A method for accurately determining age from mitochondrial DNA by specifying the relative N6-methyladenine level at a specific site

Abstract

The subject of the present invention is a molecular biological method for accurately determining the relative (normalized against an internal control) N6-methyladenine (6mA) level at selected sites of mitochondrial (mt) DNA (deoxyribonucleic acid) (mtDNA) in an tissue sample and projecting this level onto a previously constructed reference "relative mtDNA 6mA level - age" curve. In the reference curve, the relative mtDNA 6mA level is inversely correlated with age, meaning that the higher the relative mtDNA 6mA level, the younger the individual being analyzed. When the value of the relative mtDNA 6mA level intersects the reference curve, this intersection assigns the corresponding age on the X-axis. The reference curve was previously established by determining the 6mA level of a specific mtDNA site in a large number (>1000) of healthy individuals of known age. The accuracy of the measurement depends on the accuracy of the method for determining the mtDNA 6mA level. This technique is also suitable for predicting the life expectancy (how long an individual will live) of individuals of known age. The present invention is based on the recent biological discovery that the 6mA level in a specific mtDNA region is negatively correlated (inversely proportional) with the age of the individual being analyzed. This method can be applied in the following major fields: i) age determination in forensic procedures; ii) life expectancy prediction; iii) identification of the early stages of neurodegenerative processes.

Description

Technical Field

[0001] The subject of the present invention is to accurately determine the relative (normalized to an internal control) N6-methyladenine (6mA) level at selected sites of mitochondrial (mt) DNA (deoxyribonucleic acid) (mtDNA) in tissue samples and project this level onto a previously constructed reference "relative mtDNA 6mA level - age" curve. In the reference curve, the relative mtDNA 6mA level is inversely correlated with age, meaning that the higher the relative mtDNA 6mA level, the younger the individual being analyzed. When the value of the relative mtDNA 6mA level intersects the reference curve, this intersection assigns the corresponding age on the X-axis. The reference curve was previously established by determining the 6mA level of a specific mtDNA site in a large number (>1000) of healthy individuals of known age. The accuracy of the measurement depends on the accuracy of the method for determining the mtDNA 6mA level. So far, there is only one method that can accurately determine the relative 6mA level at selected genomic sites (patent application "PCR-based method for accurately determining...", file numbers P2100409 and W2200015). In this method, individual genomes isolated from a tissue sample to be tested are digested with a 6mA-dependent restriction endonuclease, the resulting genomic fragments are ligated to a linker DNA fragment called an adapter, and finally, sequence-specific PCR (polymerase chain reaction)-based DNA amplification is performed using a forward primer that is specific simultaneously to the downstream part of the linker DNA fragment and the target mitochondrial genomic site adjacent to the linker. Ligation of the linker enables direct amplification of the selected methylated (digested) adenine nucleobases. The amount of the PCR product is proportional to the relative (correlated with another mtDNA site) 6mA level determined at a specific mtDNA position.

[0002] Specifically, this innovation is a molecular biology technique that accurately determines the relative 6mA level at specific mtDNA sites within tissue samples containing multiple individual genomes, and extrapolates this level onto a previously established "mtDNA 6mA level - age" curve to accurately determine the age of the individual being analyzed. This technique is also suitable for predicting the life expectancy (how long an individual will live) of individuals whose age is known. The present invention is based on the recent biological discovery that the 6mA level in specific mtDNA regions is negatively correlated (inversely proportional) with the age of the individual being analyzed. Thus, throughout adulthood, the epigenetic process of methylation of N6-adenine in these specific mtDNA regions continuously weakens and serves as a reliable indicator of the aging rate and biological age of an organism. This method can be applied to the following major fields: 1. Age determination in forensic procedures (Since the age can be accurately determined from biological traces of unknown criminals, i.e., the remaining tissue samples, the number of suspects can be significantly narrowed down, leading to the success of the investigation process). 2. Life expectancy prediction (The life expectancy of individuals whose age is known can be predicted, and it can be found that there are significant changes in lifestyle and administrative issues (e.g., property) in light of this data). 3. Identification of the early stages of neurodegenerative processes (The rate of the aging process is different between normal individuals and patients suffering from neurodegenerative pathologies. Therefore, if the age estimated from the determined mtDNA 6mA level is significantly different from the actual age of an otherwise normal-looking individual, this may indicate the early stage of the neurodegenerative process before the onset of cognitive decline, and this early detection can facilitate the selection of effective treatment methods).

