for purely informative (eg empirical) purposes.

A further advantageous variant of the invention relates to a compact chewing mass for use as in the preceding two

Paragraphs, characterized in that it is an easily deformable compact mass on which it is also possible to chew for up to several hours, without the nucleic acids to be examined, which are bound directly or indirectly to it, being destroyed.

A further preferred embodiment of the invention relates to a compact chewing mass according to one of the preceding three Paragraphs, characterized in that it contains as base (gum base) natural resins, such as mastic or chicle, waxes and / or natural or synthetic rubbers, such as polyisobutylene. Alternatively, substances as described below for Gum Base may be included.

Another preferred variant of the invention according to the above four paragraphs relates to a compact chewing gum for the determination of single nucleotide polymorphisms, in particular those which make it possible to decide whether a therapy (this term includes both in the context of the present disclosure a prophylactic as well as a therapeutic treatment) with a certain active substance or specific active ingredients or other therapeutic methods is promising and whether or which risk exists for the occurrence of adverse drug reactions.

Yet another preferred variant relates to a method for carrying out a genetic analysis in which first oral mucosal cells and / or cell fragments are collected with a chewing gum according to the invention (in presence or absence of the subject) the cells and / or cell fragments are separated from the chewing gum be subjected to DNA extraction and an individual gene expression analysis, for example an SNP analysis, is performed.

A further preferred embodiment of the invention relates to a kit comprising a chewing gum according to the invention and further reagents required for separating cells and / or cell fragments from chewing gum, nucleic acid (in particular DNA) extraction and / or for gene analysis, in particular described above and below. Yet another preferred embodiment of the invention relates to a drug kit containing a drug

(For example, an anti-rheumatic drug, a graft rejection-preventing agent

Antibiotic, an antiviral agent, a painkiller, an anti-inflammatory agent, an antihypertensive agent, an endocrine disruptor, a lipid lowering agent, a

Cholesterol-lowering agent, a chemotherapeutic agent for the treatment of proliferative diseases or the like) and a compact chewing gland according to the invention for collecting oral mucosal cells and / or cell fragments, in particular for use and for methods as described above and below. The chewing gum can then be used to obtain appropriate samples for gene analysis (e.g., by sending it to a laboratory or donating to a doctor or the like), for example, to optimize dosage for a subject.

It is advantageous if a compact chewing mass, in particular according to one of the preceding paragraphs, is characterized in that it contains at least one abrasive. This allows a particularly good recovery of cells and cell fragments of

Oral mucosa, possibly due to the abrasive and possibly also adsorptive properties of such abrasives, without these explanations being exhaustive.

Advantageously, such an abrasive in a weight fraction of 0.01 to 20%, in particular from 0.1 to 10 wt .-%, for example from 0.1 to 5%, in particular from 0.2 to 3 wt .-%, based on the gum, includes.

The abrasive is for example selected from sparingly soluble calcium salts, aluminum oxides, silica, complex silicates, powdered plastics, bioactive glass pastes and titanium dioxide.

An important aspect of the invention also relates to the use of a chewing gum, in particular as described above and below, for obtaining nucleic acids, oral mucosal cells and / or oral mucous membrane fragments from the oral cavity of humans. Thus, corresponding methods or methods are also the subject of the present invention

Particularly important in the context of the present invention is the use of further including the determination of sequence variants of certain genes or DNA sections, which are related to the susceptibility to and / or the treatability of diseases and / or risks associated with exposure to certain substances.

For this purpose, preferably a chewing gum sample (obtained for example from a test person) is treated with a customary lysis buffer (extraction buffer) for lysing the oral mucosa cells and cell fragments, the DNA is isolated by customary methods, then amplified by conventional methods and finally, if desired, sequenced by customary methods, if the amplification does not already provide adequate detection of corresponding DNA segments with sequence variations m.

"If desired" always means that a corresponding section of the procedure does not necessarily have to be carried out, but only if this is expedient or, for example, based on previously obtained information, it is not possible to decide which variant of a sequence present, which is informative, for example, for corresponding sensibilities and / or therapeutic options.

