Abstract:
A method of assessing the age or expectation of life wherein the length of the telomeres is determined by a blot method and, optionally, by subsequently hybridizing the DNA gel-electrophoretically separated and bound to the membrane obtained through the blot method by means of a nucleic acid probe complementary to the telomeric sequence, and wherein the specimen DNA containing the telomeric DNA prior to the gel-electrophoretic separation is subjected to a full restriction digestion, characterized in that the separation of the digested DNA in the gel-electrophoresis underlying the blot method takes place at (a) &lt;5 V/cm, and (b) for the duration of at least 6 hours.

Description:
FIELD OF INVENTION  
         [0001]    The present invention relates to a process for determining the age of a person, and also the statistical life expectancy of that person.  
         BACKGROUND OF THE INVENTION  
         [0002]    No procedures have been described as yet for determining the age or the average life expectancy of human beings.  
           [0003]    In a textbook on Forensic Medicine (Arbab-Zadeh A., Prokop O., Reimann W., Rechtsmedizin für Ärzte, Juristen, Studierende und Kriminalisten [Forensic Medicine for Physicians, Jurists, Students and Criminalists], 1. edition, Stuttgart, N.Y.: Gustav-Fischer-Verlag, 1977.) the following information can be found: “Is this a child&#39;s blood or is it from an adult? At the moment this question cannot be answered from lanes of blood.” There is, however, a well established method for distinguishing the blood of an infant from that of an older child or an adult, which is checking the presence of foetal hemoglobin (Arbab-Zadeh A., supra). But forensic medicine in particular would benefit greatly from a method to determine the age of a perpetrator—when profiling that person—with a tissue sample found at the site of the crime. Inquiries with criminal prosecution and the police have also shown that there is no procedure yet to determine donor age from samples of blood or saliva. Both agencies express strong interest in such a procedure.  
           [0004]    It would be yet another advantage, if a person&#39;s statistical life expectancy could be determined from a tissue sample. This could be valuable to insurance companies.  
           [0005]    Tests have been performed to measure telomer length (precisely: length of the terminal restriction fragment), proving that telomer length decreases continuously during in vivo aging (Hastie N. D., Dempster M., Dunlop M. G., Thompson A. M., Green D. G., Allshire R. C. Telomer reduction in human colorectal carcinoma and with aging. Nature (1990) 346, 866-868; Vaziri H., Schächter F., Uchida I., Wie L., Zhu X., Effros R., Cohen D., Harley C B., Loss of Telomeric DNA during Aging of Normal and Trisomy 21 Human Lymphocytes., Am. J. Hum. Genet. (1993) 52, 661-667).  
           [0006]    The level of correlation between in vivo aging and telemore reduction, however, has not been sufficiently investigated. So, until the present invention, it has been impossible in practice to decide the age from a person&#39;s tissue sample. Moreover, a test by Levy et al. has shown that there is no age specific telomer reduction (Levy T., Agoulnik I., Atkinson E. N., Tong X. W., Gause H. M., Hasenburg A., Runnebaum I. B., Stickeler E., Möbus V. J., Kaplan A. L., Kieback D. G., Telomer Length in Human White Blood Cells Remains Constant with Age and is Shorter in Breast Cancer Patients, Anticancer Research (1998) 18, 1345-1350).  
           [0007]    Due to these contradictory statements within the field, it has been completely unclear, if measuring telomer length could be utilized at all to determine age.  
           [0008]    For the research papers mentioned above telomer length was established through telomer specific nucleic acid marked with  32 P.  
           [0009]    There is also one non radioactive kit for assessing telomer length commercially available. It is the Roche Diagnostics GmbH, Telo TTAGGG Telomer Length Assay Kit, which uses a chemiluminescent substrate to detect telomer DNA. It is, however, a disadvantage that the corresponding procedure, according to the manufacturer, results in the lower and upper edges of the telomer smear being in different places according to the amount of DNA separated in the gel, after the gel electrophoresis is finished.  
