Mitochondrial DNA deletion between about residues 12317-16254 for use in detection of cancer
An individual, breast cancer technology, used in recombinant DNA technology, DNA/RNA fragments, determination/examination of microorganisms, etc.
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Embodiment 1
[0104] Example 1: Relationship between prostate cancer and 4kb deletion in human mtDNA
[0105] Urine samples were collected from 5 patients who had been diagnosed with prostate cancer and from 5 patients whose prostate malignancies were not detected by needle biopsy methods. These samples were collected following a digital rectal examination (DRE) to facilitate the collection of prostate cells.
[0106] Once the sample was obtained, a 5ml aliquot was removed and the 2ml sample was centrifuged at 14000 xg to pellet. Remove and discard supernatant.
[0107] Resuspend the pellet in 200 μl phosphate-buffered saline. DNA extraction procedures were performed on resuspended pellets and whole urine samples using the QiaAMP DNA Mini Kit (Qiagen P / N 51304) according to the manufacturer's instructions. The resulting DNA extracts were then quantified using a NanoDropND-1000 spectrophotometer and normalized to a concentration of 0.1 ng / μl.
[0108] Samples were analyzed by quantitative ...
Embodiment 2
[0143] Example 2: Relationship between 4kb deletion in human mtDNA and breast cancer
[0144] Of the 20 breast tissue samples collected, 10 were malignant and the other 10 had benign breast disease or no abnormalities. These samples were embedded in formalin-fixed paraffin, then individually cut into 20 micron sections into individual sample tubes and extracted according to the manufacturer's protocol of the QiaAMP DNA Mini Kit (Qiagen P / N 51304). DNA was then quantified using NanoDrop ND-1000 and normalized to a concentration of 2 ng / μl.
[0145] The level of the 4 kb deletion in the samples was then determined by quantitative real-time PCR using the following protocol.
[0146] X iQ SYBR Green Supermix (Bio-Rad product number: 170-8880)
[0147] 175nmol forward primer (5'-TTGGTGCAACTCCAAAGCCACCCCTCACC-3') (SEQ ID NO: 4)
[0148] 175nmol reverse primer (5'-AGGATGGTGGTCAAGGGAC-3') (SEQ ID NO: 5)
[0149] 25μl reaction solution containing 20ng template DNA
[0150] Reactio...
Embodiment 3
[0174] Example 3: Association of Prostate Cancer with 4kb Deletions in Human mtDNA Using Needle Biopsy Samples
[0175] Prostate needle biopsy samples were obtained from 19 individuals, 9 without prostate cancer and 10 with prostate cancer. Needle biopsies were formalin-fixed paraffin-embedded (FFPE) as is standard in clinical diagnostic settings. A 10 micron section of each biopsy was placed directly into a centrifuge tube and DNA was extracted using the QiaAMP DNA Mini kit (Qiagen, p / n 51306). DNA extracts were quantified using a NanoDrop ND-1000 spectrophotometer at 260 nm. Yields ranged from 347ng to 750ng. Dilute these samples to 2ng / μl and set up the amplification reaction according to Table 9 and the following:
[0176] Table 9: Reagents and concentrations for amplification reactions
[0177]
[0178]Add nuclease-free water to a final reaction volume of 25 µl. Amplification was performed on a DNA Engine Chromo4 real-time PCR instrument (Bio-Rad Laboratories) acc...
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