Rfx

Figure 4.6 • Mechanism of a restriction endonuclease reaction.

These cuts yields an overhanging C/C "sticky" end that is relatively easy to ligate with complementary ends of other DNA molecules. A cut from an endonuclease like Haelll:

Figure 4.6 • Mechanism of a restriction endonuclease reaction.

Restriction Endonucleases84,85

The restriction endonuclease (or restriction enzyme) is probably best described as a set of "molecular scissors" in nature. Restriction endonucleases are bacterial enzymes that, as the name implies, cleave internal phosphodiester bonds of a DNA molecule. The cleavage site on a segment of DNA lies within a specific nucleotide sequence of about 6 to 8 bp. More than 500 restriction endonucleases have been discovered, and these react with more than 100 different cleavage sites. The chemical reaction of the restriction endonuclease releases the 3' end of one base as an alcohol and the 5' end as a monophosphate. The general reaction is shown in Figure 4.6.

The recognition sites for restriction endonucleases are specific palindromic sequences of DNA85 not more than 8 bp long. A number of these palindromes are listed in Table 4.2. A palindrome is a sequence of letters that reads the same way forward and backward, for instance: "A man, a plan, a canal: Panama!," "DNA-land," "Did Hannah see bees? Hannah did." Restriction endonucleases cleave DNA at palindromic sites to yield several types of cuts:

5' CCTAGl

requires more complicated methods to ligate into vector DNA. Palindromic cleavage sites for some selected restriction enzymes are given in Table 4.2. The arrow shows the cleavage site. The restriction endonucleases are a robust group of enzymes that form a toolbox for investigators working in the field of genetic engineering. About the only caveat to their use is an obvious one: They must be chosen not to make their cut inside the gene of interest.

DNA Ligases86

When the gene of interest has been excised from its flanking DNA by the appropriate restriction endonucleases and the vector DNA has been opened (using the same restriction endonuclease to break phosphodiester bonds), the two different DNA molecules are brought together by annealing. In the first step of this process, heating unwinds the double-stranded DNA of the vector. The insert or passenger DNA is added to the heated mixture, and subsequent cooling facilitates pairing of complementary strands. Then, phosphodiester bonds are regenerated, linking the two DNA molecules, vector and insert. A total of four such bonds must be reformed, two on each strand at the 5' and 3' sites. This process is termed ligation, and the enzymes that catalyze the reaction are named DNA ligases. Typically, ATP or another energy source is required to drive the ligation reaction, and linker fragments of DNA are used to facilitate coupling. There are several different types of ligation reactions that are used, depending on the type of restriction endonuclease product that was formed. The sticky-ended DNA, using complementary vector and insert ends that easily base pair at the cuts, is probably the easiest to accomplish, although methods exist to ligate the blunt-ended varieties, using DNA ligase.

The Vector84

There are several methods available for introducing DNA into host cells. DNA molecules that can maintain themselves by replication are called replicons. Vectors are subsets of replicons. In genetic engineering, the vector (carrier) is the most widely used method for the insertion of foreign, or passenger, genetic material into a cell. Vectors are genetic elements such as plasmids or viruses that can be propagated and that have been engineered so that they can accept fragments of foreign DNA. Depending on the vector, they may have many other features, including multiple cloning sites

TABLE 4.2 Palindromic Cleavage Sites

AluI AsuII Bal I BamH1 Bgl II ClaI EcoRI

AG I CT TT CGAA TGG CCA G GATCC A GATCT AT CGAT G AATTC

TABLE 4.2 Palindromic Cleavage Sites

AluI AsuII Bal I BamH1 Bgl II ClaI EcoRI

AG I CT TT CGAA TGG CCA G GATCC A GATCT AT CGAT G AATTC

EcoRV

Haelll

HhaI

HindII

HindIII

HpaII

KpnI

GATlATC

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