In a further aspect, the invention also relates to a method or method for isolating and determining oral mucosal cells or fragments thereof, in particular for obtaining nucleic acids (especially DNA) bound to a compact chewing mass, including the lysis (extraction) of above all attached thereto (and in part also enclosed therein) cells or cell debris and the subsequent amplification of the nucleic acid or, in particular, specific sections thereof, such as genes, with specific primers and transcriptases which allow the selective recognition of sequence variants of particular genes or nucleic acid segments present in the Related to the susceptibility to and / or the treatability of diseases and / or risks associated with exposure to certain substances, by binding to and amplification of appropriate sections or nucleic acids. The amplification makes it possible to determine which sequences are present (possible amplification means detection of the corresponding sequence).

In a further embodiment, the invention relates, in particular, to such a method or such a method, further comprising the partial or complete purification of the nucleic acid after lysis and before the amplification.

Yet another aspect of the invention relates to a method for carrying out a genetic analysis, in particular for testing the human body for sequence variations of specific genes or DNA segments, which are associated with susceptibility to and / or the ability to treat diseases and / or or risks in the Exposure to certain substances is used, including the extraction of appropriate nucleic acids (for example from oral mucosal cells and / or fragments thereof) from the oral cavity, in particular from oral mucosa cells and / or fragments thereof, by means of a compact chewing gauze and the subsequent extraction and amplification of the nucleic acid or in particular, specific sections thereof, such as genes, with specific primers and transcriptases which allow, in particular, an individual genome analysis, preferably the selective recognition of sequence variants of particular genes or nucleic acid sections, for the determination of physiological or pathophysiological characteristics, such as eye color, gene defects, paternity tests , genealogical studies, or the like, or in particular the selective recognition of those genes or nucleic acid segments associated with susceptibility to and / or the Therapeutic or examinations of gene polymorphisms in the context of screening procedures and for purely informative (eg empirical) purposes, in particular SNP analysis, by binding to and amplification of corresponding substances Sections or nucleic acids.

The following definitions of more general terms identify preferred more specific meanings that may be used individually or in combination with all or several of the various embodiments of the invention in lieu of the more general expressions, and thus may describe advantageous embodiments of the invention.

"Compact" means first and foremost that the gizzards predominantly on their surface a bond of

Oral mucosal cells, fragments thereof or nucleic acids

(especially DNA) - the presence of a

Sponge structure is therefore not required. The attachment is more likely due to a kind of "stickiness" or

Adulthood of the chewing mass, less on the inclusion (which, however, can not be excluded, even if it is not required). In other words, a "stickiness" rather than an absorbency is sufficient, which is surprising.

A chewing gum according to the invention includes common materials for the preparation of chews, such as chewing gums, which basically consist of an insoluble gum base and more or less soluble components.

As a base, it may contain the usual gum bases for chewing gums, if appropriate also resins, such as acacia, or waxes, such as carnauba wax.

Gum base, a water-insoluble mass, generally includes elastomers, waxes, fats, oils, fillers, texturizing agents, and may for example consist of conventional ingredients such as petroleum, lanolin, glycerm, polyethylene, Polivinylacetat, petroleum wax, stearic acid, or in particular natural resins, such as Mastic or natural gums or rubbers such as chicle, waxes and / or synthetic rubbers or gums such as ethylene-propylene rubber, polyisobutylene, isobutylene-isoprene copolymers, styrene-butadiene copolymers, polyisoprene, other polyolefins or thermoplastic elastomers (TPE) or the like, wherein one or more of said components may be present. For example, the gum base accounts for 5 to 95% of the weight of the chewing gum composition, typically, for example, 10 to 50%, often, for example, 15 to 35% of the chewing gum.

