WO2009046514A1 - Methods for non-invasive collection of skin cells for dna analysis - Google Patents

Abstract

A method for the detection of an aberration in DNA of skin cells. The method comprises contacting a skin site with a sterile swab, swabbing the skin site with the sterile swab to collect the skin cells, extracting DNA from the skin cells, and detecting the presence of the aberration in the DNA. In an alternate embodiment, the skin cells may be collected using a fine needle.

Description

METHODS FOR NON-INVASIVE COLLECTION OF SKIN CELLS FOR DNA

ANALYSIS

FIELD OF THE INVENTION:

[0001] The present invention relates to methods for the collection of human skin samples for use in the diagnosis or characterization of disease, aging, or exposure to ultraviolet radiation. More specifically, the present invention provides a non-invasive method for the collection of skin samples for genotyping and the assessment of DNA damage caused, for example, by UV radiation.

DESCRIPTION OF THE PRIOR ART

[0002] Current methods for the collection of skin samples for use in the diagnosis or characterization of diseases, such as skin cancer, include invasive or painful methods that can cause substantial discomfort to the individual being tested. Examples of current methods for the collection of skin samples include punch biopsy, tapelift, and surgical excision. In addition to the discomfort caused by the current methods for skin collection, these methods must also be performed by a medical practitioner in order to be safely conducted. Further, the costs associated with these invasive test methods make it difficult to rapidly genotype and assess DNA damage for large populations of individuals. It would, therefore, be advantageous for there to exist a non-invasive skin collection methodology that may be conducted easily and rapidly in a home, clinical or cosmetic setting.

[0003] Mitochondrial deletions associated with UV exposure

[0004] The incidence of non-melanoma skin cancer (NMSC) is increasing in populations of European origin (Severi and English, 2004). For example, one million new cases are diagnosed each year in the USA (Wesson and Silverberg, 2003) and 65,000 in the UK (figures provided by Cancer Research. UK). NMSC accounts for around 90% of skin cancers and consists of basal cell and squamous cell carcinomas (BCC and SCC, respectively). BCCs are the most common form of NMSC and arise predominantly from the basal keratinocytes of the epidermis but also from cells in hair follicles and sebaceous glands. They are locally invasive but rarely metastasize. SCCs are also derived from basal keratinocytes; however, in contrast to BCCs, SCCs may metastasize. Compared with BCC, SCC shows the greatest increase with age and is concentrated in the elderly (Severi and English, 2004). The relative density of NMSC is highest on body sites usually exposed to the sun when outdoors such as scalp, face, neck, and ears as defined by Armstrong (2004). SCC, however, differs appreciably from BCC in having a much lower density on body sites which are occasionally exposed to the sun such as shoulders, back, and chest as defined by Armstrong (2004).

[0005] Therefore, the major determinant of NMSC is the ultraviolet (UV) radiation component of sunlight that induces DNA damage. Importantly it is both the pattern (more continuous versus intermittent) and the cumulative amount of sun exposure that influences the development of NMSC (Armstrong and Kricker, 2001). To determine a reliable marker of cumulative UV exposure in human skin, the inventors and others have examined the novel idea of using mitochondrial DNA (mtDNA), rather than nuclear DNA, as a biomarker of UV- induced DNA damage (Pang et al, 1994; Berneburg et al, 1997; Birch-Machin et al, 1998; Birch-Machin, 2000).

[0006] The use of mtDNA damage as a biomarker for cumulative sun-exposure in human skin is a relatively new field of research and previous work has simply compared mtDNA damage to distinguish between sun-protected and sun-exposed skin (Pang et al, 1994; Berneburg et al, 1997; Birch-Machin et al, 1998). This approach is limited because NMSC is predominantly formed on body sites which are "usually" exposed to the sun when outdoors as opposed to sites that are "occasionally" exposed to the sun (Armstrong, 2004).

[0007] In the present Applicant's co-pending PCT application bearing publication no.WO/06/111029 (the contents of which are incorporated herein by reference), a 3895 bp deletion in human mitochondrial DNA (mtDNA) was identified as a biomarker of UV- induced DNA damage. This deletion was identified in the minor arc spanning nucleotides 547-4443. This deletion had previously been associated with Kearns Sayre Syndrome and Chronic Progressive External Opthalmoplegia (Moraes et al, 1995).

