SELEX screening experiments of single-stranded DNA libraries can be used for (1) in vitro diagnostics; (2) in vivo therapeutics, etc.
Operation method
SELEX screening of single-stranded DNA libraries
Principle
In addition to RNA libraries, SELEX technology can also be used to screen single-stranded DNA libraries. The abundance of single-stranded DNA forming high-level structures is also sufficient for screening many target molecules, and the DNA libraries are more stable and relatively easy to use.
Materials and Instruments
3' Primer Streptavidin Magnetic Beads Move I. Materials and equipment Caveat Prevents contamination of RNAase. Common Problems I. Measures to prevent RNAase contamination 1. All glassware should be dry-roasted at 180℃ for 6hr or longer before use. 2. 2. Plastic containers can be soaked in 0.1% DEPC water or rinsed with chloroform (Note: Plexiglas utensils can be corroded by chloroform, so they can not be used). 3. 3. Plexiglass electrophoresis tanks can be washed with detergent, rinsed with double-distilled water, dried with ethanol, immersed in 3% H2O2 at room temperature for 10min, and then rinsed with 0.1% DEPC water and air-dried. 4. The prepared solution should be treated with 0.1% DEPC as much as possible, at 37°C for more than 12hr. Then autoclave to remove the residual DEPC. reagents that can not be autoclaved, should be prepared with DEPC-treated sterile double-distilled water, and then filtered through a 0.22μm filter membrane to remove bacteria. 5. Operators should wear disposable masks, hats and gloves, and the gloves should be changed frequently during the experimental process. 6. Set up a special laboratory for RNA operation, and all instruments should be specialized. Commonly used RNAase inhibitors 1. Diethyl pyrophosphate (DEPC): It is a kind of strong but incomplete RNAase inhibitor. It inhibits the activity of RNAase by binding to the imidazole ring of histidine, the active group of RNAase, and denaturing the protein. 2. 2. Guanidine isothiocyanate: Currently considered the most potent RNAase inhibitor, it inactivates RNAase while cleaving tissue. It both destroys cellular structures to dissociate nucleic acids from nucleoproteins and has a strong denaturing effect on RNA enzymes. 3. oxovanadium ribonucleoside complex: a complex formed by oxovanadium ions and ribonucleosides, which combines with RNAase to form a transition state-like substance that almost completely inhibits the activity of RNAase. 4. protein inhibitor of RNAase (RNasin): acidic glycoprotein extracted from rat liver or human placenta, RNasin is a non-competitive inhibitor of RNAase and can bind to a variety of RNAases and inactivate them. 5. Others: SDS, urea, diatomaceous earth, etc. also have a certain inhibitory effect on RNA enzymes. For more product details, please visit Aladdin Scientific website.
Electrophoresis PCR Instrument Centrifuge Gel Imager Liquid Scintillator Long Wave UV Lamp UV Spectrophotometer Nucleic Acid Halo pH Meter Incubator Oscillator Clean Bench
1. 3' primers labeled with biotin at the 5' ' end.
2. Streptavidin magnetic beads (Promega).
Other equipment and reagents are consistent with the RNA library protocol.
Operation Methods
1. asymmetric PCR for ssDNA preparation
(1) Firstly, amplify the ssDNA obtained by screening into double-stranded DNA with reference to the experimental protocol of RNA library, and then purify and recover it.
(2) Determine the optimal amount of double-stranded DNA template for asymmetric PCR preparation of the ssDNA library. Different amounts of the above PCR products were used as templates for 35 cycles of asymmetric PCR amplification.
The asymmetric PCR reaction system was as follows: 10 μl of 10X PCR reaction buffer, 8 μl of dNTP mix, 2 μl of 5' primer (25 pmol/μl), 0.5 μl of 3' primer (1 pmol/μl), an unspecified amount of dsDNA template, and 1 μl of Taq DNA polymerase (2 U/μl), which was made up to 100 μl with deionized water.
PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; amplification for 35 cycles; final extension at 72 ℃ for 5 min.
The 12% denaturing PAGE containing 7 mol/L urea showed that the single-stranded DNA bands should be slightly higher than the double-stranded DNA bands, and the double-stranded DNA template that could produce a large amount of clear single-stranded DNA bands was selected as the optimal amount.
(3) Take the optimal amount of double-stranded DNA template for mass asymmetric PCR amplification, and purify it by 12% denaturing PAGE with 7mol/L urea, and then use nucleic acid quantification to determine the amount of ssDNA for the next round of screening.
(4) In the first round of screening, the input ssDNA is synthesized directly.
2. Streptavidin magnetic bead preparation of ssDNA
(1) Amplify the ssDNA obtained from the screening into double-stranded DNA with a 3' primer labeled with biotin at the 5' end, purify and recover it, and dissolve it to 20 μl with SHMCK buffer.
(2) Preparation of streptavidin magnetic beads. Take 0.5 ml of Streptavidin magnetic beads and wash it with 0.5X SSC three times, 600 μl each time, 1 min each time, and then wash it with SHMCK buffer three times, 600 μl each time, 1 min each time.
(3) Mix the double-stranded DNA with the magnetic beads, then add 40 μl of SHMCK buffer and incubate for 30 min at room temperature (mix well on the DNA mixer).
(4) Discard the supernatant after magnet adsorption, and wash with SHMCK buffer three times, 600 μl each time, 1 min each time.
(5) Add 400 μl of 0.15 mol/L NaOH, cleave the double-stranded DNA into ssDNA, and incubate at 37℃ for 15 min.
(6) After magnet adsorption, aspirate the supernatant, and add 20 μl of 3 mol/L HCl to neutralize the NaOH, and adjust the buffer system to 1X SHMCK, 0.1% gelatin, and 12 μg/μL tRNA, with the total volume of 500 μl, and then proceed to the next round of screening.
The source of this experiment is RNA Laboratory Instruction Manual, edited by Zheng Xiaofei.