Other additives, such as flavorings, e.g. those which produce a medicinal taste, e.g. Menthol, thyme, cinnamon oil, linalool, limonene or the like may also be included. The proportions by weight thereof may be, for example, each in the range of 0.001 to 5 wt .-% for the individual additives, in total, e.g. at 0.005 to 10 wt .-%, are, for example, a total of 0.01 to 3 wt .-%.

Preferably, one or more abrasives are included in the chewing gums of the invention.

An abrasive (which facilitates the abrasion and adhesion of cells of the oral mucosa and fragments of such cells and free nucleic acids by its abrasive and sometimes also adsorptive action) is selected in particular from sparingly soluble calcium salts, silicon dioxide, complex silicates, aluminum oxides, powdered plastics, bioactive glass pastes, Titanium dioxide, talc and the like, or a mixture of two or more such abrasives. One or more abrasives are advantageous in a total weight fraction of 0.01 to 20%, in particular from 0.1 to 5 wt .-%, based on the total chewing mass includes.

As sparingly soluble calcium salts, e.g. Calcium phosphates such as dicalcium orthophosphate in the form of its dihydrate or anhydrous, tricalcium phosphate, calcium pyrophosphate, insoluble alkali metal metaphosphates, calcium phosphate, hydroxyapatite or calcium carbonate (chalk).

Silicas are in particular silicic acid (aqueous silicon dioxide), including before and after in particular aqueous silica gel (= aqueous silica gel) of preferably highly dispersed silicon dioxide, for example aqueous dispersions / gels based on Syloid® (WR Grace Davison Chemicals, Columbia, USA)), Aerosil® (Degussa AG, Frankfurt, Germany), Cab-o -sil® (Cabot Corporation, Tuscola, USA), Ludox® (Du Pont Chemicals, Wilmington, USA), Quso® (Philadelphia Quartz Corp., Valley Forge, USA), Santocel® (Monsanto Co., St. Louis, USA ), Siflox® (Chem. Oker.), Silin S 100® (van Baerle & Co. Chemische Fabrik, Gernsheim, Germany), Sipernat D22® (Degussa) or Spheron®

(Presperse Inc., Piscataway, USA), especially colloidal

Silica which is present in silica gels in the form of more or less highly condensed polysilicic acids. Particular preference is given to precipitated colloidal silica gel, amorphous silica gel or pyrogenic colloidal silica gel. For precipitation, for example, the precipitation of soda waterglass with acids, in particular hydrogen chloride in an aqueous solution is suitable, wherein the gel formation preferably takes place at pH 5.3 to 6 and then the resulting silica gel is washed until, for example, a sodium chloride content of, for example, less than 1, 5 g / l is achieved. Preferably, particles are obtained down to a size of about 10 nm, which, however, can agglomerate.

As the complex silicates, there may be mentioned, for example, such as zirconium silicate or aluminosilicates such as zeolite type A (for example, as described in EP 0 002 690 and 0 003 023).

Aluminum oxides may in particular be alumina trihydrates, with a preferred particle size distribution between about 1 and about 20, preferably about 10 microns. As powdered plastics, for example, polymers, such as polyacrylates or polymethacrylates, for example, as their sodium salts or esters, with a preferred particle size distribution between 0.5 and 5 microns may be mentioned.

Titanium dioxide may also be added as a whitening agent due to its action.

Furthermore, bioactive glass pastes are usable, for example glass based on silicon dioxide, sodium oxide, calcium oxide and diphosphorus pentoxide, eg S53P4 (Abmin Technologies Ltd., Turku, Finland), which consists of 53% SiO ₂ , 23% Na ₂ O, 20%. CaO and 4% P ₂ O ₅ exists.

For example, chewing gums according to the invention may also include sweetening agents such as sugars or sugar substitutes such as sugar alcohols (eg sorbitol, xylitol and / or mannitol) or other sweetening agents such as aspartame, sucralose, acesulfamic acid salts, alitan, neolan, saccharin and its salts, cyclamic acid and its salts, glycyrrhizin , Sterol, dihydrochalcones, thaumalin, monellin or the like.