[0008] Examples in PCT publication no.WO/06/111029 demonstrate that that the frequency of occurrence of the 3895 bp mtDNA deletion is significantly different between body sites that are "usually" versus "occasionally" exposed to the sun. In addition, the examples demonstrated a link between the etiology of the 3895 bp deletion and the UV radiation component of sunlight by inducing the 3895 bp deletion in vitro with repetitive sublethal doses of a UVA+UVB light source. Skin samples for the examples provided in the PCT application were obtained by painful methods of skin collection previously known in the art. [0009] It would be desirable to have a method for rapidly collecting skin samples and genotyping individuals to identify the presence of the 3895 bp and other mtDNA deletions associated with UV skin damage.

[0010] The present invention seeks to obviate or mitigate some or all of the above- mentioned problems associated with current methods for the collection of skin samples.

SUMMARY OF THE INVENTION

[0011] In one embodiment, the present invention provides a method for the detection of an aberration in DNA of skin cells comprising;

contacting a skin site with a sterile swab;

swabbing the skin site with the sterile swab to collect the skin cells;

extracting DNA from the skin cells;

detecting the presence of the aberration in the DNA.

[0012] In one aspect, the deletion being detected is the 3895 bp mtDNA deletion described below.

[0013] In another embodiment, the present invention provides a method for the noninvasive collection of DNA or mtDNA from skin cells for use in the detection of DNA or mtDNA biomarkers, the method comprising;

-contacting a skin site with a sterile swab;

-swabbing the skin site to collect the skin cells;

-extracting the DNA or mtDNA from the skin cells.

[0014] In another embodiment, the present invention provides a method for the detection of an aberration in DNA of skin cells comprising;

collecting skin cells from the dermis and/or epidermis by piercing through a tented layer of skin with a needle, without obtaining blood, to obtain a microscopic amount of dermal and/or epidermal tissue adhered to the core of the needle; expressing the dermal and/or epidermal tissue from the core of the needle;

extracting DNA from the dermal and/or epidermal tissue;

detecting the presence of the aberration in the DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] One or more embodiments of the invention will now be described by way of example only with reference to the appended drawings wherein:

[0016] Figure 1 shows real-time PCR data relating to the 3895 bp mtDNA deletion levels in skin samples collected from the nose and the heel using the method of the present invention.

[0017] Figure 2 shows real-time PCR data relating to levels of the 3895 bp mtDNA deletion in skin cells collected from various body sites using the method of the present invention.

[0018] Figure 3 shows real-time PCR data relating to levels of the 3895 bp mtDNA deletion in skin cells collected from various body sites using the method of the present invention.

[0019] Figure 4 is a gel showing the presence of amplification products present in samples collected from various non-invasive skin collection methods.

[0020] Figure 5 is a gel showing the presence of amplification products present in samples collected from various non-invasive skin collection methods.

DETAILED DESCRIPTION OF THE INVENTION

[0021] As used herein, "cycle threshold" (C_T) is the point at which target amplification of a nucleic acid sequence rises above background, as indicated by a signal such as a fluorescence signal. The C_T is inversely related to the quantity of the sequence being investigated. [0022] As used herein, "diagnostic" or "diagnosing" means using the presence or absence of a mutation or combination of mutations as a factor in disease diagnosis or management. The detection of the mutation(s) can be a step in the diagnosis of a disease.

[0023] As used herein, "deletions" means removal of a region of DNA or mtDNA from a contiguous sequence of a nucleic acid. Deletions can range in size from one base to thousands of bases or larger.

[0024] As used herein, "mitochondria" means a eukaryotic cytoplasmic organelle that generates ATP for cellular processes.

[0025] As used herein, "mitochondrial DNA" or "mtDNA" is DNA present in mitochondria.

[0026] As used herein, "mutation" encompasses any modification or change in a DNA or RNA sequence from the wild type sequence, including without limitation point mutations, transitions, insertions, transversions, translocations, deletions, inversions, duplications, recombinations or combinations thereof. The modification or change of the sequence can extend from a single base change to the addition or elimination of an entire DNA or RNA fragment.