Background Art

[0003] N6-methyladenine (6mA) epigenetic mark The genetic material (genome) of an organism is composed of DNA (deoxyribonucleic acid), and in many cases, the genome is divided into functional units called chromosomes (chromosomes exist as complete DNA strands). DNA is constructed from nucleotides (constituent elements, each composed of a ribose, a phosphate group, and an organic base called a nucleobase), and the nucleobases differ from each other as either adenine, guanine, cytosine, or thymine. The genetic information stored in DNA is basically determined by the order of nucleotides. This is called the DNA sequence. Human genetic information (haploid genome) is composed of 3.1 billion nucleotides and is organized into 23 pairs of chromosomes, 22 pairs of autosomes, and 1 pair of sex chromosomes.

[0004] The functional unit of a chromosome is called a gene, and its products (proteins and RNA (ribonucleic acid)) manipulate the cell. At the DNA level, gene activity can be changed mainly in two ways. The first is at the gene level. In this case, specific nucleotides are changed (lost, added, or converted to another nucleotide). Such changes are related to mutations and genetic polymorphisms. The second is at the epigenetic level. In this case, the order of nucleotides remains the same, but small chemical changes, the addition of a methyl group (-CH3), affect specific nucleobases, adenine or cytosine. Adenine is mainly converted to N6-methyladenine (6mA), and cytosine is mainly converted to 5-methylcytosine (5mC) (Figure 1). Generally, 6mA has a role in gene activation (when changes occur within the genome, the affected gene is activated), while 5mC has a role in gene repression (maintaining genome stability by X chromosome inactivation, genome imprinting, and repression of transposable elements called "jumping genes"). These epigenetic changes are often caused by environmental factors and are inherited by daughter cells and subsequent generations. The 6mA mark is generated from adenine, and the process is catalyzed by the DNA methyladenine transferase enzyme (Figure 1). 6mA is reconverted to adenine by the action of the 6mA demethylase enzyme (Figure 1).

[0005] DNA N6-adenine methylation is widely observed in plants and bacteria. This process was recognized in animal and organelle (mitochondria) genomes only a few years ago (Greer et al., 2015; Zhang et al., 2015; Wu et al., 2016; Hao et al., 2022). However, recent studies have suggested that the presence of 6mA in animal and organelle genomes is merely the result of methodological artifacts (Schiffers et al., 2017; O'Brown et al., 2019; Douvlataniotis et al., 2020). Nevertheless, the biological function of the 6mA mark remains mostly unexplained.

[0006] Techniques for demonstrating DNA methylation (epigenetic mark) To prove the existence of methylated DNA nucleobases (6mA and 5mC epigenetic marks), several techniques have been developed. These techniques include single-molecule real-time sequencing (SMRT-seq), bisulfite sequencing (the genome is treated with sodium bisulfite and cytosine nucleobases are converted to uracil nucleobases, but 5mC is not altered by this compound), liquid chromatography tandem mass spectrometry (LC-MS / MS), and labeling (hybridization) with 6mA / 5mC-specific antibodies (Dahl and Guldberg, 2003; Flusberg et al., 2010; Rocha et al., 2010). These methods are quite costly (requiring expensive equipment and advanced expertise), time-consuming, and difficult to obtain (e.g., SMRT-seq is provided by only a few companies worldwide), so their application to diagnosis has not been essentially established in medicine and forensic genetics. However, the biggest drawback is that artifacts are frequently generated (OBrown et al. 2019; Schiffers et al., 2017; Douvlataniotis et al., 2020). This is mainly due to the fact that the epigenetic marks of 6mA and 5mC are widely present in the genomes of bacteria that often infect eukaryotic tissues and in eukaryotic RNA. Also, artifacts may be generated due to insufficient specificity of antibodies against 6mA and 5mC or due to technical limitations of SMRT-seq. Furthermore, the above techniques cannot identify the amount (relative level) of methylated nucleobases at specific genomic sites within the analyzed tissue sample (genomic DNA is isolated from a large number of cells), that is, they cannot identify the number of individual genomes methylated at specific genomic sites within the sample, but rather can only provide a "yes" or "no" answer regarding the methylation status of the nucleobases examined.

