A method of assessing the extent of mitochondrial DNA (MTDNA) oxidative damage

Abstract

The invention relates to a method of assessing the extent of oxidative damage on the mitochondrial DNA (mtDNA) of a test biological sample, which does not require prior isolation of mtDNA and does not underestimate the level of mtDNA lesions especially in samples with a high amount of oxidative lesions.

Description

[0001] A method of assessing the extent of mitochondrial DNA (mtDNA) oxidative damage

[0002] The present invention relates to the field of molecular biology and biochemistry. More in particular, it relates to a method of assessing the extent of oxidative damage on mitochondrial DNA (mtDNA) in animals and cell cultures.

[0003] Background of the invention

[0004] Assessment of the extent of DNA damage is important for monitoring the dynamics of damage, including the rate of occurrence and remediation by DNA repair enzymes, and for detecting and assessing environmental genotoxic risk or genotoxicity of candidate drugs.

[0005] Numerous studies indicate that mtDNA is more susceptible to various carcinogens and reactive oxygen species (ROS) than nuclear DNA (nDNA), whereby assessing the extent of damage on mitochondrial DNA is of particular importance.

[0006] While there are several commercially available assays to detect DNA damage, e.g. the DNA Damage Assay Kit (AP sites, Colorimetric) (Abeam, ab211154), to the inventors’ knowledge there is only one commercial kit for mtDNA damage analysis, i.e. the Human Real-Time PCR Mitochondrial DNA Damage Analysis Kit (Detroit R&D, cat. # DD2H). This kit seems to be based on a protocol published in Furda AM, Bess AS, Meyer JN, Van Houten B. Analysis of DNA damage and repair in nuclear and mitochondrial DNA of animal cells using quantitative RT-PCR. Methods Mol Biol. 2012;920:111-32. doi: 10.1007 / 978-1- 61779-998-3_9.

[0007] This is currently the standard protocol employed by the various research groups studying mtDNA damage. With respect to the previous protocols, the Furda protocol has the advantage of not requiring the isolation of mitochondria, thus reducing the amount of starting material needed. However, the Furda protocol has the disadvantage of being timeconsuming as it requires two steps of overnight precipitation. Additionally, the Furda protocol was found to underestimate the extent of mtDNA damage especially in samples having a high amount of lesions. The present invention is therefore aimed at overcoming the disadvantages of the prior art, in particular those of the Furda protocol.

[0008] Accordingly, a first object of the present invention is to provide a method of assessing the extent of mitochondrial DNA (mtDNA) oxidative damage which does not underestimate the level of mtDNA lesions especially in samples with a high amount of lesions.

[0009] A further object of the present invention is to provide a method of assessing the extent of mitochondrial DNA (mtDNA) oxidative damage wherein the time required for analysis is reduced as compared to the Furda protocol.

[0010] Another object of the present invention is to provide a method of assessing the extent of mitochondrial DNA (mtDNA) oxidative damage that does not require prior isolation of mitochondria from the biological sample to be analyzed.

[0011] Still another object of the present invention is to provide a method of assessing the extent of mitochondrial DNA (mtDNA) oxidative damage that does not require prior isolation of mtDNA from nuclear DNA (nDNA).

[0012] Still another object of the present invention is to provide a method of assessing the extent of mitochondrial DNA (mtDNA) oxidative damage that requires a reduced amount of starting DNA as compared to the Furda protocol.

[0013] These and other objects are achieved by the method of assessing the extent of oxidative damage on mitochondrial DNA (mtDNA) as defined in appended claim 1.

[0014] The dependent claims define further advantageous and exemplary features of the invention.

[0015] All appended claims for an integral part of the description.

[0016] Detailed description of the invention The method of the invention includes several steps, which are carried out in parallel on a test biological sample, i.e., a biological sample which contains the mtDNA to be assessed for mtDNA oxidative damage extent, and on a reference biological sample, i.e., a biological sample which contains the mtDNA against which the test sample mtDNA is assessed. The reference biological sample is selected such that the amount of oxidation-damaged mtDNA nucleobases contained therein is expected to be different from the amount of oxidationdamaged mtDNA nucleobases contained in the test biological sample. In certain embodiments, the reference biological sample is a control sample, i.e. a sample which contains mtDNA that is expected to be free or substantially free from mtDNA oxidative damage, or which contains mtDNA that is expected to contain a lower amount of oxidationdamaged mtDNA nucleobases than the test sample.

