3.2 Cutting DNA
The most important enzymes for gene cloning are the restriction endonucleases. These nucleases make internal cuts in the nucleic acid molecule only where certain sequences of nucleotides are present. Thus, they tend to cleave DNA into rather large pieces rather than small fragments.
These enzymes are found in various bacterial species. They are believed to protect the organisms from any invading foreign DNA, such as might be introduced by bacterial viruses. The DNA of the host is protected by methylation of the DNA bases that are a part of the recognition sequence.
For example, Escherichia coli K has a restriction endonuclease EcoK which is coded for by the gene hsdR. However, EcoK does not cleave the host DNA because E. coli K has a methylase coded for by the gene hsdM that methylates the bases in the region where the endonuclease would cleave unprotected DNA. Both of the enzymes require the presence of gene hsdS which is needed for DNA recognition.
Over 800 restriction endonucleases are known. The most useful for cloning work are known as Type II which generally make a staggered cut within the recognition site and across the double stranded DNA.
The enzymes are named by a three letter code indicating the organism from which they were isolated. For example, EcoRI was isolated from E. coli and BamHI from Bacillus amyloliquefaciens.
The action of EcoRI is as follows:
Notice that the enzyme recognizes a six-base pair sequence in the DNA, and the cleavage results in single-stranded ends (sticky ends) that would readily reform hydrogen bonded base pairs again with each other.
Most restriction endonucleases recognize four, five or six base pair sequences. It can be calculated that if the base sequence of DNA were random, then the expected frequency for a given sequence would be 4 (for the number of different bases to the nth power:
Expected frequency of cleavage = 4n where n is the length of the recognized sequence.
Thus, the EcoRI site might be expected to occur about every 4096 base pairs. Enzymes that recognize a four base pair sequence (such as HaeIII)would be expected to produce shorter fragments (around 256 base pairs in length). Other restriction endonucleases are shown in the table.After cleaving with restriction endonucleases, plasmid DNA and the DNA to be inserted are allowed to anneal via their sticky ends.
Figure 17. Some commonly used type II restriction endonucleases
Figure 18. The three-dimensional structure of a restriction endonuclease showing the binding of DNA