[0007] Alternatively, attempts have been made to determine 5mC levels using 5mC-dependent / sensitive restriction enzymes followed by PCR-based amplification of target sites (Luo et al., 2016, Yao et al., 2017). However, even these methods cannot accurately determine the methylation levels of specific DNA sites within tissue samples. These DNA methylation analysis techniques are still rarely used in the fields of medicine and forensic science. Recently, a new method has provided a solution, in which genomic DNA isolated from tissue samples is digested with 6mA- or 5mC-specific restriction endonucleases, the resulting DNA fragments are ligated to linker DNA fragments, and finally, representative sites are amplified by a PCR reaction mediated by a forward primer specific for both the linker sequence (10 - 15 nucleotides) and the adjacent genomic sequence (10 - 15 nucleotides). The application of linker DNA fragments enables the direct amplification of digested (methylated) DNA sequences. This is important because the methylation rate of specific nucleobases is a relatively rare event; only a small number of genomes within a tissue sample are methylated at a specific genomic location at a specific time. Using this method, the amount of PCR product reliably reflects the relative 6mA or 5mC level of the selected genomic site (Patent Application: "PCR-based method for accurate determination of...", File Numbers: P2100409 and W2200015, and the inventors have granted us permission to confirm the content of the application under the condition of strictly maintaining IP rights (NDA)). Among the individual genomic sites that make up the tissue sample being examined, the more genomes that are methylated at the selected genomic site, the greater the amount of PCR product generated.

[0008] DNA Methylation in Age Determination Over the past decade, it has been shown that the epigenetic mark 5mC is formed in an age-dependent manner in the human genome. 5mC mainly accumulates at transposable element (TE) loci during early development and plays an important role in suppressing these mutagenic sequences, thereby maintaining genome stability (Yoder et al., 1997). Dr. Steve Horvath (UCLA, USA) developed an algorithm, "Horvath's clock," that can determine the biological age of human tissue samples by specifying the 5mC content of specific genomic regions (Horvath, 2013). He demonstrated that the 5mC content of these genomic regions decreases with age. Measurement of the 5mC content in specific genomic regions is basically performed by bisulfite sequencing, and the results are used for measuring biological age with an average error of 7 to 10 years (±7 to 10 years). This is not accurate enough to apply this technology in medicine and forensic genetics, where high accuracy is required. This method is used by several companies (such as Chronomics, Altos Labs, Zymo Research, Elysium Health, Ra Pharmaceuticals Ltds, etc.) in the field of DNA diagnosis, but the results are only reference for laypersons. Furthermore, it has been found that throughout life, 5mC levels increase at specific genomic positions and decrease at other genomic positions, and the methylation process of 5-cytosine is greatly affected by environmental and physiological factors. Finally, organisms such as the nematode (Caenorhabditis elegans) and the fruit fly (Drosophila melanogaster) essentially lack the methylation phenomenon of 5-cytosine. Considering these facts, it can be said that specifying the 5mC level in specific genomic regions is not sufficient to accurately determine an individual's (biological) age. Summary of the Invention Problems to be Solved by the Invention