In addition, other toxicologically acceptable additives, such as dyes, pigments, binders (such as cellulose or starch), film formers, such as shellac, salts, such as potassium chloride, buffers (eg based on 2- [N-morpholino] ethanesulfonic acid, bis [ 2-hydroxyethyl] iminotris [hydroxymethyl] methane, N- [acetamido] 2-iminodiacetic acid, N- [carbamoylmethyl] -2-aminoethanesulfonic acid, 3- [N-morpholino] -2-hydroxypropanesulfonic acid, piperazine-N, N ' bis [2-ethanesulfonic acid], citric acid, lactic acid, tartaric acid, fumaric acid or acetic acid, which (in the case of buffer substances present as base) with a Acid, for example HCl, or (in the case of buffer substances present as acid) with a base, eg

Sodium hydroxide, to a suitable pH), in particular to adjust a pH in the range of 6 to 8, complexing agents (also for reducing the degradation of

Nucleic acids), plasticizers, humectants (such as glycerol),

Antioxidants (such as ascorbic acid (vitamin C), propyl gallate, butylated hydroxyanisole (BHA), butylated hydroxytoluene

(BHT) or tert. Butylhydroquinone (TBHQ)), dentifrices (such as fluorides, eg sodium fluoride, sodium fluoromonophosphate or oleamine fluoride), the substantivity (attachment) increasing agents (such as mucoadhesive polymers, such as partial or fully synthetic hydrogel, especially cellulose derivatives (eg, methyl, hydroxyethyl, hydroxypropyl , Carboxymethyl or sodium carboxymethylcellulose), high molecular weight variants of the polyacrylic acid (carbomer, carbopol or polycarbophil), hyaluronic acid, mussel adhesion protein or chitosan, furthermore gelatin, pectins, lectins, eg adhesins, or polyoxyethylene-polyoxypropylene copolymers (eg poloxamer) or non-polymeric compounds , in particular sucrose-octa-sulfate, in particular in the form of its aluminum salt (known as sucralfate ^® = 1 ', 2, 3, 3', 4, 4 ', 6, β'-octa-O-sulfo-beta-D- fructofuranosyl-α-D-glucopyranoside-aluminum hydroxide hydrate); or emulsifiers, or mixtures of two or more thereof, in the chewing mass.

The sweeteners and additives may be included, for example, in total in a weight proportion of 0 to 20 wt .-%, for example, from 0.01 to 8% by weight.

It is particularly advantageous if the chewing gum contains an indicator which allows the display of the (for gene analysis) sufficient collection (material acquisition) of cells, Cell fragments and / or nucleic acids by means of the chewing gum allows. This may, for example, be a suitable dye that turns over on the dissolution of a soluble component in the chewing gum, for example a pH indicator which reacts to a change in the pH or an oxygen-sensitive indicator which is oxidized in the oral cavity and thus changes its color, or an indicator that changes its color by an enzyme present in the mouth (such as amylase) or a dye that is dissolved out during chewing so that the chewing gum loses color, or a dye that activates or decolorizes by the moisture in the oral cavity or more suitable enzyme reaction or protein binding or nucleic acid binding or the like, or more such indicators and methods, or the like. Corresponding indicator materials may be included, for example, in a proportion, based on the total mass of the chewing gum, of from 0.0001 to 10, such as, for example, from 0.01 to 5% by weight.

The invention thus makes it possible in a simple manner to determine, in particular, variations with respect to sequence sections with one or more nucleic acid bases, in particular individual bases in the DNA (so-called single nucleotide polymorphisms) of a human, and thus the test persons, such as humans, certain collectives with regard to therapy with drugs and / or to attribute sensitivity to substances, ie to select the therapy or the protective measures against potentially damaging substances as well as possible by means of appropriate sequence variations as biomarkers. Such single nucleotide polymorphisms can be found, for example, at http://www.pharmgkb.org/index.jsp or at http://www.ncbi.nlm.nih.gov/projects/SNP/, which are preferred here for such polymorphisms Reference be included. For example, this allows to diagnose suitable patients for particular therapies or to identify patients for whom exposure to certain substances poses a higher risk than others. The determination of other physiological or pathophysiological characteristics, such as eye color, gene defects, the performance of paternity tests or genealogical examinations, or the like, can also be done. Furthermore, investigations of gene polymorphisms are possible in the context of screening methods and for purely informative (eg empirical) purposes.