           [0010]    There is another publication (Gomez D. E., Tejera A. M., Olivero O. A., Irreversible Telomer Shortening by 3′-Azido-2′,3′-Dideoxythymidine (AZT) Treatment, Biochemical and Biophysical Research Communications (1998) 246, 107-110), where detection of telomer length was done with a color reaction. But no tissue samples were examined, nor was age determined. Additionally, no lower and upper edge were established, since telomer length was determined only once for each individual sample.  
           [0011]    (To realize there is a defined upper and lower edge at all, one has to do several telomer detection procedures with one DNA solution in different concentrations. Only then one can be certain to see the upper and lower edge.)  
           [0012]    Moreover, blotting has apparently not worked properly, since the smears appear very pale when reproduced. Also, no information is given about the voltage used and the running time of the gel.  
           [0013]    There have been attempts by forensic institutions to infer age from telomer length (Jefferies J M C., Watson N D., Smith W E., Investigation af Donor Age Using Telomer Lengths from Simulated Biological Samples. Progress in Forensic Genetics (1999) 8, 27-29). Up to now this research has, however, not had tangible results.  
           [0014]    In summary, this makes it clear that it is as yet not possible to determine the donor&#39;s age from a tissue sample.  
           [0015]    Thus, in view of the above mentioned state-of-the-art it is an object of the present invention to enable determination and estimation of a person&#39;s age and average life expentancy. Further objects not expressly mentioned also underlie the present invention and arise from and are closely connected to the current state of the art. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]    The present invention is further described with reference to the drawing, wherein  
         [0017]    [0017]FIG. 1 is a diagram of telomer length test results.  
         [0018]    [0018]FIG. 2 shows the photographic evaluation of a southern blot procedure done in accordance with this invention. 
     
    
     DETAILED DESCRIPTION  
       [0019]    By determining telomer DNA length, it is possible to determine the sample donor&#39;s age and most likely the average life expectancy in a rather simple manner, by comparing results with standard samples according to the positioning of the signal on the southern blot.  
         [0020]    For this, the telomer DNA length of a tissue sample is being determined with a southern blot procedure and ensuing hybridization of the DNA connected to the membrane, separated by gel electrophoresis, that has been obtained through the southern blot procedure with a nucleic acid probe that is complementary to the telomer sequence.  
         [0021]    In this process, the sample DNA containing telomer DNA is subjected to a complete restriction digestion—before the gel electrophoresis separation—through a restriction enzyme, that has a specific identification point of 4-base pairs, and it is characterized as follows:  
         [0022]    (a) the separation of the digested DNA in the gel electrophoresis, which is the basis of the southern blot procedure, is being done at &lt;5 volt/cm and for a period of at least 6 hours,  
         [0023]    (b) a marked oligonucleotide probe, complementary to the telomer sequence, is being used for the specific telomer DNA detection for the hybridization,  
         [0024]    (c) the detection of the telomer DNA complex obtained from step (b) is being carried out with a color reaction and  
         [0025]    (d) comparison values are being determined through application of known samples in the gel electrophoresis, which is the basis of the southern blot procedure.  
         [0026]    Proceeding in accordance with the invention makes it possible, surprisingly, to increase the accuracy of measuring the (medium) telomer length so much, that age can be determined for some of the samples&#39; donors.  
         [0027]    Proceeding in accordance with the invention furthermore makes it possible to establish a lower and upper edge of the signal that has been obtained through the southern blot procedure, which can be used as an indication for determining length, apart from the medium telomer length.  
         [0028]    In this procedure, as opposed to the methods used until now, the exact concentration of the DNA used is surprisingly not a decisive factor, because with the invention&#39;s method, as opposed to the traditional methods for determining telomer length, the lower and upper edge of the signal has an almost consistent position on the southern blot for a large range of DNA concentrations.  
         [0029]    The southern blot procedure, very well known to experts, is used to transfer DNA from a gel, agarose or polyacrylamide for instance, after the gel electrophoresis separation, onto membranes, nylon membranes for instance, which are then accessible for other analytical processes.  