[0009] Problems Solved by Innovation We have shown that the relative 6mA levels at specific sites decrease with aging throughout adulthood in human mitochondrial DNA (mtDNA) samples (Figure 2). Although the biological significance of this phenomenon is unclear at present, the 6mA epigenetic mark at specific mtDNA sites is a powerful marker of biological age. The present invention is based on the observation that the relative 6mA level at specific sites of the mitochondrial genome decreases with the aging of the organism. To the best of our knowledge, such findings are not found in the literature. Using specific primer pairs, we determined the relative 6mA levels at multiple sites of the mitochondrial genome and found that a gradual age-dependent decrease was observed at each site analyzed. Gradually Subsequent to this, we determined the relative 6mA levels at specific sites in a large number (hundreds) of healthy individuals of known identity (age) and generated a reference "relative mtDNA 6mA level - age" curve in which the 6mA level is inversely correlated with age (Figure 3). The lower the determined 6mA level, the older the individual being analyzed. This data set is referred to as the reference "relative mtDNA 6mA level - age" curve. Determining the relative 6mA level at this specific site in the mtDNA of an individual of unknown identity and projecting the obtained value (on the Y-axis) onto the reference curve assigns the age of that individual at the intersection projected perpendicular to the X-axis (Figure 3). This method provides relatively accurate (average error of 2 - 3 years) and specific (artifact-free) data and is fast (PCR-based) and cost-effective, and thus can be used to establish epigenetic DNA diagnosis, a potentially important area in the DNA diagnostics industry.

Means for Solving the Problems

[0010] This method is applicable to the following major diagnostic fields:

[0011] This method can be applied to the following main diagnostic fields: 1. Forensic genetics. In the field of criminal behavior, it is common for biological traces (evidence, such as hair or blood droplets) of the perpetrator to be collected. In such cases, genomic DNA is isolated from the remaining traces and the DNA profile of the perpetrator is determined. The profile is often based on individual DNA polymorphisms (e.g., the number of repeats of short DNA repeats called microsatellites). Next, the results (profile) are compared with a criminal DNA database containing known perpetrators' DNA profiles, and if there is a match, the identity of the perpetrator can be recognized. However, in most cases, the DNA database does not contain a profile that matches the actually identified DNA polymorphism (i.e., the perpetrator's profile has not yet been introduced into the DNA database). In such cases, by specifying the relative 6mA level of a specific mtDNA locus from the biological trace and comparing that value with the reference "relative mtDNA 6mA level - age" curve, the age of the perpetrator can be accurately specified (e.g., 56 ± 2 years - i.e., an error limit of 2 years) (Figure 4). Considering the specified age, the number of suspects can be significantly narrowed down, greatly facilitating the success of the investigation process.

[0012] 2. Prediction of the lifespan of an individual of known age. Even within the same species, the lifespan varies among individuals. For example, if three 50-year-old people are randomly selected, theoretically the first person will live for 56 years, the second person for 66 years, and the third person will die at the age of 34. That is, even individuals of the same age can have significantly different lifespans. Using the above method, the predicted lifespan (how long one will live) of an individual of known age can be predicted. If the relative 6mA level at a specific mtDNA site in the tested person is significantly higher than the level predicted from the reference curve at a specific age, that value predicts a shorter lifespan than predicted. Therefore, the higher the relative mtDNA 6mA level at a specific age, the shorter the predicted lifespan of that person. It is important to note that this is only a "prediction". This is because, for example, due to a fatal accident, that person may die much earlier than predicted from the 6mA level. People who receive an unfavorable prediction (short average lifespan) from the test can significantly change their lifestyle (e.g., starting physical activity, avoiding stress, improving nutrition) to reverse the aging rate of their cells. Additionally, an unfavorable average lifespan can also prompt the start of preparing a will.

