Restriction endonuclease in enzyme digestion technology

Restriction endonuclease can specifically bind to a specific site in or near the DNA sequence called restriction enzyme recognition sequence to cut double-stranded DNA. It can be divided into three categories: Class I and Class III enzymes have both cleavage and modification (methylation) in the same protein molecule, and depend on the existence of ATP. Class I enzymes bind to the recognition site and randomly cleave DNA not far from the recognition site, while Class III enzymes cleave DNA molecules at the recognition site and then dissociate from the substrate. Class ⅱ consists of two enzymes: one is restriction endonuclease, which cleaves specific nucleotide sequences; The other is an independent methylase that modifies the same recognition sequence. Class ⅱ restriction enzymes have been widely used in molecular cloning and are the basis of recombinant DNA. Most class Ⅱ restriction endonucleases recognize a specific nucleotide sequence with palindrome symmetry of 4 to 6 nucleotides in length (for example, EcoRⅰI I recognizes 6 nucleotide sequences: 5'-G↓AATTC-3'), and a few enzymes recognize longer sequences or degenerate sequences. In the recognition sequence, some cleavage sites of class ⅱ enzymes are cleaved at the symmetry axis, resulting in blunt-ended DNA fragments (such as SMA ⅰ: 5'-CCCGGG-3'); Some cleavage sites are on the side of symmetry axis, which leads to DNA fragments with single-stranded protruding ends, which are called sticky ends. For example, Ecor Ⅰ Ⅰ cleaves the recognition sequence and produces two complementary sticky ends.

5'…G↓AATTC…3'→5'…GAATTC…3 '

3'…CTTAA↑G…5'→3'…CTTAAG…5 '

DNA purity, buffer, temperature conditions and restriction endonuclease itself will all affect the activity of restriction endonuclease. Most restriction enzymes are not affected by RNA or single-stranded DNA. When trace pollutants enter the restriction enzyme storage solution, it will affect its further use, so a new pipette head should be used every time the restriction enzyme is absorbed. If two kinds of restriction enzymes are used, it is necessary to pay attention to providing their respective optimal salt concentrations. If they can use the same buffer, they can be hydrolyzed simultaneously. If different salt concentrations are needed, the restriction endonuclease with low salt concentration must be used first, then the salt concentration should be adjusted, and then the restriction endonuclease with high salt concentration should be used for hydrolysis. Or, after the first digestion reaction is completed, extract with phenol/chloroform with equal volume, add 0. 1 times volume of 3mol/LNaAc and 2 times volume of anhydrous ethanol, mix well, then refrigerate at -70℃ for 30 minutes, centrifuge, dry, and re-dissolve in buffer for the second digestion reaction.