[0017] According to steps (a) and (a’) of the method of the invention, DNA is extracted from the test biological sample and from the reference biological sample, respectively. Any DNA extraction technique and protocol known per se to the skilled in the art can be used in steps (a) and (a’). The experimental examples disclosed herein mention the use of the QIAmp DNA Mini Kit (Qiagen, cat. 51304), but this is to be understood merely as an illustrative example, as the person skilled in the art may use any other known DNA extraction kit following the manufacturer's instructions.

[0018] As is known, total cellular DNA includes both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), whereby the extracted DNA contains a proportion of mtDNA and a proportion of nDNA. However, a particularly advantageous aspect of the method of the present invention is that it does not require isolation of mtDNA from the extracted DNA, nor does it require prior isolation of mitochondria from the starting biological sample.

[0019] According to steps (b) and (b’) of the method of the invention, the DNA extracted respectively from the test biological sample and from the reference biological sample is digested with the Formamidopyrimidine DNA Glycosylase (Fpg) enzyme. Fpg is a bifunctional DNA glycosylase with DNA N-glycosylase and AP lyase activities. The N- glycosylase activity releases damaged purines, including 2,6-diamino-4-hydroxy-5- formamidopyrimidine (FapyG) and 8-oxo-7,8-dihydroguanine (8oxoG), generating abasic sites (AP). The AP lyase activity cleaves the AP site, via P and 6-elimination, creating a 1 nucleotide DNA gap with 5' and 3' phosphate termini. Therefore, with Fpg the continuity of the DNA strand is not lost but gaps (are created, namely sites where the nucleobase is not present, at which the polymerase that will be used in the subsequent Long Range PCR reactions is blocked and no longer polymerizes.

[0020] After digestion with Fpg is completed, the Fpg enzyme may be inactivated by heating to high temperature (e.g., about 60°C) and optionally removed, e.g., by column purification, so as not to interfere with the subsequent method steps.

[0021] Digestion of the extracted DNA with Fpg provides a digested DNA mixture which comprises a mtDNA in which the oxidation-damaged nucleobases are replaced by gaps (mtDNA-gap).

[0022] Since the biological reference sample is selected such that the amount of oxidation-damaged mtDNA nucleobases contained therein is expected to be different from the amount of oxidation-damaged mtDNA nucleobases contained in the test biological sample, the Fpg- digested mtDNA derived therefrom is expected to contain a different amount of gaps than the Fpg-digested mtDNA derived from the test biological sample. In certain embodiments in which the reference biological sample is a control biological sample as defined above, the Fpg-digested mtDNA derived from the control sample is expected to contain a lower amount of gaps than the Fpg-digested mtDNA derived from the test biological samples, or even no gaps.

[0023] The amount of mtDNA-gap obtained by Fpg-digestion from the test biological sample and the amount of mtDNA-gap obtained by Fpg-digestion from the reference biological sample are normalized in method steps (c) and (c’). Normalization is performed by quantitative RT- PCR amplification of a unique short mitochondrial sequence having a length of between 80 to 120 bp (e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 bp) which is present in both the test mtDNA and in the reference mtDNA. Normalization allows the same amount of test mtDNA-gap and reference mtDNA-gap to be used in the subsequent method steps.

[0024] After normalization, the method of the invention provides for a Long Range PCR amplification step (step (d)), namely a PCR amplification reaction performed with a polymerase enzyme that is capable of amplifying extremely long mtDNA target sequences, such as a mtDNA target sequence having a length of about 1-16.5 kbp (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 16.5 kbp). A preferred length of the mtDNA target sequence is of 8-12 kbp. Polymerases optimized for Long Range PCR are well known to the skilled in the art, who is able to select and use them in a Long Range PCR amplification reaction without undue burden.

[0025] A polymerase used within the context of the present long range PCR should possess the following properties for the reaction to be reliable:

[0026] 1. high processivity, i.e., it should be a polymerase capable of amplifying very long fragments (classical polymerases instead amplify up to 5 kbp but not longer)

[0027] 2. high-fidelity, namely the ability to always incorporate the correct nucleotide and to stop if it recognizes the presence of a damaged base or an abasic site.