[0013] 3. Early detection of neurodegenerative processes. If the relative mtDNA 6mA level identified in an individual of known age is significantly different from the level predicted from the reference curve at a given age, that value may indicate the initial stage of the neurodegenerative process (before the onset of cognitive decline) (Figure 5). This is because the rate of the aging process in individuals affected by neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or ALS) is different (usually faster) from that in individuals not affected at the same age, and this difference can be accurately measured by the method introduced in this patent application (the higher the relative mtDNA 6mA level at a given age, the faster the rate of the aging process). Therefore, the relative 6mA level at specific mEDNA sites can be used as an early marker for various neurodegenerative pathologies. This is an important discovery. Because when a neurodegenerative disease manifests (apparent cognitive impairment), the major parts of the brain have already been affected by neuronal death, and it has been proven that the applied pharmacological treatments are mostly ineffective (dead neurons cannot be regenerated). An early (well before the onset of cognitive decline) diagnosis can provide a solution, but an appropriate marker for this has not yet been generated. When the relative mtDNA 6mA level indicates involvement in an otherwise seemingly normal (healthy) individual, it is recommended that the patient undergo further PET (positron emission tomography) or NMR (nuclear magnetic resonance) examinations to recognize the presence of the degenerative process and initiate drug therapy.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] Detailed Description of the Innovation To determine age from mtDNA, the following steps need to be carried out: i) identify the relative 6mA level at specific mtDNA sites within tissue samples taken from an unknown individual by a PCR-based approach (semi-quantitative PCR or real-time quantitative PCR); ii) extrapolate the identified 6mA level (Y-axis) and assign a value to the reference “relative mtDNA 6mA level - age” curve; iii) project this value (intersection point) onto the X-axis to assign the age of the individual. To accurately perform this series of experiments, to date, only a single method of performing the following steps is available: i) isolation of genomic DNA from tissue, ii) digestion of genomic DNA by a 6mA-specific restriction endonuclease (e.g., Dpnl which cuts DNA at the -GATC- sequence only when A is methylated: -GAMeTC-), iii) ligation of linker DNA fragments to the digested genomic DNA fragments, iv) PCR amplification of the target (digested) site using a forward primer specific to both the linker and the adjacent mtDNA sequence simultaneously, v) quantification of the PCR product, vi) comparison of the amount of the product with the amount of an internal control (this normalization gives the relative 6mA level). In this case, the PCR product accurately reflects the relative 6mA level of the selected mtDNA site. This method is described in a recent patent application titled “Accurate Determination Method Based on PCR” with file numbers P2100409 and W2200015. The owner (inventor) of this application has given our company permission to view the file (protocol) on the condition that all aspects of the IP rights are maintained.

[0020] Using this technique, we previously identified the relative 6mA levels at specific mtDNA sites (specific adenine nucleobases) in hundreds of individuals of known age (identity). From the data obtained, a reference "relative mtDNA 6mA level - age" curve was created. The subjects analyzed were healthy and of various ages. Next, the relative mtDNA 6mA levels identified in individuals of unknown identity were projected onto the reference curve, and the age of the individual on the X-axis at the intersection point was assigned (Figure 4). Based on our measurements, the deviation of the data was within an error range of 2 to 3 years (±2 to 3 years). If the relative 6mA level at a human mtDNA site is identified in an individual of known identity (age) and the value obtained is significantly different from the value indicated by the reference curve at a specific age, the difference may reflect the presence of an initial stage of a neurodegenerative process (Figure 5). In this case, the affected individual is recommended to undergo imaging tests such as PET and NMR.

Example

[0021] Method (the technical basis of which is described in a patent application entitled "PCR-based accurate determination method". File numbers: P2100409 and W2200015. The inventor has given the company permission to view the file (protocol) on the condition that all aspects of intellectual property rights are retained - confidentiality agreement): Isolation of genomic DNA from samples Isolation from blood samples was performed using the Thermo Scientific GeneJET Genomic DNA Purification Kit (♯K0721).

