DNA probes are divided into two categories: isotope-labeled probes and non-isotope-labeled probes. Isotope-labeled probes usually have high radioactivity and high sensitivity for hybridization, but they have a short service life, radioactive hazards, difficult disposal of contaminants, require special instruments and equipment, and are not suitable for general laboratories. In recent years, non-isotope labeling methods have been greatly developed, such as enzymatic labeling methods (e.g., biotin, digoxin labeling method) and chemical labeling methods (e.g., fluorescent biotin, enzyme labeling method). Non-isotope-labeled probes have a longer shelf life and avoid isotope contamination, but are not as sensitive as isotope-labeled probes.
Principle
After agarose gel electrophoresis of the DNA digestion products, it is not possible to analyze the changes in DNA fragments due to a continuous band on the gel because of the proximity of the enzyme fragment lengths, so it is necessary to use specific labeling probes to hybridize the DNA transferred to the nylon membrane.
Appliance
In RFLP analytical studies, labeling probes with 32P is a common method, however, the limited retention time of 32P, as well as the need for special protective devices, waste liquid disposal devices, has limited its use, and now in many experiments have been switched to non-isotopic labeling instead of 32P.
Operation method
basic program
Principle
For Southern hybridization analysis, insert fragments without vectors should be labeled as probes. The commonly used labeling method is time-consuming, which involves isolating the insert fragments from the plasmid or λDNA by restriction endonuclease digestion and labeling them by the incision shift method or random primer labeling method. In contrast, the PCR polymerase chain reaction method for labeling probes has several advantages. A large number of efficiently labeled insert fragments can be amplified using only a very small amount of plasmid DNA (50 ng) as template. The addition of non-radioactive substituting nucleotides (biotin dUTP, digoxin-11-dUTP base unstable) to the PCR reaction allows the synthesis of a large number of labeled probes within a few hours.
Materials and Instruments
LB medium psr142 primer FBA065 primer dATP, dGTP, dCTP solution Taq polymerase Move 1. Inoculate a single colony in a 5mL lysate tube with 1mL of LB-antibiotic medium, loosely cap the tube, and incubate overnight at 37℃ with vigorous shaking. 2. Take 800μL of bacterial solution into a 1.5mLEppendorf tube. Centrifuge at 12000r/min for 30 s. Store the remaining liquid at 4℃ or add 40μL of sterilized glycerol and mix it at -80℃. 3. Discard the supernatant. Resuspend the bacterial precipitate with 400 μL of 1% TritonX-100 with vigorous shaking. 4. Boil the bacterial suspension for 5 min and leave it on ice for 5 min. store the bacterial lysate at 4℃ or -20℃. 5. Take a micro-amplification reaction tube and add the following reagents to a final volume of 50μL. 25μL of sterilized distilled water 10μL of 50% sterilized glycerol 5μL 10×PCR reaction buffer 0.5μL 5mmoL/LdATP 0.5μL 5mmoL/LdCTP 0.5μL 5mmoL/LdGTP 1μL 2mmoL/LdTTP 1μL 0.2mmoL/L digoxin-11-dUTP base unstable 0.625μL 20μmoL/L primer 1 0.625μL 20μmoL/L primer 2 0.25μL 5U/μL Taq polymerase 5μL bacterial lysate 6. PCR amplification was performed according to the following parameters 1 cycle: 94℃,1min 35 cycles:94℃,15s 48℃,30s 72℃,3min 1 cycle:72℃,7min 7. 3μL of PCR amplification product was taken for horizontal agarose gel electrophoresis, and the concentration of the amplification product was estimated by using the standard DNA of known concentration as control. The remaining PCR amplification products were stored at 4℃. Common Problems The advantages of using the non-isotopic method include. (1) Reduced risk to the laboratory operator, no specialized disposal of laboratory waste, thus allowing the handling of large quantities of RFLP-analyzed samples. (2) Multiple, large numbers of probes can be labeled for future use. The labeled probes can be stored at -20℃ (at least one year). (3) The probes, CSPDR and membrane of the chemiluminescence detection system can be reused (3-5 times, 3-5 times, 5-8 times or more, respectively) for RFLP analysis. It is more economical compared to isotope detection methods. (4) Experimental arrangement is more convenient. This is because there is no need to be limited by the decay time of the isotope, as is the case when using 32P. (5) Exposure at room temperature is sufficient, without the need for a sensitizing screen or -80°C refrigerator. (6) Short exposure time (a few hours), i.e. a film can be rehybridized every 3d. Reagent description: 1.LB LB medium (1% trypsin, 1% NaCL, 0.5% yeast extract, pH 7.0), antibiotics, 1% TritonX-100, sterilized ultrapure water, 50% sterilized glycerol, 10×PCR reaction buffer (0.1moL/LTris-HCL, pH 8.3; 15mmoL/LMgCL2; 0.5moL/LKCL. 1mg/mL gelatin). 2.psr142 Primers. 20μmoL/L Reverse primer:5 ATTCGAGCTCGGTACC 3 20μmoL/L Forward primer:5 AGGTCGACTCTAGAGG 3 3.FBA065 Primer: 5 AGGTCGACTCTAGAGG 20μmoL/L Reverse primer:5 AACAGCTATGACCATG 3 20μmoL/L Forward primer:5 gtaaaacgacggccagt 3 4.5 mmoL/LdATP dGTP, dCTP solution, 2 mmoL/LdTTP solution, 0.2 mmoL/L digoxin-11-dUTP (base-unstable type), 5 U/μL Taq polymerase. For more product details, please visit Aladdin Scientific website.
DNA probe to be labeled
PCR instrument Electrophoresis tank and power supply Micropipettes and corresponding tips 1.5 ml Eppendorf tubes 5 ml lysis tubes Tabletop microcentrifuge 37 ℃ constant temperature shaker Constant temperature water bath -20 ℃ refrigerator -80 ℃ refrigerator 4 ℃ refrigerator