[0028] Thus, by using a high fidelty polymerase in the long range PCR step, better performances in the method of the invention are achieved. If in fact the fpg digestion step for whatever reason should not be 100% effective (and thus should it not create a gap for each damaged nucleotide identified), the high-fidelty polymerase would still prevent the insertion of a random nucleotide, the presence of which would cause extension of the ssDNA even when damaged and would distort the final assessment of the damage present on the mtDNA of interest.

[0029] According to step (d) of the method of the invention, the Long Range PCR reaction is performed on both the mtDNA-gap derived from the test biological sample (also called the “test mtDNA-gap”) and the mtDNA-gap derived from the reference biological sample (also called the “reference mtDNA-gap”). The mtDNA target sequence being amplified in the Long Range PCR reaction is a long mitochondrial sequence, having a length of approximately 1 to 16.5 kbp, preferably 8-12 kbp (e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16 or 16.5kbp), which is present on both the test mtDNA-gap and the reference mtDNA-gap. It is also important to note that the target long mitochondrial sequence contains the unique short mitochondrial sequence which was used as the target in the normalization of steps (c) and (c’). The Long Range PCR reactions performed in step d) respectively on the test mtDNA- gap and the reference mtDNA-gap result in a so-called “long mitochondrial amplicon” (having a length of 1-16.5 kbp, preferably of 8-12 kbp) as the mtDNA amplification product. However, since the amount of gaps which are present on the test mtDNA-gap is expected to be different from the amount of gaps which are present on the reference mtDNA-gap, the amount of long mitochondrial amplicon resulting from the Long Range PCR amplification of the test mtDNA-gap is different from the amount of long mitochondrial amplicon resulting from the Long Range PCR amplification of the reference mtDNA-gap. Namely, the Long Range PCR amplification performed on the test mtDNA-gap provides a first amount of the long mitochondrial amplicon, while the Long Range PCR amplification performed on the control mtDNA-gap provides a second amount of the long mitochondrial amplicon, wherein the first amount is different from the second amount and the difference between the first amount and the second amount is quantitatively related to the extent of mtDNA oxidative damage in the test biological sample. Once the Long Range PCR amplification is completed, the same amount of an internal control, namely a DNA fragment, is added to the amplification reaction mix of the test mtDNA-gap and to the amplification reaction mix of the reference mtDNA-gap. This DNA fragment is totally unrelated with the mtDNA to be analyzed. For example, the internal control is a recombinant plasmid for the expression of the GFP protein of C. elegans.

[0030] As both Long Range PCR amplification products (amplicons) are usually purified, e.g., by column purification, in order to remove all the Long Range PCR components which can interfere with the subsequent quantitative RT-PCR reaction step, the internal control is employed to “normalize”, i.e. to ensure that the subsequent quantification step is performed on the same amount of sample, both in the case of the test amplicon and the reference amplicon.

[0031] As mentioned, to quantify the first and second amount of the long mitochondrial amplicon obtained with the Long Range PCR reactions of step d), a quantitative RT-PCR is used (step e). The amplification target to be amplified is the unique short mitochondrial sequence which is present in the long mitochondrial amplicon obtained from both the test mtDNA-gap and the reference mtDNA-gap. This quantitative RT-PCR amplification is performed with the concomitant amplification of the internal control, which, as mentioned above, is used to confirm that the mtDNA fragments amplified during the Long Range PCR are not lost during the purification step.

[0032] The method of the invention may be used for assessing the extent mtDNA oxidative damage in any biological sample of interest. For example, the biological sample to be analyzed for the extent of mtDNA oxidative damage is a cell line (for example, a human or an animal cell line) or a biopsy. In the case of a biopsy a preferred embodiment is a human biopsy.

[0033] The cell line is any cell line whose degree of mitochondrial DNA oxidative damage the skilled in the art wishes to analyze for any reason, for example a cancer cell line (such as HeLa, 0CI-AML2, SF767, JHH6 or Huh7) or a cell line that was previous exposed to a ROS-inducing agent. A biopsy is a tissue specimen that is removed from a living body, preferably a human body, to discover the presence, cause, or extent of a disease, for example cancer.

[0034] In a preferred embodiment which is described in more detail in the experimental part herein below, the test biological sample is a biopsy of tumor liver tissue from a human patient with hepatocellular carcinoma and the reference biological sample is a biopsy of non-tumor liver tissue from the same human patient.