[0027] The present invention provides a non-invasive method for the collection of skin samples for genotyping or diagnostic tests. The method involves the use of a sterile swab, such as those used in the collection of buccal cells or cotton-tip swabs. The sterile swab is removed from its packaging and is rubbed on a skin site of interest. Preferably, the site is swabbed approximately 15 times in order to ensure that a sufficient number of skin cells are collected for genotyping or diagnostic purposes. Although the present invention is described below with reference to a specific example, the method may also be used to collect skin samples for the diagnosis or characterization of disease, aging, or exposure to ultraviolet radiation, and the identification of mutations associated therewith.

[0028] Following the swabbing of the skin, the swab is deposited into a sterile tube. Buffer may be added to the tube as necessary in order to maintain the integrity of the genetic material (i.e. DNA) contained therein. The DNA is then extracted utilizing well known methods in the art. [0029] The method of the present invention may be used for widespread skin screening for both medical and cosmeceutical purposes. The method of the present invention may be used to measure various biomarkers associated with skin cancer (both non-melanoma skin cancer and melanoma). The ability to assess the level of DNA damage in an individual's skin due to UV radiation at any time point and from any external anatomical location provides the foundation for a unique and informative screening test for skin health.

[0030] The collection materials used in the method of the present invention may be packaged, depending on the desired application, into a consumer kit or a medical kit to be used in a clinical environment. Such kits could not only include the sterile swabs, but other materials necessary for genotyping (eg. the identification of mutations).

[0031] In example 1 provided below, one embodiment of the method of the present invention is used to collect skin cells for the quantification of biomarkers associated with damage caused by UV radiation. Specifically, the method of the present invention was used to collect skin samples for testing for deletions in the human mitochondrial genome, namely the 3895 bp mtDNA deletion identified in PCT application no. WO/06/ 111029. The 3895 bp deletion has a sequence corresponding to SEQ ID NO:1. The example shows that skin cells collected via the non-invasive method of the present invention provides sufficient mtDNA for obtaining results comparable to mtDNA obtained via previous skin collection methodologies.

[0032] In another embodiment of the present invention, a very small gauge needle (28 or 29 gauge) is used to collect skin cells for the purpose of genetic investigation, hi this embodiment, skin cells are collected from the dermis and epidermis of a subject by piercing through a tented layer of the skin such that little or no blood is drawn, but a microscopic amount of dermal and epidermal tissue is adhered to the inner core of the needle. The skin may be tented by raising the skin using, for example, fingers, tweezers, or other forms of clamp. The skin material is contained in the needle until it is extracted for further processing (ie. DNA extraction). To express the skin sample from the needle, phosphate buffered saline is deposited into the column of the needle and then forced through with the plunger into a sterile tube. DNA is extracted utilizing well known methods in the art. As illustrated by example below, this minimally invasive method for the collection of a skin sample yields sufficient DNA or mtDNA for the assessment of DNA or mtDNA damage, for example, caused by UV radiation. As with the previous embodiment, this method of obtaining skin samples is safe and painless. Further, as illustrated below, allows for sufficient DNA or mtDNA to be collected for conducting accurate assays.

[0033] Example 1 : Analysis of 3895 bp human mtDNA deletion.

[0034] The method of the present invention was used to analyze the 3895 bp mtDNA deletion identified in PCT application no. WO/06/ 111029. Collection and extraction of the mtDNA was conducted as provided below.

[0035] 1. Skin samples were collected by swabbing a skin site approximately 15 times with a sterile swab. Skin samples were collected from heel (n= 41), nose (n= 43), inner arm (n= 20), ear (n= 5), shoulder (n= 5), buttock (n= 5), and back (n= 5).

[0036] 2. mtDNA was extracted using a commercially available kit (QiaAMP™ DNA Micro Kit, product no. 56304, Qiagen, Maryland USA) according to the manufacturer's protocol.

[0037] 3. Double stranded DNA was quantified using the HS-DNA Quant-it™ dsDNA HS Assay Kit (product no. Q32851, Invitrogen, California USA) on the Qubit™ Fluorometer (product no. Q32857), Invitrogen, California USA).

[0038] 4. The level of the 3895 bp deletion was then quantified by real-time PCR (rt- PCR) using the iQ Sybr Green Supermix™ (product no. 170-8882, Bio-Rad, California USA) and the following primers:

Forward 5'-CTGCTAACCCCATACCCCGAAAATGTTG-S' (SEQ ID NO: 2); Reverse 5 '-GAAGGATTATGGATGCGGTTGCTTGCGTGAG -3 ' (SEQ ID NO: 3).