         [0030]    For example, the DNA that is attached to the membrane can be hybridized with specific DNA probe molecules. The specificity of the hybridization reaction is being controlled through the choice of reaction conditions, in which stringent (high content of salt in the hybridization buffer, low temperature) to very stringent hybridization conditions (low content of salt in the hybridization buffer, high temperature) are being used.  
         [0031]    Normally the DNA probe molecules are being marked in some way. For example, the probe molecules could be marked through radioactivity, or carry a residue of biotin, or they could be connected to enzymes, antibodies or other proteines and/or peptides.  
         [0032]    The probe molecules that have been specifically hybridized to the DNA on the membrane are then being detected through an agent, which interacts in some way with the marking connected to the probe molecule.  
         [0033]    In the case of a radioactive marking it is sufficient to do an auto radiograph, where an x-ray film is being blackened by the radioactive marking in the place, where the specific probe molecules are connected to the membrane. The signal obtained on the x-ray film can then be allocated to the DNA that has been separated by gel electrophoresis.  
         [0034]    In other cases for instance, clear substrates are being added that can interact with enzymes and release a colored product, that may then be documented through photography.  
         [0035]    All this is well known among experts, and this performance example as well as others of the southern blot procedure used in accordance with the invention can be found in numerous textbooks, of which only a few will be mentioned here: Sambrook J:, Fritsch E F., Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, New York: Cold Spring Habour; Bertram S., Gassen H G. (1991), Gentechnische Methoden, eine Arbeitsanleitung für das molekularbiologische Labor [Methods in Genetic Engineering, an Operation Manual for the Molecular Biological Laboratory], Stuttgart, Jena, N.Y.: Fischer G.; Cornel M. (2000), Der Experimentator: Molekularbiologie [The Experimenter; Molecular Biology], Heidelberg, Berlin: Spektrum Akad. Verlag.  
         [0036]    For the purposes of this invention, it is particularly important that the separation through gel electrophoresis of the sample DNA, which has been treated with the restriction enzyme, is being run slowly, that is with &lt;5 volt/cm, and over a long period of time, that is &gt;4 hours. Thus a good separation of the sample DNA is guaranteed.  
         [0037]    Furthermore, the procedure should be carried out using color reactions for the detection of the specific complex of telomer DNA and probe DNA.  
         [0038]    Especially when there is only very little sample material and thus little DNA, as is often the case in forensics, it makes sense to stretch out the color reaction as long as possible, i.e. several days or even weeks. With this concept the intensity of the coloring can be brought up to a sufficient level. Color reaction should be construed as follows within the scope of this invention: Detection of the telomer smears is being done through a visible chemical substance. This detection is called chromogene detection.  
         [0039]    In contrast to this, the detection of telomers in the indirect detection process is being done with some sort of radiation, either radioactive (P 32 ) or non radioactive (chemiluminescence, Roche), whereby radiation always has to be documented (for example with an x-ray film).  
         [0040]    It is therefore clearly understandable to the expert, that for marking the oligonucleotides it is favorable to use such systems suitable for creating a specific color reaction and also well known in the field. Examples of suitable dyes are 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) for the alkaline phosphatase. For the horseradish peroxidase chloro naphthol is a suitable dye. Also, processing should be done without vortex mixing, if possible, so the DNA is not exposed to strong shear forces unnessecarily. The largest part of the isolated DNA should have a molecule length of over 30,000 base pairs.  
         [0041]    Moreover, it is presumably favorable to use high concentrations of the marked oligo- or polynucleotide, complementary to the telomer sequence, for exmaple 1 μg/ml, which is roughly ten times of the concentraion commonly used.  
         [0042]    Oligo- or polynucleotide means: &gt;6 base pairs, and include nucleic acid mimics, like paptide nucleic acids. In a favored performance method of the present invention an agarose gel is used, which has an agarose concentration of 1.3-1.6% (w/w) in a lower section and 1-1.4% (w/w) in an upper section; the gel electrophoresis is to be run for at least 8 hours, with the lower section being the one that determines the size of the lower edge of the telomer smear, and the upper section being the one that determines the size of the upper edge of the telomer smear. It has been shown that the separation works even better then and the lower and upper edge of the signal can be defined even clearer.  
