Detailed Introduction to Common Enzymes Used in Gene Cloning

The basic technology of genetic engineering is artificial cutting, splicing, and combination of genes. A gene is a DNA molecule with a certain function. To accurately cut out the DNA linear molecular fragments of different genes, various restriction endonucleases are required. To connect different fragments together, DNA ligase is required. To synthesize a gene or one of its fragments, DNA polymerase is required. Therefore, enzymes are indispensable tools in DNA recombination technology, and the enzymes used in genetic engineering are collectively referred to as tool enzymes.

Tool enzymes can be divided into three categories in terms of their uses: restriction endonucleases, ligases and modification enzymes. Among them, restriction endonucleases (restriction enzyme) are a large class of enzymes (up to thousands). Gene recombination uses these tool enzymes to carry out a series of enzymatic reactions on DNA molecules to realize the cutting and joining of DNA molecules in vitro. Therefore, the discovery of tool enzymes provides a very important technical basis for gene manipulation.

From the perspective of the development history of molecular biology, the discovery and application of nucleic acid restriction enzymes have played an incalculable role in the development of the discipline. The first experiment to clone foreign genes in Escherichia coli was completed in 1973. Stanley Cohen and Herbert Boyer used restriction endonucleases as a molecular scalpel.

Restriction endonucleases are a type of nucleic acid hydrolases in prokaryotes that can recognize specific base sequences in double-stranded DNA. The restriction and modification system of prokaryotes is like the immune system of higher animals, relying on a pair of restriction endonuclease and methylase activities that recognize the same sequence to resist foreign DNA invasion. When the genome replication is completed, the next round of DNA replication is modified by methylase (to methylate a specific sequence), avoiding recognition and hydrolysis by the corresponding restriction enzymes. The invading phage will be destroyed due to future modification, thereby protecting the bacteria from phage infection. Various bacteria can synthesize one or several sequence-specific endonucleases. The function of these enzymes is to cut DNA through the recognition of specific sequences to limit the intrusion of exogenous DNA into their own cells, so this endonuclease is called a restriction enzyme.

According to the characteristics of the enzyme's recognition and cleavage sequence, the catalytic conditions and whether it has the activity of modifying the enzyme, it is divided into three categories: I, II, and III:

Class I restriction endonucleases are bifunctional enzymes with modification activity (methylation) and endonuclease activity. When it acts, it needs to consume ATP, which can recognize a specific nucleotide sequence and cut the double strands of DNA molecules about 1000 nucleotide pairs from the recognition point. But the nucleotide sequence of the cut is random.

Class II restriction endonucleases only have endonuclease activity, can recognize a specific nucleotide sequence with a palindrome structure, and cut the double strand at a fixed position within the sequence. It does not need to hydrolyze ATP to provide energy.

Class III restriction endonucleases also have both modification activity and endonuclease activity, with a specific recognition sequence, but not a symmetric palindrome sequence. It cuts the double strand at a fixed position of 24-26 nucleotide pairs next to the recognition sequence, but these nucleotide pairs are arbitrary.