[0035] In another preferred embodiment, the quantitative RT-PCR reactions of steps (c) and (e) are carried out with a forward primer of SEQ ID NO:1 (5’-CCC CAC AAA CCC CAT TAC TAA A-3’) and a reverse primer of SEQ ID NO:2 (5’-TTG GTC GTG GTT GTA GTC COS’) which are designed to amplify a unique short mitochondrial sequence which is present on human mtDNA.

[0036] In a further preferred embodiment, the Long Range PCR reactions of step (d) are carried out with a forward primer of SEQ ID NO: 3 (5’-TCT AAG CCT CCT TAT TCG AGC CGA- 3’) and a reverse primer of SEQ ID NO:4 (5’-TTT CAT CAT GCG GAG ATG TTG GAT GG-3’) which are designed to amplify a long mitochondrial target sequence which is present on human mtDNA-gap.

[0037] In a further preferred embodiment, the quantitative RT-PCR amplification of the internal control is carried out with a forward primer of SEQ ID NO:5 (5’-TCC GTC ACA TCG AGG AC -3’) and a reverse primer of SEQ ID NO:6 (5’-CGT CTC CGA TTG GGG TGT TT -3’) which are designed to amplify the C. elegans GFP plasmid 15ABEXQP_mGFP_pMK-RQ.

[0038] The following examples are provided by way of illustration only and are not intended to limit the scope of the invention as defined in the appended claims.

[0039] Examples

[0040] Example 1: mtDNA damage analysis in cultured cells

[0041] The analysis was carried out according to the flowchart in Figure 1.

[0042] 1.1 Cell culture and treatments

[0043] HeLa, SF767 and HUH7 cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (FBS). 0CI-AML2 cells were grown in alpha-MEM (Minimum Essential Medium) supplemented with 20% fetal bovine serum (FBS). JHH6 cells were grown in Williams E medium supplemented with 10 % fetal bovine serum (FBS). All media were completed with 100 U / mL penicillin and 10 pg / mL streptomycin. Cells were incubated at 37°C with 5% CO2. To generate mtDNA damage by inducing the production of mtROS, Antimycin A (AMA) treatments were performed in the different culture media corresponding to each cell line without FBS, for 30 minutes, with the established doses of AMA (Barchiesi A, et al (2020) Mitochondrial oxidative stress induces rapid intermembrane space / matrix translocation of apurinic / apyrimidinic endonuclease 1 protein through TIM23 complex. J Mol Biol 432(24): 166713. Santos JH, Meyer JN, Mandavilli BS & Van Houten B (2006) Quantitative RT-PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol Biol 314: 183-199).

[0044] 1.2 DNA extraction from cultured cells [step 1 in Figure 1 ]

[0045] Trypsinized cells were harvested and counted. DNA extraction was performed starting from IxlO4cells and using the QIAamp DNA Mini Kit (Qiagen, cat. 51304). 20 pL of Proteinase K and 200 pL of Buffer AL were added to the resuspended cells and incubated at 56°C for 10 min. After 200 pL ethanol addition, the mixture was applied to the purification column. Following manufacture’s instruction two washes were performed with solutions AW1 and AW2 and finally DNA was eluted in 50 pL of water.

[0046] 1.3. Fpg digestion [steps 2 and 3 in Figure 1 ]

[0047] Formamidopyrimidine DNA Glycosylase (Fpg) enzyme (New England BioLabs, cat. M0240S) was diluted in its IX buffer to a final concentration of 1.6 U / pL. A reaction was prepared with 50 pL of DNA, 10 pL of Fpg buffer 10X, 20 ng of BSA, 2 pL of diluted Fpg and water to 100 pL final volume. The reaction was incubated at 37°C for 30 min. Enzyme activity was heat inactivated at 60°C for 10 min. 233 pL of ethanol were added to the mix and the sample was applied to a new purification column of the QIAmp DNA Mini Kit and processed as described before to remove Fpg before proceeding with the following steps of the protocol.