         [0043]    In another favored performance method of this invention different amounts of the DNA to be examined (i.e. as 1 μl, 5 μl and 10 μl) are being applied next to one another, since especially in high DNA concentrations and long reaction periods of the detection reaction, even those parts of the lane, where no hybridization has taken place, could be colored. It could then be difficult to define the lower and upper edge of the smear.  
         [0044]    The just mentioned coloring of parts of the lane, where there are no telomers present, happens first in the lane where the largest amount of DNA was applied. As long as the upper and lower edge in all lanes have the same length, one can be sure not to have been fooled by this artefact.  
         [0045]    In accordance with the invention the procedure can be carried out with all human tissue samples, from which DNA can be isolated. This will preferably be samples of, saliva and/or blood, skin, hair. Samples of other tissues or secretions can be used as well.  
         [0046]    As mentioned above, in order to define age it is simplest to compare the sample with one being tested in the same procedure. In another equally favored performance method of this invention statistical data are being collected for all cohorts in question, in order to define a calibration line, which is the basis of determining age.  
         [0047]    For defining a person&#39;s life expectancy especially, this is particularly convenient, since otherwise a very large number of comparison samples would have to be carried along in the test.  
         [0048]    [0048]FIG. 2 shows the photographic evaluation of a southern blot procedure done in accordance with this invention. The specific test conditions as well as the occupied lanes are shown in example 1.  
         [0049]    The following example serves to illustrate the invention, should however not be construed as a limitation.  
       EXAMPLE 1  
       [0050]    The genomic DNA collected from a saliva or blood specimen, 200 μl, was isolated by conventional methods (Sambrook, I.; Fritsch E F., Maniatis T. (1989), Molecular Cloning: A Laboatory Manual, New York: Cold Spring Habour; Bertam S., Gassen H G (1991), “Gentechnische Methoden, eine Arbeitsanleitung für das molekularbiologische Labor”, Stuttgart, Jena, N.Y.: Fischer G.).  
         [0051]    (The specific method employed is not relevant; what alone is important is the fact that the DNA still is of a high molecular weight (the bulk of the un-cut DNA after isolation still has more than 30,000 base pairs, preferably 100,000 pairs, i.e. it was not exposed to excessive shearing forces). Accordingly, the specimens, during processing, should not be vortexed. Simple shaking or finger snapping will be adequate.  
         [0052]    Thereafter, the DNA was completely cut by the restriction enzyme Alu I (Roche Diagnostics GmbH) having an identification sequence of four base pairs (genomic digestion). The restriction enzyme Alu I does not cut within the telomer as does the majority of enzymes. The cut DNA was electrophoretically separated in an agarose gel (1.3% w/w) at a 30 V voltage (corresponding to 1 V/cm) over 20 hours. Then the gel (10×10 cm) was blotted equally by conventional methods, using a vacuum-blotter (Pharmacia) at 40 mbar:  
         [0053]    After applying vacuum, 15 ml of a depurination solution (0.25 N HCl) was added to the surface of the gel. After 30 min the depurination solution was replaced by 15 ml of a denaturation solution (1.5 M NaCl; 0.5 M NaOH) for 30 min. Similarly, the denaturation solution was replaced by 15 ml of a neutralizing solution (1.0 M Tris; 2.0 M NaCl adjusted with HCl to pH 5.0) for 30 min.  
         [0054]    Then the neutralizing solution was replaced by 20 ml 20×SSC for 60 min. From time to time an aliquot of 20×SSC was added in order to insure, that the gel be permanently covered by liquid. After completion of the transfer, the membrane was removed and washed in 10×SSC for 1 min. Then the DNA was fixed on the membrane by UV-radiation (125 mJ).  
         [0055]    Then Prehybridization (for at least one hour) was performed in 20 ml of a prehybridization solution (5×SSC, blocking reagent (Roche) 1% (added from 10% stock solution), N-lauroylsarcosine 0.1%, SDS 0.02%) at 42° C. in a thermostat-controlled hybridization incubator.  