[0022] Digestion of genomic DNA with DpnI restriction endonuclease Add the following components to an Eppendorf tube: 1 μg of Dpnl enzyme (10 U / μl, ThermoFisher Scientific, ERI 701) 4 μl of Tango buffer (10x) 15 μl of H20 20 μl of genomic DNA solution (minimum concentration: 10 ng / μl) (Final volume: 40 μl / sample) → Incubate at 37 °C for 20 minutes Inactivation of DpnI: Incubate the sample at 80 °C for 20 minutes.

[0023] Ligation of linker DNA and digested genomic DNA fragments Add the following components to the inactivation mixture (40 μl): 4 μl of T4 ligase (5 U / μl, ThermoFisher Scientific, EL001 l) 5 μl of ATP (2 mM) (ThetnoFisher Scientific, 100 mM, R0441) 5 μl of genomic linker (at 100x concentration) 4 μl of Tango buffer 22 μl of H20 Final volume of the mixture: 80 μl Incubate the sample at 40 °C overnight, Inactivation of ligase: Incubate the sample at 800 °C for 20 minutes DNA sequence of the linker: 5'-TAG ATC TGA CCT AAC GGT AAG AGA GTT TCA TAA TAt ttt ttt ttt ttT AT-TAT GAA ACT CTC TTA CCG TTA GGT CAG ATC TA-3'

[0024] PCR reaction 6mA sample (Dpnl) - Forward (left) primer: 5'-ATG AAA CTC TCT TAC CGT TAG GTC AGA TCT Atc aac-3' and Reverse (right) primer: 5'-tga acg ttg gcc tgc ctt gc-3' Reaction mixture: 10 μl of ABI master mix (2x) 3.5 μl of digested / ligated DNA solution (template) 3 μl of primer mix (5 - 5 μM) 3.5 μl of H20 Final volume: 20 μl / sample

[0025] PCR conditions (DpnI): 1. Initial denaturation: 95°C, 30 seconds 2. Denaturation: 95°C, 10 seconds 3. Annealing and extension: 60°C, 45 seconds 4. Repeat steps 2 and 3 for 37 cycles 5. Store samples at 4°C

[0026] Control (PvuII) - Forward (left) primer: 5'-tga atg aaa tga agc gag aag gga agt tta gag-3' and Reverse (right) primer 5'-tga acg ttg gcc tgc ctt gc-3' Reaction mixture: 10 μl of ABI master mix (2x) 1 μl of DNA solution (template) 3 μl of primer mix (5 - 5 μM) 6 μl of H20 Final volume: 20 μl

[0027] PCR conditions (PvuII): 1. Initial denaturation: 95°C, 60 seconds 2. Denaturation: 95°C, 10 seconds 3. Annealing and extension: 60°C, 45 seconds 4. Repeat steps 2 and 3 for 27 cycles 5. Store samples at 4°C

[0028] Gel recording Run samples on a 1% agarose gel at 80 mV and photograph with a Kodak camera.

[0029] Novelty of the Invention 1. The recognition that the relative 6mA level at specific sites of the mitochondrial genome increases proportionally with age. Based on this discovery, the age can be accurately determined from mtDNA isolated from an individual of unknown identity. 2. The recognition that the relative 6mA level at specific mtDNA sites predicts lifespan. 3. The recognition that the relative 6mA level at specific mtDNA sites indicates the initial stage of the neurodegenerative process.