A corresponding complete diagnostic procedure therefore preferably includes in the strictest sense the step of a deductive medical decision phase as a purely intellectual measure, the preceding steps of

Processing of sample chewing gum and analysis of

Nucleic acid and possibly the specific sampling by means of the gum on a human. However, in another aspect, the invention also relates to the pure use of

Chewy masses for nucleic acid (especially DNA) recovery, regardless of the presence of the subject. Also stored

Samples from different volunteers can therefore be examined appropriately (for example, statistically or with regard to subjects).

Also corresponding methods are the subject of the invention, as well as corresponding methods of use.

Preferably, the processing of the chewing gum in the uses and methods of the invention can be such that a (for example, from a subject in a suitable container, such as a plastic bag, a

Tube, vials or the like) of a chewing gum according to the invention while avoiding contamination

(For example, under conditions customary for cell culture, such as under prints, on cell banks or the like) or GMP compliant is worked up.

For sequencing, which is only necessary if the detection of sensitivity and / or treatment-relevant sequence variations is not already successful by the amplification, conventional methods, such as, for example, methods using biochips, can be used.

In particular, methods can be used in which by means of interesting primers (for example, for certain interesting for sequence variations sections, as for certain diseases or treatment options relevant

SNPs) gene amplifications (for example by means of polymerase

Chain reaction method, such as PCR) performed and the nucleases are then sequenced accordingly obtained.

The lysis (also denoted as extraction, since it ultimately serves the extraction of nucleic acids, especially DNA) is preferably carried out by liquid homogenization (eg with Dounce or Potter Elvjehem homogenizers), ultrasound, freezing methods, mortar and pestle, shaking with particles such as glass beads or the like - corresponding methods, solutions, devices and the like are known and, for example, in the manual of Thermo Fisher Scientific Pierce (Thermo Scientific Pierce® Cell Lysis Technical Handbook, 2008), instructions from Quiagen (Hilden, Germany) or below or described in the examples. For example, the lysis / extraction can be carried out as described in the examples. Alternatively, external lysis and purification using other purification systems such as the MagNa Pure® LC system from Roche Diagnostics GmbH, Mannheim, Germany (hereinafter Roche) is conceivable:

Brief explanation of the isolation principle of the MagNA Pure LC: 1. Complete cell lysis and release of the DNA after addition of the lysis buffer. Proteins are denatured. 2. Addition of proteinase K and digestion of cellular proteins.

3. addition of magnetic particles (MGP); Binding of the DNA to the silica surface of the MGPs by the chaotropic salt concentrations, isopropanol, and the large ionic strength of the lysis / binding buffer.

4. Repeated washing of MGPs with wash buffers to remove unbound substances such as proteins and reduction of chaotropic salt concentration.

5. Elute the purified DNA at 70 ° C. ;

For this purpose, an external proteinase K digestion is first carried out.

For this purpose, 200 μl proteinase K (Roche DNA isolation kit I) are incubated for 1 h at 37 ° C with occasional shaking.

Then the 200 μl supernatant is transferred to the reaction vessels of the MagNa Pure® LC device. This is followed by the start of the program DNA I Blood_Cells High Performance.

Alternatively, a short external lysis without proteinase K can be made as follows: 200 μl lysis buffer (Roche DNA isolation kit I) is added, and it is incubated for 5 min at RT.

Transfer of the 200 μl supernatant into the reaction vessels of the MagNa Pure LC followed by the start of the program DNA I Blood_Cells High Performance.

Common methods of amplification include those named Polymerase Chain Reaction (PCR). Corresponding methods and protocols can be found, for example, in the following documents and / or examples.