[0048] 1.4. Quantitative RT-PCR [steps 4 and 8 in Figure 1 ]

[0049] Quantitative RT-PCR were performed with a CFX96 Real-Time System (Bio-Rad) using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, cod. A25741). For the short fragment amplification and the final quantitative RT-PCR the following primers were used: forward 5’-CCC CAC AAA CCC CAT TAC TAA A-3’ (SEQ ID NO:1); reverse 5’- TTG GTC GTG GTT GTA GTC CG-3’ (SEQ ID NO:2). For the standard amplification the following primers were used: forward 5’-TCC GTC AC A TCG AGG AC -3’ (SEQ ID NO:5); reverse 5’-CGT CTC CGA TTG GGG TGT TT -3’ (SEQ ID NO:6). 1.5. Long Range PCR [steps 5, 6 and 7 in Figure 1 ] mtDNA lesions were detected by Long Range PCR, using the following primers: Mitolong Forward: 5’-TCT AAG CCT CCT TAT TCG AGC CGA-3’ (SEQ ID NOG) and Mitolong Reverse: 5’-TTT CAT CAT GCG GAG ATG TTG GAT GG-3’ (SEQ ID NO:4) which amplified an 8.9 Kbp mitochondrial fragment. Amplification was performed with Platinum™ SuperFi™ DNA Polymerase (Thermo Fisher Scientific, cod. 12351010) diluted 1:10, using the following protocol:

[0050] 2 minutes at 94°C;

[0051] 18 cycles

[0052] 1) denaturation for 15 seconds at 94°C;

[0053] 2) annealing for 10 seconds at 66°C;

[0054] 3) extension for 5 minutes and 30 seconds at 68°C;

[0055] 10 minutes at 68°C.

[0056] After the Long Range PCR reaction, 100 pg of standard were added and then the PCR product was purified with QIAquick PCR Purification Kit (Qiagen, cat. 28104). 250 pL of buffer PB were added to the sample and applied to the purification column. A wash was performed with 750 pL of buffer PE and the purified PCR product was eluted in 50 pL of water. Finally, a quantitative RT-PCR was performed as described in 1.4 to quantitatively evaluate the amount of mtDNA amplified with the Long Range PCR and the internal standard.

[0057] Example 2: mtDNA damage analysis in human liver tissue

[0058] The analysis was carried out according to the flowchart in Figure 1.

[0059] 2.1 Samples from human tumor tissue specimens and adjacent non-tumor tissues

[0060] Needle biopsies of paired HCC (hepatocellular carcinoma) and adjacent non-tumor liver tissues from patients undergoing HCC resection were obtained from the Department of Medicine, General Surgery and Transplantation of the University of Udine, Udine, Italy and stored in 20% glycerol Isolation Buffer (IB) [10 mM Tris / MOPS, 1 mM EGTA / Tris, 200 mM Sucrose]. None of the patients had received any local or systemic anticancer treatments before the surgery. Diagnosis of HCC was performed in all cases by preoperative imaging (CT or MRI scan) or by liver biopsy when requested. Hepatic serology, a-fetoprotein (AFP), routine laboratory assessment of liver and renal function were also performed. The presence of suspected (based on radiologic features) neoplastic main branch portal thrombosis was considered a contraindication to surgery. After hospital discharge all patients were followed up and monitored for tumor recurrence by monthly assessments of serum AFP and by US, CT or MRI scan every 3 - 6 months. HCC recurrence was in all cases diagnosed by CT or MRI imaging according to international guidelines.

[0061] Approval from the Ethical Committee and informed consent

[0062] This study was approved by the Unique Regional Ethical Committee, August 24, 2019, Protocol number 18659, and informed consent was obtained from each patient.

[0063] 2.2 DNA extraction from human liver needle biopsies [step 1 in Figure 1 ]

[0064] DNA was extracted from needle biopsies using the QIAMP DNA Mini Kit (Qiagen, cat. 51304), following the manufacturer’s instructions for DNA isolation from tissues. The needle biopsies were suspended in 180 pL of Buffer ATL supplemented with 20 pL of Proteinase K and incubated at 56°C until dissolution. 200 pL of AL were added, and samples were incubated for 10 min at 70°C. 200 pL of ethanol were included in each sample before loading into the QIAamp Mini spin columns. Following manufacture’s instruction, two washes were performed with solution AW1 and AW2. Finally, the DNA was eluted in 100 pL and quantified at Nanodrop.

