Quantitative analysis of trace DNA by PCR method
Quantitative analysis of trace DNA by PCR method
PCR is used to quantify the amount of a particular DNA sequence from 1 to 20,000 molecules per sample. In addition, it can assist in assessing the presence of contaminating sequences that contribute to the failure of such experiments. Source: Compact Molecular Biology Laboratory Guide, 5th Edition
Operation method
basic program
Materials and Instruments
DNA Move 1) Put the cell or tissue samples into microcentrifuge tubes with screw tops, add 100 μL of protease digestion buffer for every 2X1O6 cells, and add 20 mg/ml of proteinase K to a final concentration of 100 μg/ml, and place the samples at 50℃ overnight. It is usually necessary to set up several samples without viral sequences as negative controls, taking care to avoid contamination of cell or tissue samples with other unrelated DNA sequences. It is best to process the samples in order of increasing likelihood of the target sequence. Extraction should be performed away from processing PCR products and other large amounts of DNA plasmids. Wear disposable gloves when handling and change them frequently. 2) Blow gently up and down using a 200 μL micro capillary pipette to mix the sample. Add 100 μL of buffer to equilibrate the phenol and mix gently; add 100 μL of chloroform/isoamyl alcohol (24:1) and mix gently; centrifuge at high speed for 5 min and transfer the aqueous phase (containing DNA) into a new centrifuge tube. Screw-top microcentrifuge tubes and microcapillary tips minimize aerosol contamination from microcentrifuges and automated pipettes. One pipette is required for each reagent. Positive pressure pipettes with disposable disposable tips and stoppers are easier to use than microcapillary pipettes, although they are more expensive. Centrifuges that have been used to centrifuge PCR products or large amounts of plasmid DNA should be avoided. 3) Extract the organic phase again with protease digestion buffer (about 1/2 of the original volume), centrifuge at high speed for 5 min at room temperature, and transfer the aqueous phase into the aqueous phase obtained in step 2. 4) The aqueous phase was extracted twice with an equal volume of chloroform/isoamyl alcohol (24:1), and each time centrifuged at high speed for 5 min at room temperature to separate the aqueous phase from the organic phase. 5) The final aqueous phase was added 10 mol/L ammonium acetate to a final concentration of 2.5 mol/L, followed by 2.5 times the volume of pre-cooled anhydrous ethanol and gently mixed. Place on dry ice for 30 min. centrifuge at high speed for 15 min at 4°C. The precipitate was washed once with 70% ethanol, centrifuged and dried. 6) Resuspend the precipitate with TE at pH 7.5 (~2 X 1O6 cell DNA extracts dissolved in 100, 5 ~ 100 ng DNA/μL). Store at 4°C. 7) Take small portions of the solution and standard DNA and electrophoresis them simultaneously on an agarose gel, or estimate the DNA content by quantification with ethidium bromide spotting. 8) Prepare several tubes for addition of DNA from non-infected cells or tissues. Add 110 ng of DNA to 1 tube, 90 ng of DNA to I tube, and 100 ng of DNA to the rest of the tubes. Use these tubes for a series of 10-fold dilutions of the target sequence in the following way: add a known amount (e.g., 20,000 molecules) of the target-sequence-containing DNA to the 1,000-ng tubes and mix. Then mix. Take 1/10 into another 100 ng tube and mix, then take another 1/10 into another 100 ng tube, and so on until the tube contains <10 molecules of the target sequence, then take 1/10 from this tube and add to 90 ng DNA tubes, which should have a volume of <71 μL per tube. 9) For each amplification reaction, prepare separate screw-top microcentrifuge tubes containing 24 μL of reaction mixture and add sufficient sterile water to make a final volume of 100 μL (after addition of DNA and Taq DNA polymerase, steps 10 and 12). After mixing, cover each tube with 100 μL of mineral oil to ensure that the surface of the reaction mixture is completely covered. 10) Open only those tubes that will contain the same samples and add 100 ng of sample DNA to each corresponding tube, then cap and centrifuge slightly to mix. After adding the other negative controls, the sample DNA should be added in increasing order of likelihood of containing the target sequence, starting with the virus-free control and ending with the serial dilution tubes. 