There are more than 100 kinds of restriction enzymes, each enzyme has its specific nucleotide sequence recognition specificity, and the enzyme activity needs Mg + 2 to activate. Different enzymes also have many differences: some enzymes require the activation of other cofactors such as ATP in addition to Mg 2+; the distance between the cleavage site and the recognition sequence is different; and some endonucleases also have methylation. Based on these differences, restriction enzymes can be divided into I, II and III types.
Type II restriction enzymes require only divalent magnesium ions
Activation, the enzyme cuts double-stranded DNA within its recognition sequence, and the various DNA fragments produced have the same terminal structure, and most type II enzymes can provide sticky ends, which is conducive to re-ligation of fragments, most type II enzyme The identified sequence has a reverse symmetric structure, or palindrome structure. The identification and incisions of EcoRI and HindIII are as follows:
EcoRI: G ↓ AATT C HindIII: A ↓ AGCT T
T TCGA ↑ AC TTAA ↑ G
The number of fragments cut by the restriction enzyme on the circular plasmid DNA is the same as the number of nicks. Therefore, by identifying the number of fragments in the electrophoresis gel after digestion, the number of cuts can be inferred; from the fragment mobility, the size of the digested fragments can be determined. Using linear DNA of known molecular weight as a control, the relative molecular size of unknown DNA with the same molecular shape can be roughly measured by comparison of electrophoretic mobility.
The relative molecular weight of plasmid DNA (Mr) is generally in the range of 106 ～ 107. For example, the relative molecular weight of plasmid pBR322 is 2.8 × 106. There are three configurations in the cell: ①Covalent closed-loop DNA, often in the form of supercoil; If one or more of the two strands break at one or more places, the molecule can rotate to eliminate the tension of the strand. This relaxed molecule is called open-loop DNA; It is cut at the same site, cannot form a loop, and is completely opened into a linear shape, referred to as linear DNA. If the relative molecular weight of plasmid DNA is to be determined, it is best to hydrolyze the plasmid with a single nick to obtain linear DNA fragments. The electrophoresis speeds of the three configurations of plasmid DNA during electrophoresis are: covalent closed-loop DNA> linear DNA> open-loop DNA.
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