[0039] In this example, the pair of amplification primers are used to amplify a target region indicative of the presence of the 3895 bp deletion. The forward primer overlaps a spliced region of mtDNA after deletion of the 3895 bp sequence has occurred (ie. a splice at a position between 547 and 4443 of the mtDNA genome). Therefore, extension of the overlapping primer to create the correct size amplification product can only occur if the 3895 bp section is deleted.

[0040] In the step of quantifying the 3895 bp deletion, the RT-PCR reaction was set up as follows: 12.5ul of iQ Sybr Green Supermix™; 350nmol forward primer (SEQ ID NO: 2); 350nmol reverse primer (SEQ ID NO: 3); 5ul of template (approximately 0.5ng dsDNA); water to 25ul;

[0041] Cycling parameters:

Step 1. 95°C for 3 minutes; Step 2. 95°C for 30 seconds; Step 3. 67.5°C for 30 seconds; Step 4. 72⁰C for 30 seconds; Step 5. Plate Read

45 cycles of steps 2-5

Melting Curve 55-110⁰C reading every 3 seconds at 1°C intervals

Hold at 10⁰C for 10 minutes.

[0042] The results of these assays are shown in figures 1 to 3 and demonstrate a clear distinction between skin swabs taken from areas rarely exposed to sunlight/UV radiation (ie. heel and buttocks) and those usually exposed (ie. nose and ear). Levels of the 3895 bp deletion are significantly elevated in areas receiving a higher level of UV radiation such as the nose and shoulder when compared to areas generally protected from UV radiation such as the heel and the buttocks.

[0043] As shown in figures 1 and 2, the real time PCR cycle thresholds (C_T) for the 3895 bp deletion indicate that there is a higher incidence of the deletion in skin sites usually (nose or ear) or occasionally (shoulder or back) exposed to UV radiation compared to those sites that are rarely exposed (heel or buttocks).

[0044] Figure 3 shows that mtDNA collected from skin cells obtained from sites that are usually exposed to UV radiation (e.g. nose or ears) are characterized by increased levels of the 3895 bp deletion marker than mtDNA collected from skin cells obtained from sites rarely exposed to UV radiation (e.g. heel or inner arm). [0045] These results also show the effectiveness of collecting skin samples in accordance with the present invention, in order to obtain sufficient mtDNA to conduct the assays. As such, the non-invasive skin collection methods of the present invention are similarly effective for obtaining mtDNA for analysis as invasive methodologies, for example, the methods used in the Applicant's PCT publication no.WO/06/111029.

[0046] Example 2: Comparison of Skin Collection Methods

[0047] Five different non-invasive skin collection methodologies were tested in order to identify which, if any, would yield sufficient quantity and quality of nucleic acids for molecular analyses such as quantitative real-time PCR. The five methods tested were:

• Tapelift using surgical tape;

• Biore® adhesive strip;

• Sterile swab wetted with 8% mandelic acid;

• Sterile swab wetted with distilled water; and

• Wax strip.

[0048] The tapelift, Biore strip and wax strip were applied to the surface of the skin following the application of 70% isopropanol to sterilize the area. Firm pressure was applied and then the tape or strip was removed quickly. The swabs were first deposited in a sterile solution of either 8% mandelic acid, or distilled water and then rubbed firmly on the skin site of interest after the skin had been cleaned with 70% isopropanol.

[0049] Following the collection of skin cells from 3 individuals each collection medium was deposited into 200ul phosphate buffered saline solution (PBS) and incubated overnight at 56°C.

[0050] All of the samples were then subjected to nucleic acid extraction using the Qiagen's QiaAMP™ DNA Mini Kit , buccal swab protocol (product no. 51304). The purified samples were quantified using the NanoDrop™ ND- 1000 Spectrophotometer to determine if the extraction procedure was successful. [0051] Table 1 Determination of Quantity of DNA Extracted from Skin Collected by Five Non-invasive Methods

[0052] When considering both nucleic acid concentration as well as the purity of the sample, the most consistent results were achieved for the swab samples using either water or mandelic acid as a wetting agent, or the wax samples.