11) Denature at 94°C for 10 min on a water bath or automatic thermal cycler. 12) Open the tube (containing the same sample) and add 5 μL of 0.8 U/μL Taq DNA polymerase. Cap tightly and repeat steps 10~12 for the next set of the same samples. 13) centrifuge the tubes slightly at 55°C for 2 min (replication) and 72°C for 3 min (extension). Perform the PCR reaction according to the following parameters. 29 cycles: 1 min 94°C (denaturation) 1 min 55℃ (denaturation) 1 min 72℃ (extension) 1 cycle: 7 min 72°C (extension) Final storage at 4°C 15) A small portion of the reaction (1/10 reaction volume) is taken for electrophoresis on a suitable agarose or polyacrylamide gel, which should include standard molecular mass lanes of DNA capable of visualizing both ethidium bromide staining and radioautography. The gel is stained and photographed. After the gel has been stained with ethidium bromide, DNA fragments corresponding to PCR products derived from specific DNA from the host chromosome should be readily visible and the efficiency of each amplification reaction can be estimated. Do not be surprised if you see other bands, especially large molecular mass bands, that are some non-specific PCR products. 16) Transfer the DNA on the gel to a nitrocellulose or nylon membrane, and immobilize the DNA to the membrane by UV cross-linking if desired. Pre-hybridization and hybridization with end-labeled, oligonucleotide probes specific to the target sequence are analyzed by radioautography or Phosphorimagei imaging. If the efficiency of each amplification reaction is similar, then the expected result should be that the intensity of appropriately sized labeled bands should diminish in reconstructed serial dilution samples, that there should be no bands of the same size in negative controls, and that the intensity of the bands in experimental group samples will vary. Depending on the tightness of the probe and hybridization and the conditions of the membrane wash, nonspecific adsorption of the probe to a large amount of internal control product may be seen. It is also common to see some small amount of PCR product that has a slightly greater or lesser mobility than the main specific PCR product, especially at high copy levels. Assuming that the efficiency of each amplification reaction is similar, the number of molecules of the target sequence in each sample can be estimated by comparison with serially diluted samples. 17) For further quantitative analysis, the used probe can be stripped from the membrane by heating in boiling water for 15 min and hybridized with a probe specific for the host's single-copy sequence. The signal is quantified by densitometric scanning (follow the densitometer manufacturer's instructions for use) and a standard curve is calculated from a series of dilutions, correcting for the signal of the host sequence. The number of molecules in the experimental group of samples is determined by comparison with the standard curve. Ideally the standard curve should be linearly related to the logarithm of the signal intensity of the radiation autoradiography to the logarithm of the amount of DNA. However, a perfect linear relationship may not be necessary, nor is it possible that the slope is 1. At the high end of the standard curve (0.1 to 1 copy per cell), it may be a plateau. If this causes difficulties in quantification, the number of amplification cycles must be reduced or other parameters changed. For more product details, please visit Aladdin Scientific website.
Protease digestion buffer 20 mg ml proteinase K (stored at -20°C) with 50 mmol L Tris . Cl 10 mmol L EDTA pH 7.4 buffered phenol (stored at room temperature) 24 : 1 (V V) chloroform isoamyl alcohol 10 mol L acetic acid pressed Ice-cold 100% ethanol 70% ethanol TE buffer pH 7.5 Reaction mixing system Mineral oil 0.8 U μL Taq DNA polymerase Oligonucleotide primer for hybridization
Autoclaved screw-top microcentrifuge tubes Micro capillary pipettes or positive pressure pipettes with disposable disposable tips and stoppers or use of micropipettes with cartridge tips