The usual methods for sequencing include, for example, the methods according to 2.1 Maxam and Gilbert, the Danger Sox method, pyrosequencing, sequencing by hybridization, automated DNA sequencing methods, corresponding methods based on BIOCHIP methods (bioarrays, see, for example, DNA microarrays : A Molecular Cloning Manual (Paperback), David Bowtell (Editor), Joseph Sambrook (Editor), CoId Spring Harbor Laboratory Press, 1st Edition (2002)), or the like.

Common methods for lysis, purification, amplification and sequencing are found in well-known texts, such as Short Protocols in Molecular Biology (2 volumes), Frederick M. Ausubel (Editor), Roger Brent (Editor), Robert E. Kingston (Editor), David D. Moore (Editor), JG Seidman (Editor), John A. Smith (Editor), Kevin Struhl (Editor), 52 edition (November 5, 2002), ISBN 10: 0471250929 or ISBN 13: 978-0471250920 ; Molecular Cloning: A Laboratory Manual (Library Binding), J. Sambrook (Author), Joseph Sambrook (Author), T. Maniatis (Author), CoId Spring Harbor Laboratory Press (December 1989), see also www. cshprotocols. org as an online manual; Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench (Handbook) (Spiral-bound, Jane Roskams (Author), Linda Rodgers (Author), CoId Spring Harbor Laboratory Press (2002); or Genetic Engineering Methods (Paperback) by Hans Günter Gassen (Editor), Gangolf Schrimpf (Editor), Spektrum Akad. VIg., Hdg. ; Edition: 2nd A. (March 2002), or as described in the examples.

Examples:

The following examples illustrate the invention without limiting it.

Example 1: Sample collection / chewing mass:

The chewing mass consists of a conventional chewing gum mass (chewable mass) as a matrix, to which are added materials which ensure a better binding of nucleic acids from the cells of the oral mucosa to the matrix during the chewing process.

Specifically, this means: Recipe a): Chewy mass without sugar, with the following ingredients

(in descending portion): Chewing gum base (Gum

Base), xylitol, sorbitol, tricalcium phosphate, flavor, mannitol,

Acacia Senegal, talc, potassium chloride, aspartame,

Sodium monofluorophosphate, shellac, Cera Alba, carnauba, olus, limonene, linalool.

Recipe b)

Chewing gum base 25% by weight

Xylitol 1% by weight sorbitol 52% by weight

Mannitol 5% by weight

Acacia 2% by weight

Aspartame 0.8% by weight Carnauba wax 12% by weight

Silica 2% by weight

Thyme 0.2% by weight

Recipe c)

Chewing gum base 23% by weight

Xylitol 1% by weight

Sorbitol 52% by weight

Mannitol 5% by weight Acacia 2% by weight

Aspartame 0.8% by weight

Carnauba wax 12% by weight

Silica 4% by weight

Thyme 0.2% by weight

Recipe d)

Chewing gum base 26.5% by weight

Xylitol 1% by weight

Sorbitol 52% by weight mannitol 5% by weight

Acacia 2% by weight

Aspartame 0.8% by weight

Carnauba wax 12% by weight

Silica 0.5% by weight of thyme 0.2% by weight

Recipe e)

Chewing gum base 24% by weight

Xylitol 1% by weight sorbitol 52% by weight

Mannitol 5% by weight

Acacia 2% by weight

Aspartame 0.8% by weight Carnauba wax 12% by weight

Hydroxyapatite 3% by weight

Thyme 0.2% by weight

Recipe f)

454 g Chicle * (from the pharmacy)

1362 g of sugar

Essential oils 1.75 to 3.5 g (from the pharmacy)

10 g of titanium dioxide

Recipe g):

58 g gum base (from the pharmacy)

105 g of sorbitol powder (from the pharmacy)

32 g sorbitol liquid (70%) (from the pharmacy) 8 g glycerin (from the pharmacy)

3 g of flavors

2 g glass paste S53P4 (Abmin Technologies Ltd., Turku, Finland)

Use / Application:

A piece of gum (about 1.2 g) [here recipe a)] is chewed by the subject for 2 min.

