merged oligonucleotide mutagenesis assay
merged oligonucleotide mutagenesis assay
An important feature of this method is the direct cloning of single-stranded, abbreviated oligonucleotides into homologous double-stranded molecules after their conversion into conventional vectors. Since different oligonucleotides can hybridize through the palindromic structure of their 3' ends, the oligonucleotides actually act as reciprocal primers, extending in the presence of the E. coli DNA polymerase I klenow fragment.
Operation method
Generation of large numbers of mutations in small DNA sequences
Materials and Instruments
Escherichia coli Move 1. Design oligonucleotides that contain a palindromic structure consisting of eight nucleotides at the 3' end and a recognition site for a restriction endonuclease; if possible, the 5' end should also contain a sequence with a restriction endonuclease site. The middle region should contain the target mutagenic region. For more product details, please visit Aladdin Scientific website.
DNA polymerase dNTP glycerol NaCl EDTA SDS anhydrous ethanol
Water bath Centrifuge Spectrophotometer
Figure 1. Design of the abbreviated oligonucleotide2. Oligonucleotide synthesis. A homogeneous solution of one nucleotide precursor is used for synthesis when no mutation is required at the site, while a specific mixture of nucleotide precursors is used to control the synthetic reaction when mutation is required at the site.3. Purify each nucleotide by HPLC and/or denaturing polyacrylamide gel electrophoresis containing 7 mol/l urea, adjusting the concentration to 1 mg/ml with water.
4. Add 200 pmol (1~2 μg) of oligonucleotide to a 500 μl microcentrifuge tube, add water to 7 μl and incubate for 5 min.5. Add 1 μl of 10×DNA polymerase 1 buffer, lower the temperature to a temperature suitable for hybridization of the 3' end of the palindromic structure, and hold for not less than 60 min.
6. Add 2 μl of 2.5 mmol/l mixture of the four dNTPs, 5 U of klenowase, and 10 μCi of any one of the [ α-32P ], and incubate at 23°C for 1 h. Add another 5 U of klenowase and continue to incubate for 2 h or overnight.7. Add 1 μl of 0.5 mol/l EDTA to terminate the reaction, add TE buffer to a total volume of 50 μl, and add sodium acetate to a final concentration of 0.3 mol/l. Equilibrate the phenol extraction with buffer, and precipitate the DNA with anhydrous ethanol. the DNA was resuspended in 20 μl of TE buffer. The DNA was resuspended in 20 μl of TE buffer. 2 μl of the DNA was set aside for analysis by denaturing polyacrylamide gel electrophoresis.
8. Digest double-stranded oligonucleotides for more than 2 h in a 30 μl reaction volume with 10-40 U of an enzyme recognizing the outer restriction site per μg of the nucleoside linkage, or cut the oligonucleotides with an enzyme recognizing the inner restriction site if there is no restriction site at the 5' end of the oligonucleotide.9. Remove 2 μl for denaturing polyacrylamide gel electrophoresis, extract the remainder with buffer-equilibrated phenol, precipitate with anhydrous ethanol, resuspend in not less than 10 μl of TE buffer, and purify by non-denaturing polyacrylamide gel electrophoresis.10. Cut off the gel where the double-stranded DNA is located and elute it with gel elution buffer, resuspend it with 20 μl of TE buffer, and store it at -20 °C.
11. Digest the double-stranded oligonucleotide with a restriction endonuclease that recognizes the medial cleavage site (located at the 3' end of the original oligonucleotide) to generate a mixture of homologous double-stranded oligonucleotides whose 5' and 3' ends are suitable for ligating them into a conventional vector. The mixture is set aside for denaturing polyacrylamide gel electrophoresis, and the remainder is extracted with phenol, precipitated with ethanol, and resuspended in 20 μl of buffer.
12. 2 μl of the liquid retained from steps 7, 9, and 11 is electrophoresed on a denaturing polyacrylamide gel to confirm that the desired products have been produced in each step of the reaction.
13. Set up a series of ligation reactions, each containing a constant amount of carrier and a portion of the above dilutions, by serial dilution with TE buffer from 10 to 10,000-fold serial dilutions of double-stranded oligonucleotides.
14. Transform the ligation reactants into appropriate E. coli using conventional transformation methods. The resulting DNA is analyzed by restriction endonuclease digestion and DNA sequencing.