[0065] 2.3 Fpg digestion of DNA extracted from human liver needle biopsies [steps 2 and 3 of Figure 1 ]

[0066] After a Nanodrop quantification, 1,5 pg of DNA isolated from the needle biopsy were prepared for the Fpg digestion, following the manufacturer’s indications as described in Example 1. The reaction was purified with the QIAmp DNA Mini Kit and processed as described in Example 1 to remove Fpg reaction components.

[0067] 2.4 Quantitative RT-PCR [step 4 and 8 in Figure 1 ]

[0068] A quantitative RT-PCR with a CFX96 Real-Time System (Bio-Rad) using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, cod. A25741) was performed to amplify a fragment of 84 bp on the mitochondrial DNA of each sample analysed. The primers reported in paragraph 1.4 of Example 1 were used. Based on this quantitative RT- PCR, mtDNA content was evaluated and an equal amount of mtDNA for each sample was prepared to perform the Fong Range PCR.

[0069] 2.5 Long Range PCR in human liver needle biopsies [steps 5, 6 and 7 in Figure 1 ] mtDNA lesions were quantified by Long Range PCR, using the primers disclosed in paragraph 1.5 of Example 1. DNA was amplified using Platinum™ SuperFi™ DNA Polymerase (Invitrogen) performing the protocol described for the mtDNA damage detection on cells. After the Long Range PCR reaction, 100 pg of standard were added and then the PCR product was purified with QIAquick PCR Purification Kit (Qiagen, cat. 28104). 250 pL of buffer PB were added to the sample and applied to the purification column. A wash was performed with 750 pL of buffer PE and the purified PCR product was eluted in 50 pL of water. Finally, a quantitative RT-PCR was performed with the primers reported in paragraph 1.4 of Example 1 to quantify the relative mtDNA damage detected on the samples analysed.

[0070] Reference Example 3: mtDNA damage analysis in human liver tissue according to the Furda protocol

[0071] The analysis was carried out according to the flowchart in Figure 2.

[0072] 3.1 DNA extraction from human liver specimens [step 1 in Figure 2 ] DNA was extracted from 15-20 mg of patient biopsies using the QIAGEN Genomic-tip 20 / G. Samples were incubate in 2 mL G2 solution completed with RNase A (final concentration of 200 pg / mL) and Proteinase K as indicated by the company, followed by 2 hours incubation at 50°C. Genomic-tip columns were equilibrated with 2 mL of buffer QBT. Samples were then applied to the corresponding column and the flow-through discarded via gravity. Three washes were performed with 1 mL of wash buffer QC and finally DNA samples were eluted in 2 mL of QF buffer and precipitated over / night (O / N) with isopropanol.

[0073] 3.2 Fpg digestion of DNA isolated from human liver specimens [steps 2, 3, 4 and 5 in Figure 2]

[0074] The day after, DNA was centrifuged at 15000 x g for 1 hour at 4°C, washed with 70% ethanol and pelleted at 15000 x g for 45 min at 4°C and resuspended in 50 pL of TE (Tris 20 mM, EDTA 0.5 mM). After a Nanodrop quantification, 10 pg of DNA isolated from the needle biopsy were prepared for the Fpg digestion, following the manufacturer’s indications. DNA was precipitated over / night with isopropanol. After precipitation, DNA was centrifuged at 15000 x g for 1 hour at 4°C, washed with 70% ethanol and pelleted at 15000 x g for 45 min at 4°C and finally resuspended in 50 mL of TE (Tris 20 mM, EDTA 0.5 mM). DNA concentration was adjusted to 3 ng / pL, quantifying the samples by using Quant-iT™ PicoGreen™ dsDNA Reagent.

[0075] 3.3 Long Range PCR and not-quantitative Short Range PCR in human biopsies [steps 6 and 7 in Figure 2 ] mtDNA lesions were quantified by Long Range PCR with Mitolong Forward and Mitolong Reverse primers; Mitoshort Forward: 5-CCC CAC AAA CCC CAT TAC TAA ACC CA-3’ (SEQ ID NO:7) and Mitoshort Reverse: 5’-TTT CAT CAT GCG GAG ATG TTG GAT GG-3’ (SEQ ID NO:8) were also used to amplify a 221 bp mitochondrial fragment. DNA was amplified with Platinum™ SuperFi™ DNA Polymerase (Invitrogen) using the already described condition for the Long Range PCR (paragraph 1.5 of Example 1) and the following protocol for the Short Range PCR: 2 minutes at 94°C, 18 cycles of denaturation for 15 sec at 94°C, annealing 45 seconds at 60°C, extension for 45 seconds at 72°C and a final extension for 10 minutes at 72°C. To ensure quantitative conditions a sample with the 50% of template amount was included in each amplification and, as negative control, a sample without the template was used. PCR products were quantified in triplicate by using Quant- iT™ PicoGreen™ dsDNA Reagent (Invitrogen). The Mitoshort fragment was used to calculate the Relative amount of mtDNA copies and to normalize the lesion frequencies calculated with the Mitolong fragment.