Storage after chewing and removing the chewing mass at room temperature has been tested up to 20 days and is possible. Also storage at 40 ° C was tested for two days. In both cases the analysis was still possible.

Example 2: Isolation of the DNA from a chewing gum

The extraction of genomic DNA from chewing gum (formulation a) from Example 1) after chewing by a subject is carried out manually. The following solutions are presented:

1. 600μl PBS (Phosphate Buffered Saline) 2. 20μl Protein Kinase K (Qiagen, Hilden, Germany) 3. 600μl Lysis Buffer: AL Lysis Buffer (Qiagen)

Subsequently, 10 min. incubated at 56 ° C.

Then 600 μl of absolute ethanol are added and it is vortexed.

700 μl of the pre-lysate obtained are pipetted into a homogenizer for lysing and homogenized there.

Then follow 2 min. Centrifugation at maximum speed (Heraeus Biofuge, 13,000 rpm). The 700 μl obtained are pipetted into a QIAamp column (Qiagen, (in 2 ml tube)) The column is centrifuged at 8000 rpm 1 mm, then it becomes placed in a fresh tube (or decanted).

The steps from pipetting on a QIAmp column are repeated.

500 μl of AW1 buffer (guanidium-containing buffer, Qiagen) are pipetted into the column, and it is for 1 min. centrifuged at 8000 rpm. Then the column m is placed a new tube and the old discarded (or decanted).

500μl of AW2 buffer (Wash Buffer; Qiagen) are then pipetted into the column and it becomes 3mm. centrifuged at 13000 rpm. Then put the column in a fresh tube and discard the old one.

The solution is left for 1 min. Centrifuged at maximum speed (Heraeus Biofuge, 13,000 rpm).

For this purpose, the column is placed in a 1.5 ml Eppendorf tube (polypropylene tube, Eppendorf, Hamburg, Germany) (the tube is discarded).

150 μl AE buffer (protective buffer for DNA with TRIS and EDTA, Qiagen) (or aqua, dist.) Are pipetted into the column. Then 1 min. incubated at RT and then 1 min. centrifuged at 8000 rpm.

Finally, the DNA samples thus obtained are analyzed for CYP 2D6, CYP 2C9 and CYP 2C19 genes and their polymorphisms according to Taq real-time polymerase chain reaction (TaqMan Real-Time PCR)

It should be checked whether the test subject is accessible for a pain therapy with codeine, because it has been shown that he was not found a sufficient analgesic effect.

For "set up" genotyping ("GENO"):

Forward (reverse), reverse (reverse) primer and both probes are mixed in the ratio 1: 1: 1: 1 (primers and probes are from Applied Biosystems Inc., Foster City, USA, (hereinafter AB) as follows:

(accessible via: https: // products. appliedbiosystems.com / ab / en / US / adirect / ab? cmd =

ABGTKeywordSearch & catID = 600769)

Implementation (set-up) of the GENO:

2.5 μl Taqman® (Roche, Basle) Genotyping Master Mix 0.25 μl Primer 2.25 μl DNA (-10 ng / μl) or 2.25 μl H ₂ O for control (No template Control) -> total 5 μl

Program:

50 ° C 2 min 95 ⁰ C for 15 min 92 ° C 15 s

60 ° C 1 min temperature cycle 35x

Genotyping result for the tested subject:

CYP 2D6 * 4 * 4 * 4 poor metabolizer

CYP 2C9 * 2 * 1 * 1 normal to extensive metabolizer

CYP 2C19 * 2 * 1 * 2 medium metabolizer

Therefore, the patient is not able to receive sufficient pain therapy with codeine, since the patient has a markedly slowed down degradation via CYP 2D6 and the active metabolite morphine is formed too slowly.