         [0056]    After that the prehybridization solution was discarded and replaced by 15 ml of a hybridization solution (prehybridization solution and oligonucleotide probe (TTAGGG) 4  (1 μg/ml) labelled with digoxigenin). Before adding the same, the hybridization solution was preheated for 10 min at 90° C. but was not allowed to boil. After hybridization (overnight) the hybridization solution was frozen to be re-used in the next experiment.  
         [0057]    The non-hybridized oligonucleotides freely contained in the hybridizing solution are removed by a number of washing steps: 1 st  washing in 5×SSC (0.75 M NaCl, 0.0075M sodium citrate, ph-value 7.0) for 15 minutes at a temperature of 60° C.; 2 nd  washing in 4×SSC for 15 minutes at 60° C.  
         [0058]    The telomers were then rendered visible by means of a labelling and detection reaction responding to the substance used for labelling purposes; preferably, the DIG DNA Labelling and Detection Kit from Roche Diagnostic (formerly Boehringer Mannheim).  
         [0059]    As a DNA size standard (e.g. λ-Hind) normally is also separated during the gel electrophoresis, the telomer size could be determined by the distance covered.  
         [0060]    As the telomer length is not identical for all cells within a cell population or even for all chromosomes within one cell, the telomers do not appear as individual bands but rather as smears. The center of the smear is then assumed to be the (average) telomer length.  
         [0061]    The test result is shown in FIG. 1. The analysis and the sequence of the specimens are set out in Table 1.  
         [0062]    Now, the position of the telomer smears can be seen by the naked eye. It will thus already be possible to associate a large number of persons to the old or to the young group. However, in order to obtain precise molecular weights the telomer smears will have to be analyzed by a densidometer or a scanner. The Image Station 440 cf from Kodak in combination with the 1D Image Analysis Software likewise from Kodak, can be suitably used. Moreover, it can be useful to take a picture of the membrane and to scan the same. In particular, telomer smears of a low intensity can be analyzed more accurately in this way.  
         [0063]    [0063]FIG. 1 shows the exact results for all blood specimens and shows the superiority of my method.  
                                                                                             TABLE 1                           lane (top)   1   2   3   4   5   6   7   8   9   10   11               Age   λ-   18   64   60   18   19   20   23   32   65   λ-       (years)   Hind                                       Hind       Average       14.000   6.300   6.500   8.800   9.900   9.900   8.700   8.700   8.200       telomer       length (bp)                    lane (below)   1   2   3   4   5   6   7               Age (years)   λ-Hind   65   59   57   24   63   λ-Hind       Average telomer       6.400   6.800   11.200   13.000   6.800       length (bp)       Top border (bp)       25.800   21.300   25.200   30.600   27.900       Lower border (bp)       2.900   2.900   4.000   4.000   3.300                  
 
         [0064]    Unfortunately, no upper or lower borders could be determined for the upper series as the colour of the background of the lane is too intensive. The only person that would be wrongly associated in this test would be the one in lane 4, bottom. It has relatively long telomers although that person is already 57 years of age.  
                                                                                                                                                               TABLE 2                       lane   1       3   4   5   6   7   8   9   10                                (top            Amount   λ-Hind   10 μl   5 μl   2 μl                       λ-Hind       Average       8.500   8.500   7.100       telomer length       (bp)       Top border       30.000   31000   30.000       (bp)       Lower border       3.900   4.000   4.000       (bp)            (below)            cell line                   MM6   MM6   MM6   U87   U87           Amount   λ-Hind               10 μl   2 μl   5 μl   10 μl   5 μl   λ-Hind                  
 
         [0065]    In lanes 2,3 and 4, top, different quantities of one and the same DNA preparation from blood (approximately 1 μg/μl DNA) were applied. It can be clearly seen that the upper and the lower border of the telomer smear, in all three lanes, have almost identical values.  
         [0066]    Similarly, lanes 5 through 9 clearly reveal in respect of the telomer smears recovered from cell cultures that the smears have the same sizes. In view of the intensive background colouring, a numerical determination will involve substantial inaccuracies.