[0053] Next, a PCR was performed on all samples to determine if amplification inhibitors were present or significant degradation of the sample had occurred during processing. Samples were amplified according to the following conditions:

[0054] The primers used were mitochondrial DNA primers having the sequences provided below:

12s primer sequence forward 5 '-CGTTCCAGTGAGTTCACCCTC-S' (SEQ ID NO: 4) 12s primer sequence reverse R 5'-CACTCTTTACGCCGGCTTCTATT-S ' (SEQ ID NO: 5) [0055] The amplification reactions were cycled on a DNA Engine Tetrad (Bio-Rad) according to the following protocol:

1. 94°C for 2 minutes

2. 94⁰C for 30 seconds

3. 64°C for 30 seconds

4. 72°C for 30 seconds

5. Repeat steps 2-4 39 times

6. 4⁰C HOLD

[0056] Amplification products were then electrophoresed on a 2% agarose gel and stained with ethidium bromide. The amplification results are provided in figure 4, where the top half of the gel contains:

Lane 1 500ng lOObp GeneRuler SM0323 (Fermentas)

Lane 2 Biore from Individual 1

Lane 3 Swab with water from Individual 1

Lane 4 Swab with mandelic acid from Individual 1

Lane 5 Surgical tape from Individual 1

Lane 6 Wax from Individual 1

Lane 7 Biore from Individual 2

Lane 8 Swab with water from Individual 2

Lane 9 Swab with mandelic acid from Individual 2

Lane 10 Surgical tape from Individual 2

Lane 11 Wax from Individual 2

Lane 12 empty

Lane 13 Negative amplification control

Lane 14 Positive amplification control

Lanes 15-18 empty

[0057] And where the bottom half of the gel contains:

Lane 1 Biore from Individual 3

Lane 2 Swab with water from Individual 3

Lane 3 Swab with mandelic acid from Individual 3

Lane 4 Surgical tape from Individual 3 Lane 5 Wax from Individual 3

Lane 6 500ng lOObp GeneRuler SM0323 (Fermentas)

Lane 7 Biore extract negative control

Lane 8 Swab with water extract negative control

Lane 9 Swab with mandelic acid extract negative control

Lane 10 Surgical tape extract negative control

Lane 11 Wax extract negative control

Lane 12 empty

Lane 13 Negative amplification control (duplicate loading to Lane 13 above)

Lane 14 Positive amplification control (duplicate loading to Lane 14 above)

Lane 15-18 empty

[0058] Results

[0059] No mtDNA was amplified from mtDNA collected from skin cells harvested using the Biore strips. The swabs for both the water and the mandelic acid amplified, though the water swab amplified more brightly. The surgical tape amplified sporadically. The wax amplified brightly however the extract negative control in Lane 11 of the bottom half of the gel was contaminated likely as a result of the non-sterile nature or handling difficulties associated with the wax.

[0060] With all factors considered this example demonstrated that the use of a sterile swab is the preferred method of collection of a non-invasive skin sample. The swab can be dry or wetted with various liquids to facilitate collection or buffering of the sample.

[0061] Example 3: Comparison of Additional Skin Collection Methods

[0062] In this example five additional methods for the non-invasive collection of skin samples were tested in order to identify which, if any, would yield sufficient quantity and quality of nucleic acids for molecular analyses such as quantitative real-time PCR.

[0063] From a single individual, skin samples were collected twice using the following methods:

• scraping of skin using a sterile surgical blade

• scraping of skin using a wooden scraper • sticky surface of an adhesive pad (CapSure™ Clean-up Pad, Arcturus)

• film from LCM MacroCap™ (Arcturus)

• heated film from LCM MacroCap™ (Arcturus)

[0064] The skin was first prepared by cleansing with a 70% isopropanol wipe. The wooden scraper and the surgical blade were passed firmly over the skin surface to remove skin cells and then deposited into a centrifuge tube. The adhesive pad and films were pressed firmly against the skin without rubbing to collect skin cells.

[0065] The multiple collections were processed using two different nucleic acid extraction methods. The first set was extracted using a proteinase K digestion as is well known in the art while the second set was extracted using the QiaAMP DNA Mini Kit (Qiagen 51304) .

[0066] The samples processed with the Qiagen kit were then quantified using the NanoDrop ND- 1000 Spectrophotometer. Those in the PK digestion set were not as they were not cleaned up enough to facilitate this type of quantification.

