style="font-size:15px;"> 7 The products are digested with appropriate restriction enzymes that flank the protein coding sequences and the mutated sequences are cloned into a plasmid vector to test for activity.
Figure 3.50 A random insertion protocol to introduce random mutations into a gene of interest. The inserted DNA is shown in yellow. Adapted from Murakami et al., Nat. Biotechnol. 20:76–81, 2002.
With this approach, it is possible to insert any small DNA fragment (carried on an adaptor) into the randomly cleaved single-stranded DNA, with the result that genes with a much greater number of modified nucleotides may be generated than by error-prone PCR. The mutations that are developed by this procedure may be used to select protein variants with a wide range of activities.
Random Mutagenesis with Degenerate Oligonucleotide Primers
In addition to introducing a specific nucleotide substitution into a gene, overlap extension PCR can be used to incorporate any of the four nucleotides at defined positions to generate all the possible amino acid changes in a particular region of a protein. This pattern of sequence degeneracy is achieved by programming an automated DNA synthesis reaction to add a low level (usually a few percent) of each of the three alternative nucleotides each time a particular nucleotide is added during the synthesis of an oligonucleotide primer (Fig. 3.51). In this way, the oligonucleotide primer preparation contains a heterogeneous set of DNA sequences that will generate a series of mutations that are clustered in a defined portion of the target gene. The degenerate oligonucleotides are employed as “internal” PCR primers to amplify the left and right portions of the target gene in separate reactions (Fig. 3.47). Mixing, denaturing, and annealing the left and right fragments produces some DNA molecules that overlap by complementarity and can be extended by DNA polymerase to produce a library of altered genes that have mutated sites in the region of the overlap of the degenerate oligonucleotides.
Figure 3.51 Chemical synthesis of oligonucleotide primers with any of the four nucleotides at defined positions. In this case, the flask with G phosphoramidite consists of a mixture of nucleotides, such as 94% G, 2% A, 2% C, and 2% T, leading to a mixture of oligonucleotides that may have A, C, or T at the sites where G is the specified nucleotide.
Mutagenesis using degenerate oligonucleotides confers two advantages over targeted mutagenesis: (1) Detailed information regarding the roles of particular amino acids in the functioning of the protein is not required; (2) Unexpected mutants encoding proteins with a range of interesting and useful properties may be generated because the introduced changes are not limited to one amino acid. Of course, should none of the mutants yield a protein with the properties that are being sought, then it may be necessary to repeat the entire procedure with a set of degenerate primers that is complementary to a different region of the gene.
DNA Shuffling
Some biologically important proteins, such as α-interferon (IFN-α), are encoded by a family of several related genes, with each protein having slightly different biological activity. If all, or at least several, of the genes or cDNAs for a particular protein have been isolated, it is possible to recombine portions of these genes or cDNAs to produce hybrid or chimeric forms (Fig. 3.52). This “DNA shuffling” is done with the expectation that some of the hybrid proteins will have unique properties or activities that were not encoded in any of the original sequences. Also, some of the hybrid proteins may combine important attributes of two or more of the original proteins (e.g., high activity and thermostability).
Figure 3.52 Amino acid changes may be introduced into a protein by either random mutagenesis or error-prone PCR, both of which cause single-amino-acid substitutions, and by DNA shuffling, in which genes are formed with large regions from different sources.
The simplest way to shuffle portions of similar genes is through the use of common restriction enzyme sites (Fig. 3.53). Digestion of two or more of the DNAs that encode the native forms of similar proteins with one or more restriction enzymes that cut the DNAs in the same place, followed by ligation of the mixture of DNA fragments, can potentially generate a large number of hybrids. For example, two DNAs, each of which has three unique restriction enzyme sites, can be recombined (shuffled) to produce 14 different hybrids in addition to the original DNA (Fig. 3.53).
Figure 3.53 The 14 different hybrid genes that can be generated by combining restriction enzyme fragments from two genes from the same gene family that have three different restriction sites in common. RE, restriction enzyme.
Another way to shuffle DNA involves combining several members of a gene family, fragmenting the mixed DNA with deoxyribonuclease I (DNase I), selecting smaller DNA fragments, and amplifying these fragments by PCR (Figure 3.54). During PCR, gene fragments from different members of a gene family cross-prime each other after DNA fragments bind to one another by complementary base pairing in regions of high homology. The final full-length products are amplified by PCR using terminal primers. After 20 to 30 PCR cycles, a panel of hybrid (full-length) DNAs will be established (Fig. 3.54). The hybrid DNAs are then cloned to create a library that can be screened for the desired activity. Although DNA shuffling works well with gene families—it is sometimes called molecular breeding—or with genes from different families that nevertheless have a high degree of homology, the technique is not especially useful when proteins have little or no homology. Thus, the DNAs must be very similar to one another or the PCR will not proceed. To remedy this situation and combine the genes of dissimilar proteins, several variations of the DNA-shuffling protocol have been described.
Figure 3.54 Some of the hybrid DNAs that can be generated during PCR amplification of three members of a gene family.
One procedure that was developed to combine the genes of dissimilar proteins and that does not rely on PCR amplification of DNA fragments is called nonhomologous random recombination. In this procedure (Fig. 3.55), DNAs from different sources (either defined or random DNA sequences, or a mixture of both) are combined and then partially digested with DNase I. These DNA fragments, which include a wide variety of sizes, are made blunt ended by digestion with the enzyme T4 DNA polymerase. This enzyme both fills in 5′ overhanging nucleotides and degrades 3′ overhanging nucleotides. The DNA fragments are then mixed with a synthetic DNA fragment that forms a hairpin loop and contains a specific restriction enzyme site. The entire mixture is ligated by the addition of the enzyme T4 DNA ligase that results in the formation of extended mosaic DNA molecules of variable lengths with a hairpin at each end. Ligation of the hairpins prevents further addition of fragments (concatemerization) to the molecules. The average length of these hairpin structures is dictated by the ratio between the blunt-ended DNA and the DNA hairpins added to the ligation reaction.