[0030] References Dahl C, Guldberg P. DNA methylation analysis techniques. Biogerontology 4, 233-250 (2003). PMID: 14501188 Douvlataniotis K, Bensberg M, Lentini A, Gylemo B, Nestor CE. No evidence for DNA N 6-methyladenine in mammals. sci. Adv. 2020 Mar 18;6(12):eaay3335. PMID: 32206710 Flusberg BA, Webste DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Koriach J, Turner SW.Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat.Methods 7, 461-465 (2010). PMID: 20453866 Greer EL, Blanco MA, Gu L, Sendinc E, Liu J, AristizabaJ-Corrales D, Hsu C-H, Aravind L, He C, Shi Y. DNA Methylation on N6-Adenine in C. elegans. cell 161. 868-878(2015). PMID: 25936839 Hao Z, wu T, Cui X, zhu P, Tan C, Dou X, Hsu KW, Lin YT, Peng PH, Zhang LS. et al. (2020) N(6)-deoxyadenosine methylation in mammalian mitochondrial DNA. Mol. Cell 78, 3 82-395 e388. PMID: 32183942 Horvath S. DNA methylation age of human tissues and cell types. Genome Biol.2013;14(10):R115. PMID: 24138928 Luo G-z, wang F, Weng X, Chen K, Hao Z, Yu M, Deng X, Liu J, He C. Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing. Nat. Commun. 7, 11301 (2016). PMID: 27079427 O'Brown ZK, Boulias K, wang J, wang sy, O'Brown NM, Hao Z, Shibuya H, Fady P-E, Shi Y, He C, Megason SG, Liu T, Greer EL. Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA. BMC Genomics 2019 Jun 3;20(1):445.PMID: 31159718 Rocha MS, Castro R, Rivera I, Kok R, Smulders YM, Jakobs C, de Almeida IT, Blom HJ. Global DNA methylation: comparison of enzymatic- and non-enzymatic-based methods.Clin. Chem. Lab. Med. 48, 1793-1798 (2010). PMID: 20979561 Schiffers S, Ebert C, Rahimoff R, Kosmatchev O, Steinbacher J, Bohne A-V, Spada F, Michalakis S, Nickelsen J, Muller M. Carell T. Quantitative LC-MS Provides No Evidence for m 6 dA or m 4 dC in the Genome of Mouse Embryonic Stem Cells and Tissues. Angew Chem Int Ed Engl. 2017 sep 1 1268-1 1271. PMID·. 28371147 wu TP, wang T, Seetin MG, Lai Y, Zhu S, Lin K, Liu Y, Byrum SD, Mackintosh SG, Zhong M, Tackett A, Wang G, Hon LS, Fang G, Swenberg JA, Xiao AZ. DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature. 2016 Apr 21 ;532(7599):32933. PMID: 27027282 Zhang G, Huang H, Liu D, Cheng Y, Liu X, Zhang W, Yin R, Zhang D, Zhang P, Liu J, Li C, Liu B, Luo Y, Zhu Y, Zhang N, He S, He C, wang H, Chen D. N6-methyladenine DNA modification in Drosophila. cell 161, 893-906 (2015). PMID: 25936838 Yao B, Cheng Y, wang Z, LY, Chen L, Huang L, Zhang W, Chen D, wu H, Tang B, Jin P. DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress. Nat. Commun. 8, 1122 (2017). PMID: 29066820 Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 1997 Aug;13(8):335-40. PMID: 9260521

Claims

**Claim 1** A method / procedure for accurately determining age from mitochondrial DNA (mtDNA) by discriminating the relative N6-methyladenine (6mA) level at a specific site of mtDNA. **Claim 2** The method / procedure according to claim 1, wherein the organism to be examined is any eukaryotic species. **Claim 3** The method / procedure according to claim 1 or 2, wherein the determination of the relative 6mA level at a specific mtDNA site is achieved by any suitable molecular technique. **Claim 4** The method / procedure according to any one of claims 1 to 3, wherein the target site is any mtDNA sequence. **Claim 5** The method / procedure according to any one of claims 1 to 4, wherein the type of tissue sample to be examined is arbitrary. **Claim 6** The method / procedure according to any one of claims 1 to 5, wherein the population in which the measurement of the relative 6mA level at a specific mtDNA site is achieved is arbitrary. **Claim 7** The method / procedure according to any one of claims 1 to 6, wherein the purpose of the analysis is to identify the age of an individual of unknown identity. **Claim 8** The method / procedure according to any one of claims 1 to 7, wherein the purpose of the analysis is to predict the lifespan of an individual of known age. **Claim 9** The method / procedure according to any one of claims 1 to 8, wherein the purpose of the analysis is to identify the initial stage of a neurodegenerative process.

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