Restriction Endonuclease Standardized-the BamH I

Standardized formulation of the restriction endonuclease BamHIincluded classification and name of BamHI, technical specifications,test methods, inspection rules and signs, packaging, transport andstorage. This study focused on determination of biological activity ofthe restriction endonuclease and detection of the purity of the enzyme,designed the test methods of the technical indicators. These are the corecontents of the standardization work of the restriction endonucleases.In the determination of biological activity of the restrictionendonuclease, the lambda DNA, which has been thoroughly studied,was chosen as the substrate of digestion. In view of recognition sites ofthe restriction endonucleases on the lambda DNA, it can be used as thesubstrate of other restriction endonucleases, too. So it has a wide rangeof versatility. The multiplex PCR technique was used in thisexperimental approach, through rational design, the biological activity ofthe restriction endonuclease was more accurately determined.In the experiment of overnight digestion with the enough restrictionendonuclease, other non-specific nucleases were detected. If the electrophoretic bands of the substrate digested were the same as thetypical patterns of electrophoretic bands, there is no other non-specificnuclease mixed in the restriction endonuclease.Cutting-ligation-recutting assay mainly detected exonuclease,phosphatase and other restriction endonuclease. These enzymes woulddestroy the integrity of the sticky ends generated by the restrictionendonucleases. The DNA with incomplete sticky ends could not besmoothly connected together, even if incorrectly connected, therecognition sequences of the restriction endonucleases would not exist,so the DNA incorrectly connected could not be cutted by the restrictionendonuclease. Therefore, this experiment was able to detect theseenzymes in the restriction endonuclease.The experiment of Blue-white screening in gene function,α-complementary phenomena, detected trace amounts of enzymeswhich destroyed the sticky ends in the restriction endonucleases, forexample the exonuclease. In vivo, proteins with biological functiononly could be translated by the complete DNA sequences. Thetranslation frameshift mutation would occur in the DNA sequences witha few bases missing, then it would not translate into the proteins withthe original biological function. This detection method was based onthis principle, coupled with the color change of the α-complementaryphenomenon, so the test results were to be obvious.