[0067] Table 2 Determination of Quantity of DNA Extracted from Skin Collected by Further Collection Methods (Qiagen extracted DNA)

[0068] Samples were amplified according to the protocol provided in example 2. Amplification products were then electrophoresed on a 2% agarose gel and stained with ethidium bromide. Results are shown in figure 5, where the gel contains: Lane 1 500ng of lOObp GeneRuler (SM0323)

Lane 2 Negative Amplification control Lane 3 Positive amplification control Lane 4 PK buffer wood scrape Lane 5 PK buffer surgical blade scrape

Lane 6 PK buffer CapSure pad

Lane 7 PK buffer MacroCap

Lane 8 PK buffer MacroCap heated

Lane 9 PK buffer wood scrape negative extraction control

Lane 10 PK buffer surgical blade scrape negative extraction control

Lane 11 PK buffer CapSure negative extraction control

Lane 12 PK buffer MacroCap negative extraction control

Lane 13 PK buffer MacroCap heated negative extraction control

Lane 14 QiaAMP surgical blade scrape

Lane 15 QiaAMP CapSure pad

Lane 16 QiaAMP wood scrape

Lane 17 QiaAMP surgical blade scrape negative extraction control

Lane 18 QiaAMP CapSure pad negative extraction control

Lane 19 QiaAMP wood scrape negative extraction control

Lane 20 QiaAMP reagent negative control

[0069] The surgical blade scrape and the MacroCap™ were amplified using the PK buffer, while the CapSure™ pad amplified well using the QiaAMP™ kit. When compared to skin swabbing, the amount of mtDNA collected and the amount of amplified product obtained using the methods tested in this example were not found to be as effective.

[0070] Example 4: Collection of Skin Samples using Needle

[0071] Needles were used to collect skin samples from 5 different body sites of 9 individuals. The body sites included the eyebrow, earlobe, nape of the neck, hand, and heel. Using a needle as described above, the skin was pinched or tented between the thumb and forefinger of the sample collector's hand. The needle was passed through the skin, drawing little or no blood. The skin sample was extracted from the needle by depositing phosphate buffered saline into the column of the needle and then forcing the sample from the needle with a plunger into a sterile tube. DNA was then extracted from this volume containing the skin tissue using the QiaAMP™ DNA Mini Kit™ (Qiagen product no. 51304). [0072] The samples were then amplified in order to identify the 3895bp mtDNA deletion. The reaction conditions and cycle parameters for this example were the same as for example 1 provided above. The results are presented in Table 3.

[0073] Table 3: Results for Skin Samples collected via Needle

[0074] It is clear that the material obtained through this collection method is sufficient for molecular analyses such as real-time PCR. Specifically, the amplification product indicative of the 3895 bp mtDNA deletion has been detected and quantified as evidenced by Table 3. Therefore, the collection of skin samples via the needle collection method yields sufficient DNA for use in an assay of this kind.

[0075] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention as outlined in the claims appended hereto.

Claims

Claims:

1. A method for the detection of an aberration in DNA of skin cells comprising;

contacting a skin site with a sterile swab;

swabbing the skin site with the sterile swab to collect the skin cells;

extracting DNA from the skin cells;

detecting the presence of the aberration in the DNA.

2. The method according to claim 1 wherein the DNA is mitochondrial DNA (mtDNA).

3. The method according to claim 1 wherein the detection of the aberration is conducted using real-time PCR.

4. The method of claim 1 wherein the aberration is selected from the group consisting of deletions, substitutions, and insertions.

5. The method of claim 2 wherein the aberration is a 3895 bp mtDNA deletion between nucleic acids 546 to 4444 of the mtDNA genome.

6. A method for the detection of an aberration in DNA of skin cells comprising;

collecting skin cells from the dermis and/or epidermis by piercing through a tented layer of skin with a needle, without obtaining blood, to obtain a microscopic amount of dermal and/or epidermal tissue adhered to the core of the needle;

expressing the dermal and/or epidermal tissue from the core of the needle;

extracting DNA from the dermal and/or epidermal tissue;

detecting the presence of the aberration in the DNA.

7. The method according to claim 6 wherein the DNA is mitochondrial DNA (mtDNA).

8. The method according to claim 6 wherein the detection of the aberration is conducted using real-time PCR.

9. The method of claim 6 wherein the aberration is selected from the group consisting of deletions, substitutions, and insertions.

9. The method of claim 7 wherein the aberration is a 3895 bp mtDNA deletion between nucleic acids 546 to 4444 of the mtDNA genome.

10. The method of claim 5 wherein the needle is a 28 gauge or 29 gauge needle.