Claims

claims 1. Compact chewing mass for use xn a method for obtaining nucleic acids from the oral cavity of a human body in the context of a diagnostic procedure on a human body. 2. The compact chewing mass for use according to claim 1, wherein the diagnostic method for testing the human body is suitable for sequence variations of specific genes or DNA segments which are related to a determination of physiological or pathophysiological characteristics, for paternity testing or genealogy studies, or in particular Related to the susceptibility to and / or the ability to treat diseases and / or risks associated with exposure to certain substances, or to further investigate gene polymorphisms within Screenmg procedures and for purely informative (eg empirical) purposes. Compact chewing gum for use according to one of claims 1 or 2, characterized in that it contains as base natural resins, such as mastic or chicle, waxes and / or natural or synthetic rubbers or latices, such as polyisobutylene. 4. Compact chewing composition according to one of claims 1 to 3 for the determination of emzelnukleotid polymorphisms (Single Nucleotide polymorphisms). 5. Compact chewing mass according to one of claims 1 to 4, characterized in that it includes an abrasive. 6. Compact chewing mass according to claim 5, characterized in that it contains the abrasive in a proportion by weight of 0.1 to 5%, in particular from 0.2 to 3 wt .-%, based on the chewing mass. 7. A compact chew according to claim 5 or 6, wherein the abrasive is selected from complex silicates, sparingly soluble calcium salts, silica, alumina, powdered plastics, bioactive glass pastes, titanium dioxide and talc. Compact chew according to one of claims 1 to 7, characterized in that it further includes an indicator which allows the display of a sufficient collection of cells, cell fragments and / or nucleic acids by means of the chewing gum. 9. Use of a chewing gum according to any one of claims 1 to 8 for the recovery of nucleic acids, oral mucosal cells and / or oral mucosa fragments from the oral cavity of humans. 10. Use according to claim 9, further comprising the determination of sequence variants of certain genes or DNA sections, which allow a determination of physiological or pathophysiological characteristics or the performance of paternity test or genealogical examinations, or in particular in connection with the susceptibility to and / or the treatment of diseases and / or exposure to certain substances, or Furthermore, investigations of gene polymorphisms in the context of screening methods and for purely informative (eg empirical) purposes. A method of isolating and detecting nucleic acids on a compact chewing mass, including lysing cells or cell debris primarily adhered thereto, and then amplifying the nucleic acid or, more particularly, specific portions thereof, such as genes, with specific primers and transcriptases which are the selective Recognition of sequence variants of specific genes or nucleic acid sections, which allow determination of physiological or pathophysiological characteristics or the performance of paternity tests or genealogical investigations, or in particular in connection with the susceptibility to and / or the treatment of diseases and / or risks of exposure against certain substances, or further studies of gene polymorphisms in the context of screening and for purely informative (for example, empirical) purposes, by binding to and Amplification of appropriate sections or Allow nucleic acids, and if desired, the Sequencing of the recovered nucleic acid. 12. The method according to claim 11, further comprising the partial or complete purification of the nucleic acid after lysis and before the amplification. 13. Method for testing the human body for sequence variations of particular genes or DNA sections, which determine the physiological or pathophysiological characteristics or the performance or in particular associated with the susceptibility to and / or the ability to treat diseases and / or exposure to certain substances, including the extraction of corresponding oral nucleic acids by means of a compact chewing gum and the subsequent amplification of the nucleic acid or, in particular, specific sections thereof, such as genes, with specific primers and transcriptases, which comprise the selective recognition of sequence variants of specific genes or nucleic acid segments which are associated with the susceptibility to and / or the ailability of diseases by binding and amplification of corresponding sections or nucleic acids, and, if desired, the subsequent sequencing of the recovered nucleic acid. 14. A method for performing a genetic analysis, in which first oral mucosal cells and / or cell fragments are collected with a chewing gum according to any one of claims 1 to 9, the cells or cell fragments are separated from the chewing mass, subjected to DNA extraction and an individual Gene expression analysis is performed. 15. Drug kit containing a drug and a compact chewing gum according to the invention for collecting oral mucosal cells and / or cell fragments.