[0076] 4. Statistical analysis

[0077] Statistical analysis was performed using Microsoft Excel. One-way ANOVA was used for three group comparisons and Student’s t-test was used for two group comparisons, p values of less than 0.05 were considered as significant, while values less than 0.01 or lower were considered as highly significant.

[0078] Results

[0079] The results obtained with the Furta protocol and the method of the invention are shown in Figures 3-6.

[0080] Figure 3 shows two histogram graphs in which the results obtained by applying the Furda protocol on HeEa treated with AMA are compared with the results obtained with the method of the invention. In both cases, the analyses were performed on HeEa cells treated with antimycin A (AMA) for 30 minutes to induce mtDNA damage. DNA from untreated HeLa cells (CTRL) or HeLa cells treated with AMA was extracted from either 2xlOA6 cells (“Furda et al. protocol”) or lxlOA4 cells (“new protocol”) to compare the effectiveness of the method of the invention to that of the Furda protocol. A relative DNA damage on AMA- treated cells of 2.34 ± 0.39 was detected applying the Furda protocol (left). The method of the invention resulted in the detection of a relative damage of 2.11 ± 0.26 (right). Data reported are the mean of three independent biological replicates (**: p<0.01).

[0081] Figure 4 is a histogram graph showing mtDNA damage detection on HeLa treated with AMA for different time points. HeLa cells were treated with AMA for 30, 60 or 90 min (AMA). Non-treated cells were used as a control (CTRL). As expected, increasing incubation time with AMA leads to increased damage detection.

[0082] Figure 5 shows four histogram graphs in which the results obtained by applying the method of the invention on four different immortalized human cell lines are reported. Human leukemia cells (0CI-AML2) and human glioblastoma cells (SF767) were treated with 25 mM of AMA for 30 minutes to induce mtDNA damage (AMA). Hepatocellular carcinoma (HCC) JHH6 cells were treated with 200 mM of AMA for 30 minutes and HCC Huh7 cells were treated with 75 mM of AMA for 30 minutes. Not treated cells were used as a control (CTRL). (A) The detected mtDNA damage was 2.89 ± 0.93 in the AMA-treated 0CLAML2 cells, (B) 1.42 ± 0.06 in the AMA-treated SF767 cells, (C) 2.50 ± 0.25 in the AMA-treated JHH6 cells (D) and 1.71 ± 0.22 in the AMA-treated HUH7 cells. Data reported are the mean of four independent biological replicates (*: p<0.05; **: p<0.001 and ***: p<0.001).

[0083] Figure 6 is a histogram graph showing the comparison of mtDNA damage detected on DNA isolated from biopsies of 7 representative HCC patients, by applying the Furda protocol versus the method of the invention. Patients 1, 2, 3 and 4: a similar mtDNA damage was detected using both protocols. A relative DNA damage of 1.04, 1.08, 1.02, 0.94, respectively, was quantified by applying the Furda protocol, whereas the method of the invention provided a relative DNA damage of 0.67, 0.92, 1.11, 1.17, respectively. Patients 5, 6, 7: the figure shows that these patients had increased mtDNA damage. A relative DNA damage of 1.1, 1.7, 1.87, respectively, was quantified by applying the Furda protocol, whereas by applying the method of the invention in the same samples a relative mtDNA damage of 1.86, 3.3, 10.2, respectively, was quantified.

[0084] Figure 6 therefore shows that with biological samples in which the amount of oxidationdamaged mtDNA is high, the Furda protocol underestimates the damage extent, while the method of the invention does not.

[0085] Alternative primers In Examples 1 and 2 above, other primers may be used an alternative to SEQ ID Nos: 1-8 disclosed therein. Namely, alternative primers for the quantitative RT-PCR amplification of the unique short mitochondrial sequence are:

[0086] Alternative primers for the Long Range Q-PCR amplification of the test and reference mtDNA-gap are:

Claims

CLAIMS1. A method of assessing the extent of oxidative damage on the mitochondrial DNA (mtDNA) of a test biological sample, comprising the steps of:(a) extracting DNA from the test biological sample, wherein the extracted DNA includes the test mtDNA;(a’) providing a reference biological sample which is expected to contain an amount of mtDNA oxidation-damaged nucleobases that is different from the amount of mtDNA oxidation-damaged nucleobases in the test biological sample, and extracting the DNA from the reference biological sample, the extracted DNA including the reference mtDNA;(b) digesting the DNA extracted in step (a) with the DNA-formamidopyrimidine glycosylase (Fpg) to remove the oxidation-damaged nucleobases contained therein thereby generating gaps, thereby obtaining test mtDNA including gaps (test mtDNA-gap);(b’) digesting the DNA extracted in step (a’) with the DNA-formamidopyrimidine glycosylase (Fpg) to remove the oxidation-damaged nucleobases contained therein thereby generating gaps, thereby obtaining reference mtDNA including gaps (reference mtDNA- gap);(c) normalizing the amount of test mtDNA-gap to the amount of reference mtDNA-gap by quantitative RT-PCR amplification of a unique short mitochondrial sequence of about 80- 120 base pairs (bp) which is present on both the test mtDNA and the reference mtDNA;(d) subjecting the same amount of test mtDNA-gap and reference mtDNA-gap to Long Range PCR amplification of a long mitochondrial sequence contained therein having a length of 1-16.5 kbp, said long mitochondrial sequence including the unique short mitochondrial sequence of about 80-120 bp of step (b), to generate a first amount of a long mitochondrial amplicon amplified from the test mtDNA-gap and a second amount of the same long mitochondrial amplicon amplified from the reference mtDNA-gap;(e) quantifying the first and second amount of the long mitochondrial amplicon by quantitative RT-PCR amplification of the unique short mitochondrial sequence included therein and concomitant quantitative RT-PCR amplification of an internal control, the difference between the first and second amount of the long mitochondrial amplicon being quantitatively related to the extent of oxidative damage on the mitochondrial DNA (mtDNA) of the test biological sample.

2. The method of claim 1, wherein the Long Range PCR amplification is performed with a high-fidelty polymerase.

3. The method according to claim 1 or 2, wherein the internal control of step (e) is a known amount of a standard DNA sequence unrelated with the test mtDNA.

4. The method according to claim 3, wherein the standard DNA sequence unrelated with the test mtDNA is a recombinant plasmid for the expression of GFP protein of C.elegans.

5. The method according to any one of claims 1 to 4, wherein the test biological sample is a cell line sample, or a tissue biopsy, or tumor cell line sample.

6. The method according to claim 5, wherein the test biological sample is a tumor liver tissue biopsy from a patient affected by hepatocellular carcinoma and the reference biological sample is a non-tumor liver tissue biopsy from the same patient.

7. The method according to any one of claims 1 to 6, which includes a step of removal of the Fpg enzyme from the product of the digestion steps (b) and (b’).

8. The method according to any one of claims 1 to 7, which does not include a step of mitochondria isolation or a step of purification of mtDNA from the test biological sample and the reference biological sample.

9. The method according to any one of claims 1 to 8, wherein in steps (c) and (e) the quantitative RT-PCR amplification of the unique short mitochondrial sequence is performed with a forward primer of SEQ ID NO:1 and a reverse primer of SEQ ID NO:2, or with a forward primer of SEQ ID NO:7 and a reverse primer of SEQ ID NO:8, or with a forward primer of SEQ ID NO:9 and a reverse primer of SEQ ID NO: 10, or with a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12, or with a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14.

10. The method according to any one of claims 1 to 9, wherein in step (d) the Long Range PCR amplification of the test mtDNA-gap and of the reference mtDNA-gap is performed with a forward primer of SEQ ID NO:3 and a reverse primer of SEQ ID NO:4, or with a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16, or with a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18, or with a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO:20.

11. The method according to any one of claims 1 to 10, wherein in step (e) the amplification of the internal control is performed with a forward primer of SEQ ID NO:5 and a reverse primer of SEQ ID NO:6.

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